transforming-growth-factor-beta and Carcinoma--Hepatocellular

Hepatocellular carcinoma is one of the major causes of cancer-related deaths worldwide and is associated with several inflammatory mediators, since 90% of HCCs occur based on chronic hepatitis B or C, alcoholism or increasingly metabolic syndrome-associated inflammation. EMT is a physiological process, with coordinated changes in epithelial gene signatures and is regulated by multiple factors, including cytokines and growth factors such as TGFβ, EGF, and FGF. Recent reports propose a strong association between EMT and inflammation, which is also correlated with tumor aggressiveness and poor outcomes. Cellular heterogeneity results collectively as an outcome of EMT, inflammation, and the tumor microenvironment, and it plays a fundamental role in the progression, complexity of cancer, and chemoresistance. In this review, we highlight recent developments concerning the association of EMT and inflammation in the context of HCC progression. Identifying potential EMT-related biomarkers and understanding EMT regulatory molecules will likely contribute to promising developments in clinical practice and will be a valuable tool for predicting metastasis in general and specifically in HCC.

Topics: Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Liver Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Hepatitis C virus (HCV) infection is remarkably efficient in establishing viral persistence, leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). Direct-acting antiviral agents (DAAs) are promising HCV therapies to clear the virus. However, recent reports indicate potential increased risk of HCC development among HCV patients with cirrhosis following DAA therapy. CD8+ T-cells participate in controlling HCV infection. However, in chronic hepatitis C patients, severe CD4+ and CD8+ T-cell dysfunctions have been observed. This suggests that HCV may employ mechanisms to counteract or suppress the host T-cell responses. The primary site of viral replication is within hepatocytes where infection can trigger the expression of costimulatory molecules and the secretion of immunoregulatory cytokines. Numerous studies indicate that HCV infection in hepatocytes impairs antiviral host immunity by modulating the expression of immunoregulatory molecules. Hepatocytes expressing whole HCV proteins upregulate the ligands of programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and transforming growth factor β (TGF-β) synthesis compared to those in hepatocytes in the absence of the HCV genome. Importantly, HCV-infected hepatocytes are capable of inducing regulatory CD4+ T-cells, releasing exosomes displaying TGF-β on exosome surfaces, and generating follicular regulatory T-cells. Recent studies report that the expression profile of exosome microRNAs provides biomarkers of HCV infection and HCV-related chronic liver diseases. A better understanding of the immunoregulatory mechanisms and identification of biomarkers associated with HCV infection will provide insight into designing vaccine against HCV to bypass HCV-induced immune dysregulation and prevent development of HCV-associated chronic liver diseases.

Topics: Antiviral Agents; Biomarkers; Carcinoma, Hepatocellular; Hepacivirus; Hepatitis C; Hepatitis C, Chronic; Hepatocytes; Humans; Liver Cirrhosis; Liver Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) is a transmembrane pseudoreceptor structurally related to transforming growth factor (TGF)-β type 1 receptors (TGF-β1Rs). BAMBI lacks a kinase domain and functions as a TGF-β1R antagonist. Essential processes such as cell differentiation and proliferation are regulated by TGF-β1R signaling. TGF-β is the best-studied ligand of TGF-βRs and has an eminent role in inflammation and fibrogenesis. Liver fibrosis is the end stage of almost all chronic liver diseases, such as non-alcoholic fatty liver disease, and at the moment, there is no effective anti-fibrotic therapy available. Hepatic BAMBI is downregulated in rodent models of liver injury and in the fibrotic liver of patients, suggesting that low BAMBI has a role in liver fibrosis. Experimental evidence convincingly demonstrated that BAMBI overexpression is able to protect against liver fibrosis. Chronic liver diseases have a high risk of hepatocellular carcinoma (HCC), and BAMBI was shown to exert tumor-promoting as well as tumor-protective functions. This review article aims to summarize relevant studies on hepatic BAMBI expression and its role in chronic liver diseases and HCC.

Topics: Activins; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Humans; Liver Cirrhosis; Liver Neoplasms; Membrane Proteins; Transforming Growth Factor beta

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis with insulin resistance, oxidative stress, lipotoxicity, adipokine secretion by fat cells, endotoxins (lipopolysaccharides) released by gut microbiota, and endoplasmic reticulum stress. Together, these factors promote NAFLD progression from steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and eventually end-stage liver diseases in a proportion of cases. Hepatic fibrosis and carcinogenesis often progress together, sharing inflammatory pathways. However, NASH can lead to hepatocarcinogenesis with minimal inflammation or fibrosis. In such instances, insulin resistance, oxidative stress, and lipotoxicity can directly lead to liver carcinogenesis through genetic and epigenetic alterations. Transforming growth factor (TGF)-β signaling is implicated in hepatic fibrogenesis and carcinogenesis. TGF-β type I receptor (TβRI) and activated-Ras/c-Jun-N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3 to create two phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TβRI/pSmad3C signaling terminates cell proliferation, while constitutive Ras activation and JNK-mediated pSmad3L promote hepatocyte proliferation and carcinogenesis. The pSmad3L signaling pathway also antagonizes cytostatic pSmad3C signaling. This review addresses TGF-β/Smad signaling in hepatic carcinogenesis complicating NASH. We also discuss Smad phospho-isoforms as biomarkers predicting HCC in NASH patients with or without cirrhosis.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Humans; Insulin Resistance; Liver Cirrhosis; Liver Neoplasms; Non-alcoholic Fatty Liver Disease; Protein Isoforms; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Despite progress in treating or preventing viral hepatitis, a leading cause of liver cancer, hepatocellular cancer (HCC) continues to be a major cause of cancer-related deaths globally. HCC is a highly heterogeneous cancer with many genetic alterations common within a patient's tumor and between different patients. This complicates therapeutic strategies. In this review, we highlight the critical role that the Smad-mediated transforming growth factor β (TGF-β) pathway plays both in liver homeostasis and in the development and progression of HCC. We summarize the mouse models that have enabled the exploration of the dual nature of this pathway as both a tumor suppressor and a tumor promoter. Finally, we highlight how the insights gained from evaluating pathway activity using transcriptional profiling can be used to stratify HCC patients toward rational therapeutic regimens based on the differences in patients with early or late TGF-β pathway activity or activated, normal, or inactivated profiles of this key pathway.

Topics: Animals; Carcinoma, Hepatocellular; Genes, Tumor Suppressor; Humans; Liver Neoplasms; Mice; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that performs a dual role as a tumor suppressor and tumor promoter during cancer progression. Among different ligands of the TGF-β family, TGF-β1 modulates most of its biological outcomes. Despite the abundant expression of TGF-β1 in the liver, steatosis to hepatocellular carcinoma (HCC) progression triggers elevated TGF-β1 levels, contributing to poor prognosis and survival. Additionally, elevated TGF-β1 levels in the tumor microenvironment create an immunosuppressive stage

Topics: Biomarkers; Carcinogens; Carcinoma, Hepatocellular; Humans; Immune Checkpoint Inhibitors; Ligands; Liver Neoplasms; Prognosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors; Tumor Microenvironment

The most effective way to control severity and mortality rate of the novel coronavirus disease (COVID-19) is through sensitive diagnostic approaches and an appropriate treatment protocol. We aimed to identify the effect of adding corticosteroid and Tocilizumab to a standard treatment protocol in treating COVID-19 patients with chronic disease through hematological and lab biomarkers.. This study was performed retrospectively on 68 COVID-19 patients with chronic disease who were treated by different therapeutic protocols. The patients were categorized into four groups: control group represented the patients' lab results at admission before treatment protocols were applied; group 1 included patients treated with anticoagulants, Hydroxychloroquine, and antibiotics; group 2 comprised patients treated with Dexamethasone; and group 3 included patients treated with Dexamethasone and Tocilizumab.. The study paves the way into the effectiveness of combining Dexamethasone with Tocilizumab in treatment COVID-19 patients with chronic diseases.. La forma más eficaz de controlar la gravedad y la tasa de mortalidad de la enfermedad del nuevo coronavirus (COVID-19) es mediante enfoques de diagnóstico sensibles y un protocolo de tratamiento adecuado. Nuestro objetivo fue identificar el efecto de agregar corticosteroides y tocilizumab a un protocolo de tratamiento estándar en el tratamiento de pacientes con COVID-19 con enfermedad crónica a través de biomarcadores hematológicos y de laboratorio.. Este estudio se realizó de forma retrospectiva en 68 pacientes COVID-19 con enfermedad crónica que fueron tratados por diferentes protocolos terapéuticos. Los pacientes se clasificaron en cuatro grupos: el grupo de control representaba los resultados de laboratorio de los pacientes en el momento de la admisión antes de que se aplicaran los protocolos de tratamiento; el grupo 1 incluyó a pacientes tratados con anticoagulantes, hidroxicloroquina y antibióticos; el grupo 2 estaba compuesto por pacientes tratados con dexametasona; y el grupo 3 incluyó a pacientes tratados con dexametasona y tocilizumab.. El estudio allana el camino hacia la eficacia de la combinación de dexametasona con tocilizumab en el tratamiento de pacientes con COVID-19 con enfermedades crónicas.. The Child-Mother Index constitutes a potential useful risk factor indicator for statistical analyses on data after birth. The value of the Child-Mother Index based on the estimated fetal weight before birth deserves evaluation.. Six ceria supports synthesized by various synthesis methodologies were used to deposit cobalt oxide. The catalysts were thoroughly characterized, and their catalytic activity for complete methane oxidation was studied. The supports synthesized by direct calcination and precipitation with ammonia exhibited the best textural and structural properties as well as the highest degree of oxidation. The remaining supports presented poorer textural properties to be employed as catalytic supports. The cobalt deposited over the first two supports presented a good dispersion at the external surface, which induced a significant redox effect that increased the number of Co. Some studies show that children with obesity are more likely to receive a diagnosis of depression, anxiety, or attention-deficit hyperactivity disorder (ADHD). But this does not necessarily mean obesity causes these conditions. Depression, anxiety, or ADHD could cause obesity. A child's environment, including family income or their parents' mental health, could also affect a child's weight and mental health. Understanding the nature of these relationships could help scientists develop better interventions for both obesity and mental health conditions. Genetic studies may help scientists better understand the role of the environment in these conditions, but it's important to consider both the child's and their parents’ genetics in these analyses. This is because parents and children share not only genes, but also environmental conditions. For example, families that carry genetic variants associated with higher body weight might also have lower incomes, if parents have been affected by biases against heavier people in society and the workplace. Children in these families could have worse mental health because of effects of their parent’s weight, rather than their own weight. Looking at both child and adult genetics can help disentangle these processes. Hughes et al. show that a child's own body mass index, a ratio of weight and height, is not strongly associated with the child’s mental health symptoms. They analysed genetic, weight, and health survey data from about 41,000 8-year-old children and their parents. The results suggest that a child's own BMI does not have a large effect on their anxiety symptoms. There was also no clear evidence that a child's BMI affected their symptoms of depression or ADHD. These results contradict previous studies, which did not account for parental genetics. Hughes et al. suggest that, at least for eight-year-olds, factors linked with adult weight and which differ between families may be more critical to a child's mental health than a child’s own weight. For older children and adolescents, this may not be the case, and the individual’s own weight may be more important. As a result, policies designed to reduce obesity in mid-childhood are unlikely to greatly improve the mental health of children. On the other hand, policies targeting the environmental or societal factors contributing to higher body weights, bias against people with higher weights, and poor child mental health directly may be more beneficial.. The development of an efficient photocatalyst for C2 product formation from CO. Оценка антиастенического эффекта последовательной терапии левокарнитином (ЛК) и ацетилкарнитином (АЛК) пациентов с артериальной гипертензией и/или ишемической болезнью сердца (ИБС) с астеническим синдромом (АС).. В открытое сравнительное исследование были включены 120 пациентов в возрасте 54—67 лет с артериальной гипертензией и/или ИБС с АС. Пациенты 1-й группы (. У больных 1-й группы отмечено статистически значимое уменьшение различных проявлений АС. Отличия носили достоверный характер по сравнению как с исходным уровнем, так и со 2-й группой. Установлено эндотелийпротективное действие ЛК и АЛК.. Полученные результаты свидетельствуют, что у таких коморбидных пациентов использование ЛК и АЛК уменьшает выраженность проявлений АС, а установленные эндотелиотропные свойства препаратов позволяют рекомендовать их в составе комплексной персонифицированной терапии пациентов с сердечно-сосудистыми заболеваниями.. Naproxen sodium 440 mg/diphenhydramine 50 mg combination demonstrated improvement in sleep maintenance (WASO) vs. naproxen sodium 550 mg and higher efficiency in average daily pain reduction compared with the comparison groups. The treatment was well tolerated There were no serious or unexpected adverse events reported in the study.. Сравнительный анализ эффективности и безопасности новой комбинации напроксена натрия и дифенгидрамина у пациентов с неспецифическим болевым синдромом в пояснично-крестцовом отделе спины (M54.5 «Боль внизу спины») и нарушением сна (G47.0 «Нарушения засыпания и поддержания сна [бессонница]»).. Проведено проспективное многоцентровое рандомизированное открытое сравнительное в параллельных группах клиническое исследование. Пациенты были рандомизированы в 3 группы. Больные 1-й группы получали напроксен натрия (440 мг) и дифенгидрамин (50 мг), 2-й — напроксен натрия (550 мг), 3-й — парацетамол (1000 мг) и дифенгидрамин (50 мг). Исследуемые препараты пациенты принимали однократно перед сном в течение 3 дней. Все пациенты также принимали 275 мг (1 таблетка) напроксена натрия в качестве препарата фоновой терапии. Первичным критерием эффективности было общее время бодрствования после наступления сна (WASO), измеряемое методом актиграфии. Также использовались критерии оценки продолжительности и качества сна и выраженности боли.. Анализ эффективности проведен для ITT популяции (. Применение комбинации напроксена натрия (440 мг) и дифенгидрамина (50 мг) характеризовалось более выраженным поддержанием сна по сравнению с напроксеном натрия 550 мг и более высокой эффективностью в отношении снижения интенсивности боли по сравнению со 2-й и 3-й группами. Отмечена хорошая переносимость препарата, серьезных нежелательных явлений зарегистрировано не было.

Topics: Acetaminophen; Acetylcarnitine; Acetylcholinesterase; Acids; Acinetobacter baumannii; Acinetobacter Infections; Adaptation, Psychological; Adolescent; Adsorption; Adult; Aged; Alcohol Drinking; Alzheimer Disease; Amikacin; Ammonia; Anaerobiosis; Animals; Anorexia; Anti-Bacterial Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Anxiety; Aptamers, Nucleotide; Asthenia; Attention Deficit Disorder with Hyperactivity; Bacterial Proteins; Beryllium; beta-Lactamases; Biofuels; Biomass; Biosensing Techniques; Bismuth; Blister; Body Mass Index; Body Surface Area; Boronic Acids; Brain; Breast Neoplasms; Butyrylcholinesterase; Cannabis; Carbapenems; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carboxylic Acids; Carcinoma, Hepatocellular; Cardiovascular Diseases; Carnitine; Case-Control Studies; Catalysis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Child; China; Cholinesterase Inhibitors; Clarithromycin; Clostridioides; Clostridioides difficile; Clostridium Infections; Cohort Studies; Colistin; Colitis; Colon; Coloring Agents; Coronary Artery Bypass; Creatinine; Crystalloid Solutions; Cytokines; Depression; Dextran Sulfate; Dextrans; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Diarrhea; Dietary Supplements; Diphenhydramine; Disease Models, Animal; Disease Outbreaks; Double-Blind Method; Doxorubicin; Drosophila; Drug Tapering; Dysbiosis; Electrons; Escherichia coli; Extracellular Vesicles; Fatigue; Female; Fermentation; gamma-Cyclodextrins; Gastrointestinal Microbiome; Glucose; Graft Survival; Graft vs Host Disease; Head and Neck Neoplasms; Heart Arrest, Induced; Hematopoietic Stem Cell Transplantation; High-Intensity Interval Training; Hippocampus; Humans; Hydrogen-Ion Concentration; Hypertension; Incidence; Interferon-gamma; Italy; Kinetics; Klebsiella Infections; Klebsiella pneumoniae; Lab-On-A-Chip Devices; Lactoferrin; Larva; Length of Stay; Lignin; Liver; Liver Neoplasms; Liver Transplantation; Living Donors; Low Back Pain; Lung; Lung Volume Measurements; Macrophages; Male; Melphalan; Men; Mendelian Randomization Analysis; Meropenem; Methane; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Mitochondrial Proteins; Molecular Docking Simulation; Molecular Structure; Mothers; Motivation; Mycoplasma; Mycoplasma hominis; Mycoplasma Infections; NAD; Nanocomposites; Nanoparticles; Nanotubes, Carbon; Naproxen; Neovascularization, Pathologic; Neurons; Nitrates; Nucleolin; Opuntia; Paratyphoid Fever; Phenotype; Phosphatidylinositol 3-Kinases; Phytochemicals; Plant Extracts; Pregnancy; Prevalence; Prospective Studies; Proto-Oncogene Proteins c-akt; Pulmonary Disease, Chronic Obstructive; Rats; Rats, Wistar; Resveratrol; Retrospective Studies; Rifampin; Risk Factors; RNA, Messenger; Selenium; Sleep; Social Behavior; Soil; Soil Pollutants; Squamous Cell Carcinoma of Head and Neck; Staphylococcus aureus; Structure-Activity Relationship; Suicidal Ideation; Suicide; Superoxide Dismutase-1; Surveys and Questionnaires; Swimming; Syndrome; Tannins; Temperature; Transforming Growth Factor beta; Transplantation Conditioning; Treatment Outcome; Triple Negative Breast Neoplasms; Troponin T; Tumor Microenvironment; United Kingdom; Ureaplasma; Ureaplasma urealyticum; Urinary Tract Infections; Viscum; Waste Disposal Facilities; Wastewater; Water; Water Pollutants, Chemical; Wolfiporia; Young Adult

At the heart of hepatocellular carcinoma (HCC) lies disruption of signaling pathways at the level of molecules, genes, and cells. Non-coding RNAs (ncRNAs) have been implicated in the disease progression of HCC. For instance, dysregulated expression of circular RNAs (circRNAs) has been observed in patients with HCC. As such, these RNAs are potential therapeutic targets and diagnostic markers for HCC. Long non-coding RNAs (lncRNAs), a type of ncRNA, have also been recognized to participate in the initiation and progression of HCC. Transforming growth factor-beta (TGF-β) is another element which is now recognized to play crucial roles in HCC. It has been implicated in many biological processes such as survival, immune surveillance, and cell proliferation. In HCC, TGF-β promotes disease progression by two mechanisms: an intrinsic signaling pathway and the extrinsic pathway. Through these pathways, it modulates various microenvironment factors such as inflammatory mediators and fibroblasts. An interesting yet-to-be resolved concept is whether the HCC-promoting role of TGF-β pathways is limited to a subset of HCC patients or it is involved in the whole process of HCC development. This review summarizes recent advancements to highlight the roles of circRNAs, lncRNAs, and TGF-β in HCC.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Disease Progression; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Liver Neoplasms; RNA, Circular; RNA, Long Noncoding; Transforming Growth Factor beta; Tumor Microenvironment

Primary liver cancer is one of the leading causes for cancer-related death worldwide. Transforming growth factor beta (TGF-β) is a pleiotropic cytokine that signals through membrane receptors and intracellular Smad proteins, which enter the nucleus upon receptor activation and act as transcription factors. TGF-β inhibits liver tumorigenesis in the early stage by inducing cytostasis and apoptosis, but promotes malignant progression in more advanced stages by enhancing cancer cell survival, EMT, migration, invasion and finally metastasis. Understanding the molecular mechanisms underpinning the multi-faceted roles of TGF-β in liver cancer has become a persistent pursuit during the last two decades. Contextual regulation fine-tunes the robustness, duration and plasticity of TGF-β signaling, yielding versatile albeit specific responses. This involves multiple feedback and feed-forward regulatory loops and also the interplay between Smad signaling and non-Smad pathways. This review summarizes the known regulatory mechanisms of TGF-β signaling in liver cancer, and how they channel, skew and even switch the actions of TGF-β during cancer progression.

Topics: Carcinoma, Hepatocellular; Disease Progression; Epithelial-Mesenchymal Transition; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Neoplasm Invasiveness; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Therapeutic attempts to treat hepatocellular carcinoma (HCC) frequently result in a poor response or treatment failure. The efficacy of approved drugs and survival expectancies is affected by an ample degree of variability that can be explained at least in part by the enormous between-patient cellular and molecular heterogeneity of this neoplasm. Transforming growth factor-β (TGF-β) is hyperactivated in a large fraction of HCCs, where it influences complex interactive networks covering multiple cell types and a plethora of other local soluble ligands, ultimately establishing several malignancy traits. This cytokine boosts the invasiveness of cancerous epithelial cells through promoting the epithelial-to-mesenchymal transition program, but also skews the phenotype of immune cells toward a tumor-supporting status. Here, we discuss recent strategies pursued to offset TGF-β-dependent processes that promote metastatic progression and immune surveillance escape in solid cancers, including HCC. Moreover, we report findings indicating that TGF-β reduces the expression of the proinflammatory factors CCL4 and interleukin-1β (IL-1β in human ex vivo treated HCC tissues. While this is consistent with the anti-inflammatory properties of TGF-β, whether it is an outright tumor promoter or suppressor is still a matter of some debate. Indeed, IL-1β has also been shown to support angiogenesis and cell invasiveness in some cancers. In addition, we describe an inhibitory effect of TGF-β on the secretion of CCL2 and CXCL1 by HCC-derived fibroblasts, which suggests the existence of an indirect stroma-mediated functional link between TGF-β and downstream immunity.

Topics: Animals; Carcinoma, Hepatocellular; Disease Models, Animal; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Mesenchymal Stem Cells; Mice; Neoplasm Invasiveness; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Hepatocellular carcinoma (HCC) is an inflammation-induced and chemotherapy-resistant cancer. Dysregulated signaling in the transforming growth factor beta (TGF-β) pathway plays a central role in inflammation, fibrogenesis, and immunomodulation in the HCC microenvironment. This review dissects the genetic landscape of the TGF-β superfamily genes in HCC and discusses the essential effects of this pathway on the tumor immune microenvironment. We highlight the TGF-β signature as a potential biomarker for identifying individualized immunotherapeutic approaches in HCC. An improved understanding of the detailed mechanisms of liver cancer immunogenicity and the specific role of TGF-β in mediating immunotherapy resistance in HCC will provide important insights into HCC immune escape and promote the development of biomarker-derived combination immunotherapies for HCC.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Gene Expression Profiling; Genomics; Humans; Immunotherapy; Liver Neoplasms; Molecular Targeted Therapy; Programmed Cell Death 1 Receptor; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide accounting for more than 700 thousand deaths per year. Most of the HCC develops in a cirrhotic liver, a microenvironment where fibrotic tissue replaces parenchymal cells. Thus, there is a close connection between fibrosis and HCC development. Understanding the cellular and molecular mechanisms involved in this process is a crucial step to advance in novel therapeutic or pharmacological strategies to prevent or improve the course of this malignancy. A key molecular player capable of modulating cell growth and fibrosis is the Transforming Growth Factor-beta (TGF-β). Interestingly, TGF-β seems to act like a switch, since it has dual and opposite roles during early and late phases of cancer development. Therefore to develop therapies that target TGF-β signaling pathway for HCC treatment is important to understand the underlying pathogenetic mechanisms at play with special emphasis in the crosstalk between TGF-β and other signaling pathways. In recent years, a plethora of TGR-β have been developed and some of them are under clinical investigations for testing in patients with advanced HCC. In this review, we summarize recent knowledge about the role of TGF-β signaling pathway in HCC progression.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Humans; Liver Neoplasms; Molecular Targeted Therapy; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is a cytokine essential for the induction of the fibrotic response and for the activation of the cancer stroma. Strong evidence suggests that a strong cross-talk exists among TGF-β and the tissue extracellular matrix components. TGF-β is stored in the matrix as part of a large latent complex bound to the latent TGF-β binding protein (LTBP) and matrix binding of latent TGF-β complexes, which is required for an adequate TGF-β function. Once TGF-β is activated, it regulates extracellular matrix remodelling and promotes a fibroblast to myofibroblast transition, which is essential in fibrotic processes. This cytokine also acts on other cell types present in the fibrotic and tumour microenvironment, such as epithelial, endothelial cells or macrophages and it contributes to the cancer-associated fibroblast (CAF) phenotype. Furthermore, TGF-β exerts anti-tumour activity by inhibiting the host tumour immunosurveillance. Aim of this review is to update how TGF-β and the tissue microenvironment cooperate to promote the pleiotropic actions that regulate cell responses of different cell types, essential for the development of fibrosis and tumour progression. We discuss recent evidences suggesting the use of TGF-β chemical inhibitors as a new line of defence against fibrotic disorders or cancer.

Topics: Animals; Carcinoma, Hepatocellular; Cellular Microenvironment; Extracellular Matrix; Humans; Liver Cirrhosis; Liver Neoplasms; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, and represents the second most frequently cancer and third most common cause of death from cancer worldwide. At advanced stage, HCC is a highly aggressive tumor with a poor prognosis and with very limited response to common therapies. Therefore, there is still the need for new effective and well-tolerated therapeutic strategies. Molecular-targeted therapies hold promise for HCC treatment. One promising molecular target is the multifunctional serine/threonine kinase glycogen synthase kinase 3 (GSK-3). The roles of GSK-3β in HCC remain controversial, several studies suggested a possible role of GSK-3β as a tumor suppressor gene in HCC, whereas, other studies indicate that GSK-3β is a potential therapeutic target for this neoplasia. In this review, we will focus on the different roles that GSK-3 plays in HCC and its interaction with signaling pathways implicated in the pathogenesis of HCC, such as Insulin-like Growth Factor (IGF), Notch, Wnt/β-catenin, Hedgehog (HH), and TGF-β pathways. In addition, the pivotal roles of GSK3 in epithelial-mesenchymal transition (EMT), invasion and metastasis will be also discussed.

Topics: Antineoplastic Agents; beta Catenin; Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hedgehog Proteins; Humans; Insulin-Like Growth Factor I; Liver Neoplasms; Molecular Targeted Therapy; Receptors, Notch; Signal Transduction; Survival Analysis; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most malignant tumors in Chinese people and offers poor prognosis. Tumor tissue, like normal tissue, is hierarchically differentiated. Thus, minor tumor cell populations able to differentiate, such as stem cells, sustain tumor self-renewal and proliferation. The fact that liver cancer stem cells (CSCs) with different surface markers appear heterogeneous with respect to oncogenesis and drug resistance indicates that subpopulations of surface markers preserve the hierarchical potential of differentiation during proliferation, deterioration and relapse. The epithelial to mesenchymal transition (EMT) is correlated to tumor malignancy and aggression, and hepatocytes bearing EMT have obvious hierarchical differentiation potential with respect to signaling pathways such as transforming growth factor β, Wnt/β-catenin and microRNA. Therefore, it may be more effective for early diagnosis to monitor HCC recurrence using peripherally circulating CSCs, and these may also offer potential for HCC immunotherapy or for targeting HCC treatment using these markers. Thus, we reviewed the generation, hierarchical differentiation and clinical application of hepatic CSCs.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cell Differentiation; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is the leading cause of cancer death, and hepatitis B virus (HBV) infection is one of the commonest causes in Asian countries. India has the second largest pool after China for hepatitis B-infected subjects. HBV clearance is T cell dependent, and one of the reasons for T cells hyporesponsiveness is due to mass production of regulatory T cells (Tregs) through activation of Notch signalling, which suppress CD4/CD8 T cells. Tregs are important to maintain cellular homoeostasis; however, during viral infection increase of Tregs is inversely proportional to HBV DNA titres. Tregs exert their suppressive effect either via cell-to-cell contact or through release of interleukin (IL)-2, IL-10, TGF-β and IL-35. In Chronic hepatitis B virus CHBV infection, PD-1 pathway also gets activated and is involved in promoting tolerance. However, with Tregs induction, virus-specific T cell responses also get decreased. Circulatory and intratumoural Tregs promote development of HBV-specific HCC more by decreasing and impairing the effector functions of CD8 T cells. Antiviral therapies and PD-1 blockade strategy had shown the inhibition of Tregs and reduction in HBV DNA. However, inhibition of HBV-specific Tregs is major challenge for future therapies. New cytokine blockade therapies have emerged as potential therapeutic potentials.

Topics: Carcinoma, Hepatocellular; Hepatitis B virus; Hepatitis B, Chronic; Humans; India; Interleukin-1; Interleukin-10; Interleukin-2; Liver Neoplasms; Programmed Cell Death 1 Receptor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Viral Load

Chronic intake of alcohol undoubtedly overwhelms the structural and functional capacity of the liver by initiating complex pathological events characterized by steatosis, steatohepatitis, hepatic fibrosis and cirrhosis. Subsequently, these initial pathological events are sustained and ushered into a more complex and progressive liver disease, increasing the risk of fibro-hepatocarcinogenesis. These coordinated pathological events mainly result from buildup of toxic metabolic derivatives of alcohol including but not limited to acetaldehyde (AA), malondialdehyde (MDA), CYP2E1-generated reactive oxygen species, alcohol-induced gut-derived lipopolysaccharide, AA/MDA protein and DNA adducts. The metabolic derivatives of alcohol together with other comorbidity factors, including hepatitis B and C viral infections, dysregulated iron metabolism, abuse of antibiotics, schistosomiasis, toxic drug metabolites, autoimmune disease and other non-specific factors, have been shown to underlie liver diseases. In view of the multiple etiology of liver diseases, attempts to delineate the mechanism by which each etiological factor causes liver disease has always proved cumbersome if not impossible. In the case of alcoholic liver disease (ALD), it is even more cumbersome and complicated as a result of the many toxic metabolic derivatives of alcohol with their varying liver-specific toxicities. In spite of all these hurdles, researchers and experts in hepatology have strived to expand knowledge and scientific discourse, particularly on ALD and its associated complications through the medium of scientific research, reviews and commentaries. Nonetheless, the molecular mechanisms underpinning ALD, particularly those underlying toxic effects of metabolic derivatives of alcohol on parenchymal and non-parenchymal hepatic cells leading to increased risk of alcohol-induced fibro-hepatocarcinogenesis, are still incompletely elucidated. In this review, we examined published scientific findings on how alcohol and its metabolic derivatives mount cellular attack on each hepatic cell and the underlying molecular mechanisms leading to disruption of core hepatic homeostatic functions which probably set the stage for the initiation and progression of ALD to fibro-hepatocarcinogenesis. We also brought to sharp focus, the complex and integrative role of transforming growth factor beta/small mothers against decapentaplegic/plasminogen activator inhibitor-1 and the mitogen activated protein kinase

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Ethanol; Gastrointestinal Microbiome; Humans; Liver; Liver Cirrhosis, Alcoholic; Liver Diseases, Alcoholic; Liver Neoplasms; MAP Kinase Signaling System; Plasminogen Activator Inhibitor 1; Signal Transduction; Transforming Growth Factor beta

The transforming growth factor-beta (TGF-β) family signalling pathways play essential roles in the regulation of different cellular processes, including proliferation, differentiation, migration or cell death, which are essential for the homeostasis of tissues and organs. Because of the diverse and pleiotropic TGF-β functions, deregulation of its pathways contributes to human disease. In the case of the liver, TGF-β signalling participates in all stages of disease progression, from initial liver injury through inflammation and fibrosis, to cirrhosis and cancer. TGF-β has cytostatic and apoptotic effects in hepatocytes, promoting liver differentiation during embryogenesis and physiological liver regeneration. However, high levels of TGF-β, as a consequence of chronic liver damage, result in activation of stellate cells to myofibroblasts and massive hepatocyte cell death, which contributes to the promotion of liver fibrosis and later cirrhosis. During liver tumorigenesis, TGF-β may behave as a suppressor factor at early stages; however, there is strong evidence that overactivation of TGF-β signalling might contribute to later tumour progression, once cells escape from its cytostatic effects. For these reasons, targeting the TGF-β signalling pathway is being explored to counteract liver disease progression. In this review, we aim to shed light on the state-of-the-art in the signalling pathways induced by TGF-β that are involved in different stages of liver physiology and pathology.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Hepatocytes; Humans; Inflammation; Liver Cirrhosis; Liver Neoplasms; Liver Regeneration; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (TGF)-β is a pluripotent cytokine that displays several tissue-specific biological activities. In the liver, TGF-β is considered a fundamental molecule, controlling organ size and growth by limiting hepatocyte proliferation. It is involved in fibrogenesis and, therefore, in worsening liver damage, as well as in triggering the development of hepatocellular carcinoma (HCC). TGF-β is known to act as an oncosuppressor and also as a tumour promoter in HCC, but its role is still unclear.. In this review, we discuss the potential role of TGF-β in regulating the tumoural progression of HCC, and therefore the rationale for targeting this molecule in patients with HCC.. A considerable amount of experimental preclinical evidence suggests that TGF-β is a promising druggable target in patients with HCC. To support this hypothesis, a phase II clinical trial is currently ongoing using a TGF-β pathway inhibitor, and results will soon be available.. The identification of new TGF-β related biomarkers will help to select those patients most likely to benefit from therapy aimed at inhibiting the TGF-β pathway. New formulations that may provide a more controlled and sustained delivery of the drug will improve the therapeutic success of such treatments.

Topics: Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Humans; Liver Cirrhosis; Liver Diseases, Alcoholic; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease and a risk factor for cirrhosis and hepatocellular carcinoma. The pathological features of NASH include steatosis, hepatocyte injury, inflammation, and various degrees of fibrosis. Steatosis reflects disordered lipid metabolism. Insulin resistance and excessive fatty acid influx to the liver are two important contributing factors. Steatosis is also likely associated with lipotoxicity and cellular stresses such as oxidative stress and endoplasmic reticulum stress, which result in hepatocyte injury. Inflammation and fibrosis are frequently triggered by various signals such as proinflammatory cytokines and chemokines, released by injuried hepatocytes and activated Kupffer cells. Although much progress has been made, the pathogenesis of NASH is not fully elucidated. The purpose of this review is to discuss the current understanding of NASH pathogenesis, mainly focusing on factors contributing to steatosis, hepatocyte injury, inflammation, and fibrosis.

Topics: Adipose Tissue; Animals; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Chemokines; Cytokines; Fatty Acids; Fibrosis; Gastrointestinal Microbiome; Genetic Predisposition to Disease; Hepatic Stellate Cells; Hepatocytes; Humans; Inflammation; Insulin Resistance; Kupffer Cells; Lipid Metabolism; Lipopolysaccharides; Liver; Liver Neoplasms; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Risk Factors; Signal Transduction; Transforming Growth Factor beta

The epithelial to mesenchymal transition (EMT) is a multistep biological process whereby epithelial cells change in plasticity by transient de-differentiation into a mesenchymal phenotype. EMT and its reversal, mesenchymal to epithelial transition (MET), essentially occur during embryogenetic morphogenesis and have been increasingly described in fibrosis and cancer during the last decade. In carcinoma progression, EMT plays a crucial role in early steps of metastasis when cells lose cell-cell contacts due to ablation of E-cadherin and acquire increased motility to spread into surrounding or distant tissues. Epithelial plasticity has become a hot issue in hepatocellular carcinoma (HCC), as strong inducers of EMT such as transforming growth factor-β are able to orchestrate both fibrogenesis and carcinogenesis, showing rising cytokine levels in cirrhosis and late stage HCC. In this review, we consider the significance of EMT-MET in malignant hepatocytes as well as changes in the plasticity of hepatic stellate cells for cellular heterogeneity of HCC, and further aim at explaining the current limiting insights into EMT by snapshot analyses of HCC tissues. Recent advances in the identification of clinically relevant mechanisms that impinge on important EMT-transcription factors, as well as on miRNAs causing EMT signatures and HCC progression are highlighted. In addition, we draw particular attention to framing EMT in the context of potential clinical relevance for HCC patients. We conclude that some aspects of EMT are still elusive and further studies are required to better link the clinical management of HCC with biomarkers and targeted therapies related to EMT.

Topics: Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Transforming Growth Factor beta

Primary liver cancer, mainly consisting of hepatocellular carcinoma (HCC), is one of common malignancies worldwide, and prevalent among the Chinese population. A diagnosis of early stage HCC has proven to be very difficult because of its insidious feature in onset and development. At the time of diagnosis, most HCC cases are locally advanced and/or distant metastatic, which results in difficulty to be treated and poor prognosis. For advanced HCC, systemic therapy is frequently adopted as an important palliative method. In recent years, clinical studies and observations have often reported about systemic anti-cancer therapy of advanced HCC, including molecular target therapy, systemic chemotherapy and immunotherapy. In this article, we review these treatment modalities to provide a reference for clinicians.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Deoxycytidine; Fluorouracil; Humans; Immunotherapy; Leucovorin; Liver Neoplasms; Mitogen-Activated Protein Kinase Kinases; Molecular Targeted Therapy; Organoplatinum Compounds; Transforming Growth Factor beta

Alcohol abuse predisposes individuals to the development of hepatocellular carcinoma (HCC) and synergistically heightens the HCC risk in patients infected with hepatitis C virus (HCV). The mechanisms of this synergism have been elusive until our recent demonstration of the obligatory role of ectopically expressed TLR4 in liver tumorigenesis in alcohol-fed HCV Ns5a or Core transgenic mice. CD133+/CD49f+ tumor-initiating stem cell-like cells (TICs) isolated from these models are tumorigenic in a manner dependent on TLR4 and NANOG. TICs' tumor-initiating activity and chemoresistance are causally associated with inhibition of TGF-β tumor suppressor pathway due to NANOG-mediated expression of IGF2BP3 and YAP1. TLR4/NANOG activation causes p53 degradation via phosphorylation of the protective protein NUMB and its dissociation from p53 by the oncoprotein TBC1D15. Nutrient deprivation reduces overexpressed TBC1D15 in TICs via autophagy-mediated degradation, suggesting a possible role of this oncoprotein in linking metabolic reprogramming and self-renewal.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Ethanol; GTPase-Activating Proteins; Humans; Liver Neoplasms; Mice; Neoplastic Stem Cells; Proto-Oncogenes; Toll-Like Receptor 4; Transforming Growth Factor beta

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are common primary liver cancers worldwide. However, the survival and prognosis of ICC are much poorer than those of HCC, indicating the different molecular characteristics and mechanisms between ICC and HCC. To identify differentially expressed (DE) genes between ICC and HCC or combined hepatocellular-cholangiocarcinoma (CHC), we performed integrated analysis of publicly available microarray Gene Expression Omnibus (GEO) datasets by MetaOmics. Three GEO datasets comprising 32 ICC biochips, 77 HCC biochips, and 34 CHC biochips were available for the data integration. We identified 7313 DE genes between ICC and HCC, including 3650 upregulated genes and 3663 downregulated genes. The S100 family members on chromosome 1q21 were extensively upregulated in ICC, and S100A11 had the greatest degree of upregulation in ICC. Based on the DE genes, combined gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed the enhanced pathways of local adhesion, ECM-receptor interaction, and regulation of action cytoskeleton, suggesting the enhanced communication between ICC and the microenvironment. Additionally, development-related genes and development-related pathways, including the Notch, Wnt, and TGF-β signaling pathways, were shown to be active prominently in ICC. Taken together, we identified the characteristically upregulated or downregulated DE genes and pathways in ICC compared with HCC or CHC. These DE genes and pathways supply new transcriptomics evidence for ICC and could help identify new therapeutic targets.

Topics: Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Cholangiocarcinoma; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Microarray Analysis; Neoplasm Proteins; Prognosis; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is a major health problem. Mortality owing to liver cancer has increased in the past 20 years, with recent studies reporting an incidence of 780,000 cases/year. Most patients with hepatocellular carcinoma are still diagnosed at intermediate or advanced disease stages, where curative approaches are often not feasible. Among the treatment options available, the molecular targeted agent sorafenib is able to significantly increase overall survival in these patients. Afterwards, up to 7 randomized phase III clinical trials investigating other molecular therapies in the first-line and second-line settings have failed to improve survival. Potential reasons for this include intertumor heterogeneity, issues with trial design and a lack of predictive biomarkers of response. Advance in our knowledge of the human genome has provided a comprehensive picture of commonly mutated genes in patients with HCC including mutations in the TERT promoter, CTNNB1, TP53 and ARID1A along with other amplifications (FGF19, VEGFA) or homozygous deletions (p16) as the most frequent alterations. This knowledge points toward specific drivers as candidate for druggable therapies. Thus, progressive implementation of proof-of-concept and enrichment might improve results in clinical trials testing of molecular targeted agents. Ultimately, these studies are aimed at long-term to improve current standards of care and influenced clinical decision-making and practice guidelines.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Hepatocellular; Clinical Trials, Phase II as Topic; Fibroblast Growth Factors; Humans; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Proto-Oncogene Proteins c-met; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sorafenib; Transforming Growth Factor beta

Liver fibrosis is a pathological consequence of chronic liver diseases and results from the progressive accumulation of altered extracellular matrix, highly enriched in type I and III fibrillar collagens. In advanced stages, fibrosis leads to cirrhosis, defined by abnormal liver architecture and altered vascularization. Clinical consequences of cirrhosis are failure in the synthetic function of the liver, portal hypertension, high susceptibility to infection and high risk to develop hepatocellular carcinoma (HCC). The TGF-β family of cytokines plays essential roles in many cellular processes, including growth inhibition, cell migration and invasion, extracellular matrix remodelling and immune suppression, being involved in the maintenance of tissue homeostasis. However, TGF-βs are often continuously overexpressed in disease states, such as fibrosis, inflammation and cancer, and they play pivotal roles in the sequence of events leading to end-stage of chronic liver diseases. Reactive oxygen species (ROS) are critical intermediates in liver physiology and pathology. When the equilibrium between ROS generation and the antioxidant defence of the cell is disrupted, it results in an oxidative stress process. The NADPH oxidase (NOX) family has emerged in the last years as important source of ROS in liver pathologies. Interestingly, NOXes mediate TGF-β actions in liver cells, such as regulation of hepatocyte growth and death, as well as activation of hepatic stellate cells to myofibroblasts, key executers of the fibrotic process. In this review we will update the relevant and differential roles of NOX isoforms during liver fibrosis and hepatocarcinogenesis, their cross-talk with the TGF-β pathway and their potential as therapeutic targets for these diseases.

Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Cirrhosis; Liver Neoplasms; NADPH Oxidases; Reactive Oxygen Species; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide with a multifactorial, multistep, complex process and poor prognosis. Its early diagnosis and metastasis monitoring are of the utmost importance. Hepatoma tissues synthesize various tumor-related proteins, genes, enzymes, microRNA, etc. and then secrete into the blood. Detections of circulating biomarkers are useful to find tumor at an early stage or monitor metastasis after postoperative treatment. This paper summarizes recent studies of specific biomarkers at early diagnosis or in monitoring metastasis or postoperative recurrence of HCC.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; gamma-Glutamyltransferase; Glypicans; Heat-Shock Proteins; Humans; Insulin-Like Growth Factor II; Isoenzymes; Liver Neoplasms; MicroRNAs; Neoplasm Metastasis; Telomerase; Transforming Growth Factor beta

Hepatitis B virus (HBV) which includes, fulminant, acute, chronic, asymptomatic, and occult HBV infection is the most prevalent virus that leads to human liver diseases. Chronic, asymptomatic, and occult infection can induce further sever diseases such as hepatocellular carcinoma (HCC) and cirrhosis of the liver. The underlying mechanisms that allow progression of the prolonged forms of the infection and subsequent HCC or cirrhosis of the liver are yet to be clarified. However, many researchers have suggested that immunological and genetic parameters may play important roles in the etiology of hepatitis B. Transforming growth factor beta (TGF-β) is an important cytokine with dual regulatory functions in the immune system and in the responses against viral infections. However, the pathways and mechanisms controlling these are not fully understood. The crucial roles of TGF-β in the development of Th17 and T regulatory lymphocytes, the main cell types involved in autoimmunity and destructive immune related diseases, have been documented and this provides insights into TGF-β function during hepatitis infection and subsequent HCC and cirrhosis of the liver. Recent findings also confirm that TGF-β directly alters hepatocyte function during hepatitis B, hence, the aim of this review is to address the current data regarding the association and status of TGF-β with hepatitis B infection and its related disorders including HCC and cirrhosis of the liver.

Topics: Carcinoma, Hepatocellular; Hepatitis B, Chronic; Humans; Liver Cirrhosis; Liver Neoplasms; Transforming Growth Factor beta

Perturbation of transforming growth factor (TGF)-β signaling in hepatocytes persistently infected with hepatitis viruses promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into hepatocytic fibro-carcinogenesis have emerged from recent detailed analyses of context-dependent and cell type-specific TGF-β signaling processes directed by multiple phosphorylated forms (phospho-isoforms) of Smad mediators. In the course of hepatitis virus-related chronic liver diseases, chronic inflammation, ongoing viral infection, and host genetic/epigenetic alterations additively shift hepatocytic Smad phospho-isoform signaling from tumor suppression to fibro-carcinogenesis, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma (HCC). After successful antiviral therapy, patients with chronic hepatitis can experience less risk of HCC occurrence by reversing Smad phospho-isoform signaling from fibro-carcinogenesis to tumor suppression. However, patients with cirrhosis can still develop HCC owing to sustained, intense fibro-carcinogenic signaling. Recent progress in understanding Smad phospho-isoform signaling should permit use of Smad phosphorylation as a tool predicting the likelihood of liver disease progression, and as a biomarker for assessing the effectiveness of interventions aimed at reducing fibrosis and cancer risk.

Topics: Carcinoma, Hepatocellular; Chronic Disease; Humans; Liver Cirrhosis; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta

Advanced pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC) are non-curable diseases with a particularly poor prognosis. Over the last decade, research has increasingly focused on the microenvironment surrounding cancer cells, and its role in tumour development and progression. PDAC and HCC differ markedly regarding their pathological features: PDAC are typically stromal-predominant, desmoplastic, poorly vascularized tumours, whereas HCC are cellular and highly vascularized. Despite these very different settings, PDAC and HCC share transforming growth factor-β (TGF-β) as a common key-signalling mediator, involved in epithelial-to-mesenchymal transition, invasion, and stroma-tumour dialogue. Recently, novel drugs blocking the TGF-β pathway have entered clinical evaluation demonstrating activity in patients with advanced PDAC and HCC. TGF-β signalling is complex and mediates both pro- and anti-tumoural activities in cancer cells depending on their context, in space and time, and their microenvironment. In this review we provide a comprehensive overview of the role of the TGF-β pathway and its deregulation in PDAC and HCC development and progression at the cellular and microenvironment levels. We also summarize key preclinical and clinical data on the role of TGF-β as a target for therapeutic intervention in PDAC and HCC, and explore perspectives to optimize TGF-β inhibition therapy.

Topics: Animals; Carcinoma, Hepatocellular; Carcinoma, Pancreatic Ductal; Humans; Liver Neoplasms; Molecular Targeted Therapy; Pancreatic Neoplasms; Transforming Growth Factor beta

Primary liver tumours have a high incidence and mortality. The most important forms are hepatocellular carcinoma and intrahepatic cholangiocarcinoma, both can occur together in the mixed phenotype hepatocellular-cholangiocarcinoma. Liver progenitor cells (LPCs) are bipotential stem cells activated in case of severe liver damage and are capable of forming both cholangiocytes and hepatocytes. Possibly, alterations in Wnt, transforming growth factor-β, Notch and hypoxia pathways in these LPCs can cause them to give rise to cancer stem cells, capable of driving tumourigenesis. In this review, we summarize and discuss current knowledge on the role of these pathways in LPC activation and differentiation during hepatocarcinogenesis.

Topics: Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Cell Differentiation; Cell Hypoxia; Cell Transformation, Neoplastic; Cholangiocarcinoma; Humans; Liver; Liver Neoplasms; Receptors, Notch; Stem Cells; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway

Hepatocellular carcinoma arises in patients as a consequence of long-standing preexisting liver illnesses, including viral hepatitis, alcohol abuse, or metabolic disease. In such preexisting liver diseases, TGF-β plays an important role in orchestrating a favorable microenvironment for tumor cell growth and promoting epithelial-mesenchymal transition (EMT). TGF-β signaling promotes hepatocellular carcinoma progression by two mechanisms: first, via an intrinsic activity as an autocrine or paracrine growth factor and, second, via an extrinsic activity by inducing microenvironment changes, including cancer-associated fibroblasts, T regulatory cells, and inflammatory mediators. Although there is an increasing understanding on how TGF-β signaling is associated with tumor progression in hepatocellular carcinoma, it is not clear whether TGF-β signaling is limited to a certain subgroup of patients with hepatocellular carcinoma or is a key driver of hepatocellular carcinoma during the entire tumorigenesis of hepatocellular carcinoma. Inhibitors of the TGF-β signaling have been shown to block hepatocellular carcinoma growth and progression by modulating EMT in different experimental models, leading to the clinical investigation of the TGF-β inhibitor LY2157299 monohydrate in hepatocellular carcinoma. Preliminary results from a phase II clinical trial have shown improved clinical outcome and also changes consistent with a reduction of EMT.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Clinical Trials as Topic; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Pyrazoles; Quinolines; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma is difficult to treat, primarily because the underlying molecular mechanisms driving clinical outcome are still poorly understood. Growing evidence suggests that the tissue microenvironment has a role in the biological behavior of the tumor. The main clinical issue is to identify the best target for therapeutic approaches. Here, we discuss the hypothesis that the entire tissue microenvironment might be considered as a biological target. However, the tissue microenvironment consists of several cellular and biochemical components, each of which displays a distinct biological activity. We discuss the major components of this environment and consider how they may interact to promote tumor/host crosstalk.

Topics: Carcinoma, Hepatocellular; Cell Adhesion Molecules; Disease Progression; Endothelial Cells; Extracellular Matrix; Fibroblasts; Hepatic Stellate Cells; Humans; Kalinin; Liver; Liver Neoplasms; Macrophages; Mutation; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

MicroRNAs are evolutionary conserved single stranded non-coding RNAs with immense ability to posttranscriptionally regulate gene expression via complementary base pairing with mRNAs of more than 50% protein encoding genes. They play diverse roles in physiological and pathophysiological processes such as normal development and cancer pathogenesis respectively. Recent investigations have focused on the identification and characterization of microRNAs aberrantly expressed in cancer and their target molecules that are critically involved in the initiation, progression and development of carcinogenesis as possible diagnostic, prognostic and theranostic (Integration of biomarker use as diagnostic tools and target-specific therapies to enhance selective and individualized therapy) tools to augment conventional cancer therapeutic armamentarium. In this mini review, we bring to focus the intricate interactions between microRNAs aberrantly expressed in hepatocarcinogenesis and their interactions with the transforming growth factor beta (TGF-β)/Small mother against decapentaplegic (Smad) and the mitogen activated protein kinases (MAPKs) signaling pathways. Importantly, we highlight microRNA-Specific targets as possible biomarkers for prognosis, diagnosis and as therapeutic targets for hepatocellular carcinoma (HCC) while at the same time exploring new directions for future investigations.

Topics: Animals; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Neoplasms; MicroRNAs; Prognosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Epidemiological and clinical data point to a close association between chronic hepatitis B virus infection or chronic hepatitis C virus infection and development of hepatocellular carcinoma (HCC). HCC develops over several decades and is associated with fibrosis. This sequence suggests that persistent viral infection and chronic inflammation can synergistically induce liver fibrosis and hepatocarcinogenesis. The transforming growth factor-β (TGF-β) signaling pathway plays a pivotal role in diverse cellular processes and contributes to hepatic fibro-carcinogenesis under inflammatory microenvironments during chronic liver diseases. The biological activities of TGF-β are initiated by the binding of the ligand to TGF-β receptors, which phosphorylate Smad proteins. TGF-β type I receptor activates Smad3 to create COOH-terminally phosphorylated Smad3 (pSmad3C), while pro-inflammatory cytokine-activated kinases phosphorylates Smad3 to create the linker phosphorylated Smad3 (pSmad3L). During chronic liver disease progression, virus components, together with pro-inflammatory cytokines and somatic mutations, convert the Smad3 signal from tumor-suppressive pSmad3C to fibro-carcinogenic pSmad3L pathways, accelerating liver fibrosis and increasing the risk of HCC. The understanding of Smad3 phosphorylation profiles may provide new opportunities for effective chemoprevention and personalized therapy for patients with hepatitis virus-related HCC in the future.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Viral; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; JNK Mitogen-Activated Protein Kinases; Liver; Liver Neoplasms; Phosphorylation; Receptors, Transforming Growth Factor beta; Risk Assessment; Risk Factors; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is a cancer that usually develops on a liver already compromised by cirrhosis. Study of the underlying molecular mechanisms is essential so as to improve therapeutic strategies and to develop new pharmacological agents that may prevent or improve the course of this malignancy. Transforming growth factor-beta (TGF-β) intervenes in the process of hepatic fibrogenesis and cirrhosis, two pathogenic preconditions for the formation and progression of HCC [1] [2]. In addition, TGF-β plays a crucial role in the molecular pathogenesis of HCC and may, therefore, prove to be a promising drug target. We and other authors have recently demonstrated that inhibition of the TGF-β signaling pathway results in a synergistic downstream action with an inhibitory effect on the progression of HCC. Several TGF-β inhibitors have recently been developed, most of which are still in a preclinical phase, but they may soon be available for testing in patients with HCC. However, well-designed clinical trials will be needed to evaluate the effectiveness of these new agents prior to routine use in the clinic. Aim of this article is to make a brief review of the benefits and limitations of targeting the TGF-β signaling pathway in HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Disease Progression; Drug Design; Humans; Liver Cirrhosis; Liver Neoplasms; Molecular Targeted Therapy; Signal Transduction; Transforming Growth Factor beta

In this review we will report on recent advanced in polyelectrolyte capsules for targeted drug delivery (eg of growth factor inhibitor) against epatocarcinoma. Degradable polyelectrolyte multilayers capsules (PMCs) are of particular interest for cancer therapy since under physiological conditions they can be enzymatically degraded upon cell interaction. Small bioactive molecules such as TGF-Beta inhibitors can be incorporated inside them. Nano-to-microscale delivery systems can enhance efficacy at single cell level for targeted therapy. Layer-by-layer (LbL) self-assembled capsules are novel carriers maximizing drug administration and improving antimetastatic activity of TGF-Beta inhibitors in Hepatocellular Carcinoma (HCC).

Topics: Animals; Antineoplastic Agents; Capsules; Carcinoma, Hepatocellular; Drug Carriers; Drug Delivery Systems; Electrolytes; Humans; Liver Neoplasms; Nanoparticles; Particle Size; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) represent the majority of hepatic malignancies and are among the most frequent causes of cancer deaths worldwide with a rising incidence in western countries. Upon progression of liver cancer, the epithelial to mesenchymal transition (EMT) is considered a key process that drives intrahepatic metastasis. EMT is the transformation of epithelial cells to a mesenchymal phenotype exacerbating motility and invasiveness of various epithelial cell types. In this review we focus on EMT in hepatic fibrosis, HCC and CCC that is governed by the transforming growth factor (TGF)-β signaling. This cytokine has been shown to play diverse and conflicting roles in malignant development, acting as a tumor-suppressor in early cancerogenesis but enhancing tumor dissemination in later stages of tumor progression. Importantly, TGF-β can induce EMT in a variety of cancers including HCC and CCC, even though the complex molecular mechanisms underlying this process are not yet fully understood. We aim at collecting recent findings on the impact of TGF-β-induced EMT in liver carcinoma progression and at discussing new insights on promising drugable targets for future therapeutic approaches against CCC and HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cholangiocarcinoma; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Liver Cirrhosis; Liver Neoplasms; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. It arises from modulation of multiple genes by mutations, epigenetic regulation, noncoding RNAs and translational modifications of encoded proteins. Although >40% of HCCs are clonal and thought to arise from cancer stem cells (CSCs), the precise identification and mechanisms of CSC formation remain poorly understood. A functional role of transforming growth factor (TGF)-β signalling in liver and intestinal stem cell niches has been demonstrated through mouse genetics. These studies demonstrate that loss of TGF-β signalling yields a phenotype similar to a human CSC disorder, Beckwith-Wiedemann syndrome. Insights into this powerful pathway will be vital for developing new therapeutics in cancer. Current clinical approaches are aimed at establishing novel cancer drugs that target activated pathways when the TGF-β tumour suppressor pathway is lost, and TGF-β itself could potentially be targeted in metastases. Studies delineating key functional pathways in HCC and CSC formation could be important in preventing this disease and could lead to simple treatment strategies; for example, use of vitamin D might be effective when the TGF-β pathway is lost or when wnt signalling is activated.

Topics: Animals; Carcinoma, Hepatocellular; Disease Models, Animal; Disease Progression; Humans; Liver; Liver Neoplasms; Mice; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (TGF)-β is a central regulator in chronic liver disease, contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver damage-induced levels of active TGF-β enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Further evidence points to a decisive role of cytostatic and apoptotic functions mediated on hepatocytes, which is critical for the control of liver mass, with loss of TGF-β activities resulting in hyperproliferative disorders and cancer. This concept is based on studies that describe a bipartite role of TGF-β with tumor suppressor functions at early stages of liver damage and regeneration, whereas during cancer progression TGF-β may turn from a tumor suppressor into a tumor promoter that exacerbates invasive and metastatic behavior. We have delineated this molecular switch of the pathway from cytostatic to tumor promoting in further detail and identify activation of survival signaling pathways in hepatocytes as a most critical requirement. Targeting the TGF-β signaling pathway has been explored to inhibit liver disease progression. While interfering with TGF-β signaling in various short-term animal models has demonstrated promising results, liver disease progression in humans is a process of decades with different phases in which TGF-β or its targeting may have both beneficial and adverse outcomes. We emphasize that, in order to achieve therapeutic effects, targeting TGF-β signaling in the right cell type at the right time is required.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Disease Progression; Humans; Liver Neoplasms; Mice; Rats; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Cadherins; Carcinoma, Hepatocellular; Catenins; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; MicroRNAs; Transforming Growth Factor beta

One of the main complications in patients with liver fibrosis is the development of hepatocellular carcinoma (HCC). An understanding of the molecular mechanisms leading to HCC is important in order to be able to design new pharmacological agents serving either to prevent or mitigate the outcome of this malignancy. The transforming growth factor-beta (TGF-β) cytokine and its isoforms initiate a signaling cascade which is closely linked to liver fibrosis, cirrhosis and subsequent progression to HCC. Because of its role in these stages of disease progression, TGF-β appears to play a unique role in the molecular pathogenesis of HCC. Thus, it is a promising target for pharmacological treatment strategies. Recent studies have shown that inhibition of TGF-β signaling results in multiple synergistic down-stream effects which will likely improve the clinical outcome in HCC. We also review a number of TGF-β inhibitors, most of which are still in a preclinical stage of development, but may soon be available for trial in HCC patients. Hence, it is anticipated that there will soon be new agents available for clinical investigations to evaluate the role of the TGF-β-associated signaling in this deadly cancer.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta

MicroRNAs (miRNAs) are small non-coding RNAs of 19-23 nucleotides that negatively regulate gene expression through binding to the 3'-untranslated regions of target messenger RNAs (mRNAs). Although the miRNA family constitutes only a minor fraction of the human genome, they hold fundamental importance in diverse physiological and developmental processes due to their pleiotropic effects on the post-transcriptional regulation of many vital genes. This class of regulatory RNAs has also emerged as important players in carcinogenesis; most, if not all, cancer types have abnormal miRNA expression patterns. In hepatocellular carcinoma (HCC), miRNA dysregulation plays a key role in mediating the pathogenicity of several etiologic risk factors and, more importantly, they promote a number of cancer-inducing signaling pathways. Recent studies have also demonstrated their potential values in the clinical management of HCC patients as some miRNAs may be used as prognostic or diagnostic markers. The significance of miRNAs in liver carcinogenesis emphasizes their values as therapeutic targets, while technological advances in the delivery of miRNA has shed new possibilities for their use as novel therapeutic agents against HCC.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Genetic Therapy; Humans; Liver Neoplasms; MicroRNAs; Receptor Protein-Tyrosine Kinases; Risk Factors; Signal Transduction; Transforming Growth Factor beta

Traditional wisdom holds that intact immune responses, such as immune surveillance or immunoediting, are required for preventing and inhibiting tumor development; but recent evidence has also indicated that unresolved immune responses, such as chronic inflammation, can promote the growth and progression of cancer. Within the immune system, cytotoxic CD8(+) and CD4(+) Th1 T cells, along with their characteristically produced cytokine IFN-γ, function as the major anti-tumor immune effector cells, whereas tumor associated macrophages (TAM) or myeloid-derived suppressive cells (MDSC) and their derived cytokines IL-6, TNF, IL-1β and IL-23 are generally recognized as dominant tumor-promoting forces. However, the roles played by Th17 cells, CD4(+) CD25(+) Foxp3(+) regulatory T lymphocytes and immunoregulatory cytokines such as TGF-β in tumor development and survival remain elusive. These immune cells and the cellular factors produced from them, including both immunosuppressive and inflammatory cytokines, play dual roles in promoting or discouraging cancer development, and their ultimate role in cancer progression may rely heavily on the tumor microenvironment and the events leading to initial propagation of carcinogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cytokines; Humans; Liver Neoplasms; Macrophages; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) occurs subsequent to liver injury, where regenerative hepatocytes develop into a dysplastic nodule and then early HCC, supporting the multistep hepatocarcinogenesis theory. Molecular alterations such as the p53 mutation, p16 gene silencing, and AKT signaling activation are found in the late stage of HCC progression. The overexpression of some marker molecules is observed at the early stage. Transforming growth factor-β (TGF-β), a potent inhibitor of cell proliferation, is frequently overexpressed in HCC, although the role of TGF-β signaling during HCC development remains controversial. We previously reported that HCC cells show TGF-β receptor-dependent growth inhibition in response to TGF-β. Also, reduced TGF-β receptor II in HCC correlates with intrahepatic metastasis and shorter time-to-recurrence, suggesting a role of TGF-β signaling in tumor suppression. In contrast, TGF-β overexpression in HCC is known to correlate with malignant potential, suggesting a role in tumor promotion. Enhanced formation of stroma is a feature of advanced HCC, and TGF-β also promotes the proliferation of stromal fibroblasts. The microenvironment produced via tumor-stromal interactions may be the key to the modulation of the dual roles of TGF-β signaling in HCC progression.

Topics: Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Humans; Liver Neoplasms; Models, Biological; Signal Transduction; Transforming Growth Factor beta

The two dominant models of carcinogenesis postulate stochastic (clonal evolution) or hierarchic organization of tumor (cancer stem cell model). According to the latter, at the germinal center of tumor evolution is a cancer stem cell (CSC) which, similar to normal adult stem cells, possesses the capacity of self-renewal and a differentiation potential. Over the past few years, compelling evidence has emerged in support of the hierarchic cancer model for many solid tumors including hepatocellular cancers. The CSCs are posited to be responsible not only for tumor initiation but also for the generation of distant metastasis and relapse after therapy. These characteristics are particularly relevant for a multi-resistant tumor entity like human hepatocellular carcinoma and may herald a paradigm shift in the management of this deadly disease. Identification and detailed characterization of liver CSCs is therefore imperative for improving prevention approaches, enhancing early detection, and extending the limited treatment options. Despite the current progress in understanding the contribution of CSCs to the generation of heterogeneity of tumors, the molecular complexity and exact regulation of CSCs is poorly understood. This review focuses on the genetic and epigenetic mechanisms that regulate and define the unique CSC properties with an emphasis on key regulatory pathways of liver CSCs and their clinical significance.

Topics: AC133 Antigen; Aldehyde Dehydrogenase; Antigens, CD; Antigens, Neoplasm; beta Catenin; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Division; Cell Separation; Epigenesis, Genetic; Epithelial Cell Adhesion Molecule; Genes, myc; Glycoproteins; Hedgehog Proteins; Humans; Liver Neoplasms; MicroRNAs; Models, Biological; Neoplasm Metastasis; Neoplastic Stem Cells; Nuclear Proteins; Peptides; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; Receptors, Notch; Repressor Proteins; RNA, Neoplasm; Signal Transduction; Thy-1 Antigens; Transforming Growth Factor beta; Wnt Proteins

Although the existence of cancer stem cells (CSCs) was first proposed over 40 years ago, only in the past decade have these cells been identified in hematological malignancies, and more recently in solid tumors that include liver, breast, prostate, brain, and colon. Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood, including the precise cell(s) of origin, molecular genetics, and the mechanisms responsible for the highly aggressive clinical picture of hepatocellular carcinoma (HCC). Exploration of the difference between CSCs from normal stem cells is crucial not only for the understanding of tumor biology but also for the development of specific therapies that effectively target these cells in patients. These ideas have drawn attention to control of stem cell proliferation by the transforming growth factor beta (TGF-beta), Notch, Wnt, and Hedgehog pathways. Recent evidence also suggests a key role for the TGF-beta signaling pathway in both hepatocellular cancer suppression and endoderm formation, suggesting a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as HCC.

Topics: beta Catenin; Biomarkers; Carcinoma, Hepatocellular; Cell Lineage; Embryonic Stem Cells; Hedgehog Proteins; Humans; Liver; Liver Regeneration; Liver Transplantation; Living Donors; Male; Neoplastic Stem Cells; Prognosis; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Hepatocellular carcinoma (HCC) is a major health problem, being the sixth most common cancer world-wide. Dysregulation of the balance between proliferation and cell death represents a pro-tumorigenic principle in human hepatocarcinogenesis. This review updates the recent relevant contributions reporting molecular alterations for HCC that induce an imbalance in the regulation of apoptosis. Alterations in the expression and/or activation of p53 are frequent in HCC cells, which confer on them resistance to chemotherapeutic drugs. Many HCCs are also insensitive to apoptosis induced either by death receptor ligands, such as FasL or TRAIL, or by transforming growth factor-beta (TGF-beta). Although the expression of some pro-apoptotic genes is decreased, the balance between death and survival is dysregulated in HCC mainly due to overactivation of anti-apoptotic pathways. Indeed, some molecules involved in counteracting apoptosis, such as Bcl-X(L), Mcl-1, c-IAP1, XIAP or survivin are over-expressed in HCC cells. Furthermore, some growth factors that mediate cell survival are up-regulated in HCC, as well as the molecules involved in the machinery responsible for cleavage of their pro-forms to an active peptide. The expression and/or activation of the JAK/STAT, PI3K/AKT and RAS/ERKs pathways are enhanced in many HCC cells, conferring on them resistance to apoptotic stimuli. Finally, recent evidence indicates that inflammatory processes, as well as the epithelial-mesenchymal transitions that occur in HCC cells to facilitate their dissemination, are related to cell survival. Therefore, therapeutic strategies to selectively inhibit anti-apoptotic signals in liver tumor cells have the potential to provide powerful tools to treat HCC.

Topics: Aflatoxin B1; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Death; Cell Division; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Kinetics; Liver Neoplasms; Mice; Mice, Transgenic; Receptors, Death Domain; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Hepatocellular carcinomas (HCCs) constitute a therapeutic challenge with mostly unfavorable outcome. This may reflect our incomplete understanding of disease pathogenesis, e.g. the elucidation of tumorigenic signaling pathways. Knowledge gathered hitherto focuses on genetic alterations that result in the loss of tumor suppressor functions as well as amplification and mutation of cancer genes. Further evidence points to a decisive role of cytostatic and apoptotic functions mediated on hepatocytes by transforming growth factor (TGF)-beta. These effects are critical for the control of liver mass with loss of TGF-beta activities resulting in hyperproliferative disorders and cancer. This concept is based on studies that describe a bipartite role of TGF-beta with tumor suppressor functions at early stages of liver damage and regeneration, whereas during cancer progression TGF-beta may turn from a tumor suppressor to a tumor promoter that exacerbates invasive and metastatic behavior. Consequently and most importantly, the oncogenic potential of recent therapeutic approaches against profibrogenic TGF-beta effects needs to be carefully delineated and a cancer therapy with specific targets disrupting the TGF-beta signaling cascade may be envisioned. In line with this concept, we and others found overexpression of TGF-beta antagonist Smad7 in the majority of HCC samples, providing a mechanism for hepatocytes to escape TGF-beta-dependent growth control in the process of cancerogenesis.

Topics: Carcinoma, Hepatocellular; Disease Progression; Hepatitis, Chronic; Humans; Liver Cirrhosis; Liver Neoplasms; Transforming Growth Factor beta

Scientists at the National Institutes of Health have reported that increased coffee consumption is associated with a slower progression of fibrogenesis in patients with chronic and particularly alcoholic liver disease and a reduced incidence of heptocellular carcinoma. However, a causal mechanistic explanation was pending. New results indicate that the methylxanthine caffeine--a major component of coffee and the most widely consumed pharmacologically active substance in the world--might be responsible for this phenomenon, because it inhibits the synthesis of connective tissue growth factor (CTGF/CCN2) in liver parenchymal and nonparenchymal cells, primarily by inducing degradation of Smad2 (and to a much lesser extent Smad3) and thus impairment of transforming growth factor beta (TGF-beta) signaling. CTGF and TGF-beta play crucial roles in the fibrotic remodeling of various organs, and, ultimately, carcinogenesis. This article summarizes the clinical-epidemiological observations as well as the pathophysiological background and provides suggestions for the therapeutic use of (methyl)xanthine derivatives in the management of fibro-/carcinogenic (liver) diseases.

Topics: Acetylgalactosamine; Animals; Caffeine; Carcinoma, Hepatocellular; Collagen Type I; Connective Tissue Growth Factor; Humans; Liver; Liver Neoplasms; Proteasome Endopeptidase Complex; Smad2 Protein; Theophylline; Transforming Growth Factor beta; Xanthines

Topics: Animals; Carcinoma, Hepatocellular; Hepatic Stellate Cells; Hepatitis; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Myeloid Differentiation Factor 88; NF-kappa B; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta

Topics: Cadherins; Capsid Proteins; Carcinoma, Hepatocellular; Cell Transdifferentiation; Epithelial Cells; Hepacivirus; Humans; Liver Neoplasms; Mesoderm; Neoplasm Proteins; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with a median survival of 6-16 m. Factors responsible for the poor prognosis include late onset diagnosis, underlying cirrhosis and resistance to chemotherapy; 40% of HCCs are clonal and therefore potentially arise from progenitor/stem cells. New insights are provided from several signaling pathways, such as STAT3, NOTCH, hedgehog and transforming growth factor-beta (TGFbeta), which are involved in stem cell renewal, differentiation, survival, and are commonly deregulated in HCC. Control of stem cell proliferation by the TGFbeta, Notch, Wnt and Hedgehog pathways to suppress hepatocellular cancer and to form the endoderm suggest a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as hepatocellular cancer.

Topics: Carcinoma, Hepatocellular; Hedgehog Proteins; Humans; Liver Neoplasms; Models, Biological; Neoplastic Stem Cells; Receptors, Notch; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

Signaling of transforming growth factor beta (TGF-beta) is mediated through a heteromeric complex of two types of transmembrane receptors and downstream intracellular proteins known as Smads. Alterations of TGF-beta signaling underlie various forms of human cancer and developmental diseases. Human genetic studies have revealed both point mutations and deletions of Smad2 or Smad4 in several types of cancers. However, the role of Smad3 in tumorigenesis is not clear. Recent data indicate that Smad3 also functions as a tumor suppressor by inhibiting cell proliferation and promoting apoptosis. In addition, Smad3 is essential for TGF-beta-mediated immune suppression, and it plays an important role in regulating transcriptional responses that are favorable to metastasis. Therefore, through regulating different transcriptional responses, Smad3 functions as both a negative and positive regulator of carcinogenesis depending on cell type and clinical stage of the tumor.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Humans; Liver Neoplasms; Mice; Mutation; Neoplasm Metastasis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

To discuss exhanstively: complex molecular and cellular mechanism in Hepatocellular Carcinoma (HCC); effect of chronic inflammation and cirrhosis, accompained by regenerative process, on the development of HCC; genetic instability of liver cells of regenerating nodules; the relative role of hepatitis C virus (HCV) and hepatitis B virus (HBV) in hepatocarcinogenesis; tumorigenicity of aflatoxin B1 (AFB1); gene expression profiles in HCC; liver tumors and host defense; future perspectives of HCC treatment.. We reviewed the most important studies on HCV.. HCC is an aggressive malignancy with poor prognosis and is one of the most common tumor in the world. In the majority of cases, HCC is found in conjunction with cirrhosis of the liver. Chronic inflammation and cirrhosis, accompagnied by regenerative process, function as a tumor promoter, providing a common pathway from chronic HBV or HCV infection to HCC. The direct etiologic role of HBV and HCV for HCC is obscure. Tumor progression may be brought about in HCC by mutation of the p53 tumor suppressor gene. The prevalences of p53 mutations is similar in HBV-associated and HCV-associated HCCs. Another mechanisms of host defense are the production of transforming growth factor beta1 (TGFbeta1), and the induction of cytotoxic T lymphocytes; the failure of there mechanisms permits the process of hepatocarcinogenesis. Treatment with alpha interferon of chronic hepatitis is necessary to delary or prevent the progression to liver cirrhosis and development of HCC. Various therapies, such radical operation, intra-arterial chemoembolization, percutaneous intratumoral ethanol injection, radio-frequency ablation, have been employed, but there is still non satisfactory treatment. Recent advances in recombinant and gene delivery thechnologies suggest that gene therapy may be a promising alternative to explore. Furthemore, immunotherapy may become a modality for patients with HCC. Clinical application of vaccine immunotherapy with NY-ESO-1 derived peptides in HLA-A2 positive HCC patients will be possible.

Topics: Aflatoxin B1; Animals; Carcinoma, Hepatocellular; Controlled Clinical Trials as Topic; Gene Expression; Genes, p53; Genetic Therapy; Hepatectomy; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Immunotherapy; Immunotherapy, Active; Interferon-alpha; Liver Cirrhosis; Liver Neoplasms; Liver Regeneration; Mice; Mice, Transgenic; Multicenter Studies as Topic; Mutation; Prognosis; Prospective Studies; Randomized Controlled Trials as Topic; Retrospective Studies; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Topics: Carcinoma, Hepatocellular; CD4 Lymphocyte Count; Hepatitis C; Humans; Interferon-gamma; Interleukin-10; Interleukin-2; Liver Cirrhosis; Liver Neoplasms; Th1 Cells; Th2 Cells; Transforming Growth Factor beta

Hepatic dendritic cells (DC) unquestionably play important roles in the induction and regulation of immune responses. Due to their paucity, functional characterisation of these important antigen presenting cells has been slow but use of DC growth factors (in particular GM-CSF and Flt3L) that markedly enhance their numbers has proved helpful in furnishing adequate study material. While there is growing evidence that DC function is affected in the pathogenesis of liver disease, most work to date has been performed on non-hepatic DC. Increasing knowledge of hepatic DC biology is likely to improve our understanding of disease pathogenesis and resistance to and therapy of liver disease.

Topics: Antigens, CD; Carcinoma, Hepatocellular; Cell Count; Chemotaxis; Dendritic Cells; Hepatitis, Viral, Human; Humans; Immune Tolerance; Immunity, Cellular; Leukocytes; Liver; Liver Diseases; Liver Neoplasms; Liver Transplantation; Lymph; Lymphoid Tissue; Phagocytosis; Phenotype; Stem Cells; T-Lymphocytes; Transforming Growth Factor beta

TGF-beta is an important factor in the regulation of liver growth. It is an inhibitor of hepatocyte DNA synthesis and may induce active cell death, e.g., to remove excessive tissue mass. Studies using transgenic mice suggest that expression in the resting liver has to be well balanced; either under- or overexpression appear to cause an increased turnover of hepatocytes and to predispose to hepatocarcinogenesis. TGF-beta overexpression is frequently observed in human hepatocellular carcinomas, probably as a late event in tumor development. In men and mice, TGF-beta overexpression appears to be associated with loss of TGF-beta responsiveness often by disruption of TGF-beta signaling. However, mechanisms as mutations in TGF-beta receptor II or Smad2 and 4 genes, frequently observed in other human cancers, have only rarely been observed in hepatocellular carcinomas. Further studies may clarify the mechanisms by which hepatocellular tumors escape TGF-beta growth control, as well as analyze possible roles of TGF-beta overexpression in immunosuppression and angiogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Death; Cell Division; Humans; Liver Neoplasms; Mice; Mice, Transgenic; Rats; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Cells termed myofibroblasts are prominent in the injury response of all epithelial tissues. They exhibit proliferation, migration, production of collagen and other extracellular matrix (ECM) molecules, and contraction, all for containing the injury and closing the wound. When the injury is limited in time, the final stage of the repair involves a dismantling of the cellular apparatus and restoration of normal tissue structure. With multiple cycles of repair, however, there is net accumulation of ECM, to the detriment of tissue structure and function. Repair-related ECM coalesces into fibrous bundles and, over time, undergoes changes that render it resistant to degradation. The result is a scar. In the skin, a scar may have cosmetic importance only. In the liver, however, extensive scarring is the setting for unregulated growth and neoplasia; also, fibrous bands disrupt normal blood flow, leading to portal hypertension and its complications. With regard to therapy for fibrosis, the first consideration is elimination of the injury factor. However, given that many liver diseases do not have effective therapies at present, strategies targeting fibrogenesis per se are under development. The main source of myofibroblast-like cells and ECM production in the liver is the perisinusoidal stellate cell, which responds to injury with a pleiotypic change termed activation. Activation is orchestrated by cytokines and the ECM itself. Among the cytokines involved in this process, transforming growth factor-beta (TGF-beta) is particularly prominent. The early changes in ECM include de novo production of a specific "fetal" isoform of fibronectin, which arises from sinusoidal endothelial cells. It is stimulated by TGF-beta and acts directly on stellate cells to promote their activation. Based on these and other advances in understanding the fundamentals of the injury response, several strategies now exist for altering fibrogenesis, ranging from agents that block TGF-beta to traditional Chinese herbal extracts. Arrest of fibrogenesis, even with underlying cirrhosis, is likely to extend life or prolong the time to transplant. Whether it reduces the risk of hepatocellular carcinoma remains to be proven. Although TGF-beta antagonists are effective anti-fibrogenic agents, they will require detailed safety testing because of the finding that several forms of epithelial neoplasia are associated with altered regulation of TGF-beta.

Topics: Animals; Carcinoma, Hepatocellular; Chronic Disease; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Fibrosis; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Transforming Growth Factor beta

The tightly controlled homeostatic mechanisms between cell growth and apoptosis that exist in normal liver tissue are disrupted during hepatocarcinogenesis. The TGF (transforming growth factor)-beta signaling system is a central component of the mechanisms by which cell growth and apoptosis are controlled in the liver. The recent delineation of the TGF-beta signaling pathway has provided a unique framework for analysis of the impact that disruption of individual components of this signaling pathway can have on apoptosis during hepatocarcinogenesis. Here we review recent data on involvement of the TGF-beta signaling pathway in the dysregulation of apoptosis frequently observed in hepatocellular carcinomas. The data indicate that disruption of the TGF-beta pathway at the pre-receptor, receptor, and post-receptor levels occurs in hepatocellular carcinomas and can cause dysregulation of apoptosis. Also, substantial evidence now exists that phosphatidylinositol-3-kinase (PI3K) may function as an important negative regulator of the TGF-beta 1-induced apoptosis in hepatocellular carcinomas. Taken together, the available evidence indicates that disruption of the TGF-beta 1-induced apoptosis as well as growth inhibition is an important and integral part of the multistage process of liver carcinogenesis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta

Topics: alpha-Fetoproteins; Autoantibodies; Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Protein Precursors; Prothrombin; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Complex molecular and cellular mechanisms exist to protect cells against tumor formation and to protect the entire organism against further development and spread of established tumors. The p53 tumor suppressor gene controls the cell cycle through at least two mechanisms, namely, mitotic arrest and apoptosis. Human hepatocellular carcinomas (HCCs) are often found to have mutant p53, or sometimes may have dysfunctional p53 as a result of its being bound by viral or cellular proteins. Another mechanism of host response is the production of transforming growth factor beta 1, which acts on receptors in normal hepatocytes to cause inhibition of DNA synthesis; abnormalities of transforming growth factor beta 1 have been documented in HCCs, but their biologic significance is unclear. Other host defense mechanisms include cellular responses to the tumor and the proliferation of substances with anticoagulant properties.

Topics: Antibody Formation; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunity, Cellular; Liver Neoplasms; Transforming Growth Factor beta

The most effective way to control severity and mortality rate of the novel coronavirus disease (COVID-19) is through sensitive diagnostic approaches and an appropriate treatment protocol. We aimed to identify the effect of adding corticosteroid and Tocilizumab to a standard treatment protocol in treating COVID-19 patients with chronic disease through hematological and lab biomarkers.. This study was performed retrospectively on 68 COVID-19 patients with chronic disease who were treated by different therapeutic protocols. The patients were categorized into four groups: control group represented the patients' lab results at admission before treatment protocols were applied; group 1 included patients treated with anticoagulants, Hydroxychloroquine, and antibiotics; group 2 comprised patients treated with Dexamethasone; and group 3 included patients treated with Dexamethasone and Tocilizumab.. The study paves the way into the effectiveness of combining Dexamethasone with Tocilizumab in treatment COVID-19 patients with chronic diseases.. La forma más eficaz de controlar la gravedad y la tasa de mortalidad de la enfermedad del nuevo coronavirus (COVID-19) es mediante enfoques de diagnóstico sensibles y un protocolo de tratamiento adecuado. Nuestro objetivo fue identificar el efecto de agregar corticosteroides y tocilizumab a un protocolo de tratamiento estándar en el tratamiento de pacientes con COVID-19 con enfermedad crónica a través de biomarcadores hematológicos y de laboratorio.. Este estudio se realizó de forma retrospectiva en 68 pacientes COVID-19 con enfermedad crónica que fueron tratados por diferentes protocolos terapéuticos. Los pacientes se clasificaron en cuatro grupos: el grupo de control representaba los resultados de laboratorio de los pacientes en el momento de la admisión antes de que se aplicaran los protocolos de tratamiento; el grupo 1 incluyó a pacientes tratados con anticoagulantes, hidroxicloroquina y antibióticos; el grupo 2 estaba compuesto por pacientes tratados con dexametasona; y el grupo 3 incluyó a pacientes tratados con dexametasona y tocilizumab.. El estudio allana el camino hacia la eficacia de la combinación de dexametasona con tocilizumab en el tratamiento de pacientes con COVID-19 con enfermedades crónicas.. The Child-Mother Index constitutes a potential useful risk factor indicator for statistical analyses on data after birth. The value of the Child-Mother Index based on the estimated fetal weight before birth deserves evaluation.. Six ceria supports synthesized by various synthesis methodologies were used to deposit cobalt oxide. The catalysts were thoroughly characterized, and their catalytic activity for complete methane oxidation was studied. The supports synthesized by direct calcination and precipitation with ammonia exhibited the best textural and structural properties as well as the highest degree of oxidation. The remaining supports presented poorer textural properties to be employed as catalytic supports. The cobalt deposited over the first two supports presented a good dispersion at the external surface, which induced a significant redox effect that increased the number of Co. Some studies show that children with obesity are more likely to receive a diagnosis of depression, anxiety, or attention-deficit hyperactivity disorder (ADHD). But this does not necessarily mean obesity causes these conditions. Depression, anxiety, or ADHD could cause obesity. A child's environment, including family income or their parents' mental health, could also affect a child's weight and mental health. Understanding the nature of these relationships could help scientists develop better interventions for both obesity and mental health conditions. Genetic studies may help scientists better understand the role of the environment in these conditions, but it's important to consider both the child's and their parents’ genetics in these analyses. This is because parents and children share not only genes, but also environmental conditions. For example, families that carry genetic variants associated with higher body weight might also have lower incomes, if parents have been affected by biases against heavier people in society and the workplace. Children in these families could have worse mental health because of effects of their parent’s weight, rather than their own weight. Looking at both child and adult genetics can help disentangle these processes. Hughes et al. show that a child's own body mass index, a ratio of weight and height, is not strongly associated with the child’s mental health symptoms. They analysed genetic, weight, and health survey data from about 41,000 8-year-old children and their parents. The results suggest that a child's own BMI does not have a large effect on their anxiety symptoms. There was also no clear evidence that a child's BMI affected their symptoms of depression or ADHD. These results contradict previous studies, which did not account for parental genetics. Hughes et al. suggest that, at least for eight-year-olds, factors linked with adult weight and which differ between families may be more critical to a child's mental health than a child’s own weight. For older children and adolescents, this may not be the case, and the individual’s own weight may be more important. As a result, policies designed to reduce obesity in mid-childhood are unlikely to greatly improve the mental health of children. On the other hand, policies targeting the environmental or societal factors contributing to higher body weights, bias against people with higher weights, and poor child mental health directly may be more beneficial.. The development of an efficient photocatalyst for C2 product formation from CO. Оценка антиастенического эффекта последовательной терапии левокарнитином (ЛК) и ацетилкарнитином (АЛК) пациентов с артериальной гипертензией и/или ишемической болезнью сердца (ИБС) с астеническим синдромом (АС).. В открытое сравнительное исследование были включены 120 пациентов в возрасте 54—67 лет с артериальной гипертензией и/или ИБС с АС. Пациенты 1-й группы (. У больных 1-й группы отмечено статистически значимое уменьшение различных проявлений АС. Отличия носили достоверный характер по сравнению как с исходным уровнем, так и со 2-й группой. Установлено эндотелийпротективное действие ЛК и АЛК.. Полученные результаты свидетельствуют, что у таких коморбидных пациентов использование ЛК и АЛК уменьшает выраженность проявлений АС, а установленные эндотелиотропные свойства препаратов позволяют рекомендовать их в составе комплексной персонифицированной терапии пациентов с сердечно-сосудистыми заболеваниями.. Naproxen sodium 440 mg/diphenhydramine 50 mg combination demonstrated improvement in sleep maintenance (WASO) vs. naproxen sodium 550 mg and higher efficiency in average daily pain reduction compared with the comparison groups. The treatment was well tolerated There were no serious or unexpected adverse events reported in the study.. Сравнительный анализ эффективности и безопасности новой комбинации напроксена натрия и дифенгидрамина у пациентов с неспецифическим болевым синдромом в пояснично-крестцовом отделе спины (M54.5 «Боль внизу спины») и нарушением сна (G47.0 «Нарушения засыпания и поддержания сна [бессонница]»).. Проведено проспективное многоцентровое рандомизированное открытое сравнительное в параллельных группах клиническое исследование. Пациенты были рандомизированы в 3 группы. Больные 1-й группы получали напроксен натрия (440 мг) и дифенгидрамин (50 мг), 2-й — напроксен натрия (550 мг), 3-й — парацетамол (1000 мг) и дифенгидрамин (50 мг). Исследуемые препараты пациенты принимали однократно перед сном в течение 3 дней. Все пациенты также принимали 275 мг (1 таблетка) напроксена натрия в качестве препарата фоновой терапии. Первичным критерием эффективности было общее время бодрствования после наступления сна (WASO), измеряемое методом актиграфии. Также использовались критерии оценки продолжительности и качества сна и выраженности боли.. Анализ эффективности проведен для ITT популяции (. Применение комбинации напроксена натрия (440 мг) и дифенгидрамина (50 мг) характеризовалось более выраженным поддержанием сна по сравнению с напроксеном натрия 550 мг и более высокой эффективностью в отношении снижения интенсивности боли по сравнению со 2-й и 3-й группами. Отмечена хорошая переносимость препарата, серьезных нежелательных явлений зарегистрировано не было.

Topics: Acetaminophen; Acetylcarnitine; Acetylcholinesterase; Acids; Acinetobacter baumannii; Acinetobacter Infections; Adaptation, Psychological; Adolescent; Adsorption; Adult; Aged; Alcohol Drinking; Alzheimer Disease; Amikacin; Ammonia; Anaerobiosis; Animals; Anorexia; Anti-Bacterial Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Anxiety; Aptamers, Nucleotide; Asthenia; Attention Deficit Disorder with Hyperactivity; Bacterial Proteins; Beryllium; beta-Lactamases; Biofuels; Biomass; Biosensing Techniques; Bismuth; Blister; Body Mass Index; Body Surface Area; Boronic Acids; Brain; Breast Neoplasms; Butyrylcholinesterase; Cannabis; Carbapenems; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carboxylic Acids; Carcinoma, Hepatocellular; Cardiovascular Diseases; Carnitine; Case-Control Studies; Catalysis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Child; China; Cholinesterase Inhibitors; Clarithromycin; Clostridioides; Clostridioides difficile; Clostridium Infections; Cohort Studies; Colistin; Colitis; Colon; Coloring Agents; Coronary Artery Bypass; Creatinine; Crystalloid Solutions; Cytokines; Depression; Dextran Sulfate; Dextrans; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Diarrhea; Dietary Supplements; Diphenhydramine; Disease Models, Animal; Disease Outbreaks; Double-Blind Method; Doxorubicin; Drosophila; Drug Tapering; Dysbiosis; Electrons; Escherichia coli; Extracellular Vesicles; Fatigue; Female; Fermentation; gamma-Cyclodextrins; Gastrointestinal Microbiome; Glucose; Graft Survival; Graft vs Host Disease; Head and Neck Neoplasms; Heart Arrest, Induced; Hematopoietic Stem Cell Transplantation; High-Intensity Interval Training; Hippocampus; Humans; Hydrogen-Ion Concentration; Hypertension; Incidence; Interferon-gamma; Italy; Kinetics; Klebsiella Infections; Klebsiella pneumoniae; Lab-On-A-Chip Devices; Lactoferrin; Larva; Length of Stay; Lignin; Liver; Liver Neoplasms; Liver Transplantation; Living Donors; Low Back Pain; Lung; Lung Volume Measurements; Macrophages; Male; Melphalan; Men; Mendelian Randomization Analysis; Meropenem; Methane; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Mitochondrial Proteins; Molecular Docking Simulation; Molecular Structure; Mothers; Motivation; Mycoplasma; Mycoplasma hominis; Mycoplasma Infections; NAD; Nanocomposites; Nanoparticles; Nanotubes, Carbon; Naproxen; Neovascularization, Pathologic; Neurons; Nitrates; Nucleolin; Opuntia; Paratyphoid Fever; Phenotype; Phosphatidylinositol 3-Kinases; Phytochemicals; Plant Extracts; Pregnancy; Prevalence; Prospective Studies; Proto-Oncogene Proteins c-akt; Pulmonary Disease, Chronic Obstructive; Rats; Rats, Wistar; Resveratrol; Retrospective Studies; Rifampin; Risk Factors; RNA, Messenger; Selenium; Sleep; Social Behavior; Soil; Soil Pollutants; Squamous Cell Carcinoma of Head and Neck; Staphylococcus aureus; Structure-Activity Relationship; Suicidal Ideation; Suicide; Superoxide Dismutase-1; Surveys and Questionnaires; Swimming; Syndrome; Tannins; Temperature; Transforming Growth Factor beta; Transplantation Conditioning; Treatment Outcome; Triple Negative Breast Neoplasms; Troponin T; Tumor Microenvironment; United Kingdom; Ureaplasma; Ureaplasma urealyticum; Urinary Tract Infections; Viscum; Waste Disposal Facilities; Wastewater; Water; Water Pollutants, Chemical; Wolfiporia; Young Adult

Bintrafusp alfa had a manageable safety profile and demonstrated preliminary clinical activity in heavily pretreated patients with solid tumors (including hepatocellular carcinoma) with no or limited treatment options. Findings from this study suggest bintrafusp alfa may be a novel therapeutic approach for patients with advanced solid tumors. Additional trials are needed to further explore safety and efficacy of bintrafusp alfa in specific tumor types.. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor-β (TGF-β) RII receptor (a TGF-β "trap") fused to a human immunoglobulin (Ig) G1 antibody blocking programmed death-ligand 1 (PD-L1). Bintrafusp alfa is designed to neutralize TGF-β signaling by "trapping" and sequestering all TGF-β isoforms, and this trap function is physically linked to PD-L1 blockade in the tumor microenvironment.. NCT02699515 was a phase I, open-label, dose-escalation study of bintrafusp alfa (3, 10, and 20 mg/kg every 2 weeks) in Asian patients with advanced solid tumors, including a hepatocellular carcinoma (HCC) safety-assessment cohort. The primary objective was safety and tolerability; the secondary objective is best overall response.. As of August 24, 2018, 23 patients (including 9 in the HCC cohort) received bintrafusp alfa. Eight patients experienced treatment-related adverse events (TRAEs). Three patients had grade 3 TRAEs (13.0%; hypoacusis, hyponatremia, hypopituitarism, increased blood creatine phosphokinase, and intracranial tumor hemorrhage); one had grade 4 hyponatremia (4.3%). No treatment-related deaths occurred. In the dose-escalation cohort, two patients had a confirmed partial response, and 3 had stable disease (SD), for an overall response rate of 14.3% and a disease control rate (DCR) of 35.7%. In the HCC cohort, one patient had SD (DCR, 11.1%). A dose-proportional pharmacokinetics profile was observed at doses of >3 mg/kg.. Bintrafusp alfa had a manageable safety profile and preliminary efficacy in heavily pretreated patients with advanced solid tumors, including HCC.

Topics: Carcinoma, Hepatocellular; Humans; Immunologic Factors; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Checkpoint inhibitors have shown modest activity in patients with advanced hepatocellular carcinoma (HCC). Herein, the authors report a prospective single-institution clinical/translational phase 2 study of pembrolizumab in patients with advanced HCC and circulating biomarkers closely related to response.. Pembrolizumab was administered at a dose of 200 mg intravenously every 3 weeks among patients who may have developed disease progression while receiving, were intolerant of, or refused sorafenib. The circulating levels of cytokines, chemokines, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and PD-L2 were correlated with response, tumor PD-L1 expression, and other clinicopathological features.. A total of 29 patients were treated and 28 patients were evaluable for response. The most common laboratory grade 3/4 adverse events were increases in aspartate aminotransferase and/or alanine aminotransferase and serum bilirubin, which for the most part were reversible. In terms of efficacy, one patient achieved a complete response and 8 patients achieved partial responses for an overall response rate of 32%. Four other patients had stable disease. The median progression-free survival was 4.5 months and the median overall survival was 13 months. Response did not correlate with prior sorafenib therapy, PD-L1 tumor staining, or a prior history of hepatitis. Correlative studies revealed that high baseline plasma TGF-β levels (≥200 pg/mL) significantly correlated with poor treatment outcomes after pembrolizumab. Tumor PD-L1 and plasma PD-L1/PD-1 levels were associated with plasma IFN-γ or IL-10.. Pembrolizumab was found to demonstrate activity in patients with advanced HCC. Toxicity generally was tolerable and reversible. A set of immunological markers in blood plasma as well as PD-L1 staining indicated that baseline TGF-β could be a predictive biomarker for response to pembrolizumab.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; B7-H1 Antigen; Carcinoma, Hepatocellular; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Interleukin-10; Kaplan-Meier Estimate; Liver Neoplasms; Male; Middle Aged; Programmed Cell Death 1 Receptor; Progression-Free Survival; Prospective Studies; Tomography, Emission-Computed; Transforming Growth Factor beta; Treatment Outcome

To assess the clinical relevance of transforming growth factor-beta1 (TGF-beta1) in hepatocellular carcinoma (HCC), urinary TGF-beta1 and serum alpha-fetoprotein (AFP) were determined in 94 patients with cirrhotic HCC, 94 age- and sex-matched patients with cirrhosis alone and 50 healthy adults. TGF-beta1 level in HCC was higher than in cirrhosis alone or in healthy controls (each P = 0.0001). There is an inverse correlation between TGF-beta1 and AFP levels (r = -0.292, P = 0.004). Significantly higher TGF-beta1 level was found in HCC patients with worsening Child-Pugh stages, diffuse HCC, tumour size > 3 cm, multilobular tumour and AFP < or = 20 ng ml(-1). TGF-beta1 level decreased after complete treatment with transcatheter arterial chemoembolization (P = 0.0001). The median survival in HCC patients with raised TGF-beta1 was shorter than those with normal TGF-beta1 (P = 0.018). Multivariate analysis indicated that TGF-beta1 and AFP were significantly correlated with the presence of HCC. In addition, TGF-beta1 could be used as a diagnostic marker for HCC, particularly in tumours with low AFP production. In conclusion, elevated urinary TGF-beta1 level is a tumour marker and predictor of poor survival for cirrhotic HCC.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Neoplasm Staging; Radioimmunoassay; Survival Rate; Transforming Growth Factor beta

Metastasis is found in most advanced hepatocellular carcinoma (HCC) patients, and it drives tumor recurrence and systemic failure. There is no effective treatment owing to its complex biological features. Many of the molecular drivers of metastasis are crucial players in normal physiology but behave unconventionally during cancer progression. Targeting these molecular drivers for therapy and differentiating them from a physiological background require a detailed examination of the novel mechanisms involved in their activation during metastasis.. Publicly available transcriptomic data such as that of TCGA-LIHC and Gene Expression Omnibus were utilized to identify novel targets upregulated in advanced and metastatic HCC. Validation of candidates was assisted by immunohistochemistry performed on tissue microarrays derived from more than 100 HCC patients. Expression of protein tyrosine kinase 7 (PTK7) was studied under the treatment of transforming growth factor-β1 and knockdown of SRY-Box Transcription Factor 9 (SOX9) to delineate upstream regulation, while CRISPR-mediated knockout and lentiviral overexpression of PTK7 in HCC cells were performed to study their functional and signaling consequences. Manipulated HCC cells were injected into mice models either by orthotopic or tail-vein injection to observe for any in vivo pro-metastatic effects.. PTK7 was discovered to be the kinase most significantly upregulated in advanced and metastatic HCC, at both transcriptomic and proteomic level. Bioinformatic analyses and functional assays performed in HCC cell lines revealed transforming growth factor-β signaling and SOX9 to be important activators of PTK7 expression. Functionally, enrichment of PTK7 expression could positively regulate metastatic potential of HCC cells in vitro and in lung metastasis models performed in immunodeficient mice. The up-regulation of PTK7 recruited the epithelial-mesenchymal transition components, zinc finger protein SNAI2 (SLUG) and zinc finger E-box-binding homeobox 1 (ZEB1).. Our study proposes PTK7 as a novel molecular driver in metastatic HCC, particularly in a transforming growth factor-β-activated microenvironment. The preferential expression of PTK7 resulted in a previously unobserved regulatory effect on the recruitment of epithelial-mesenchymal transition components, which established PTK7 as a potential determinant of specific epithelial-mesenchymal transition status. Therefore, our data support the continual development of PTK7-targeted agents as antimetastatic therapies.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Liver Neoplasms; Mice; Proteomics; Receptor Protein-Tyrosine Kinases; SOX9 Transcription Factor; Transforming Growth Factor beta; Tumor Microenvironment

Transforming growth factor beta (TGF-β) signaling pathway plays important roles in hepatocellular carcinoma (HCC) progression. Long intergenic non-protein coding RNAs (lincRNAs) are important components of TGF-β signaling pathway and perform their functions through different mechanisms. Here, we found that LINC02551 was activated by TGF-β transcriptionally and identified a 174-amino-acid peptide, Jun binding micropeptide (JunBP), encoded by LINC02551 in HCC tissues and HCC cell lines. Functional study showed that JunBP promotes HCC metastasis through binding to c-Jun and subsequent promotion of its phosphorylated activation. Activated c-Jun has higher binding affinity to SMAD3, which in turn leads to more SMAD3 recruited to the promoter region of LINC02551. We find a positive feedback among them, and this mechanism provides a novel potential prognostic biomarker and therapeutic target in HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; Micropeptides; Signal Transduction; Transforming Growth Factor beta

KIN17 DNA and RNA binding protein (Kin17) is involved in the regulation of tumorigenesis of diverse human cancers. However, its role in the cancer progression and metastasis in hepatocellular carcinoma (HCC) remains largely unknown. Bioinformatics and immunohistochemistry staining were used to investigate the expression pattern of KIN17 and its prognostic value in HCC patients. The transwell, wound-healing assay was employed to determine the effects of KIN17 on migration and invasion of HCC cells in vitro. The tail veins model was employed to determine the effects of KIN17 on lung metastasis in vivo. The biological mechanisms involved in cell migration and invasion regulated by KIN17 were determined with Western blot analysis method. KIN17 expression was significantly increased in HCC tissues compared with adjacent normal tissues, with particularly higher in portal vein tumor thrombus and intrahepatic metastasis tissues. Patients with higher KIN17 expression experienced poor overall and disease free survival. KIN17 knockdown in HuH7 and HepG2 cells significantly reduced cell migration and invasion abilities, whereas its overexpression promoted migration and invasion in MHCC-97L and HepG2 cells in vitro and in vivo. In HuH7 and HepG2 cells, KIN17 knockdown inhibited the TGF-β/Smad2 pathway. In contrast, KIN17 overexpression stimulated TGF-β/Smad2 pathway in MHCC-97L and HepG2 cells, along with the genes involved in the epithelial-mesenchymal transition. These findings suggest that KIN17 promotes migration and invasion in HCC cells by stimulating the TGF-β/Smad2 pathway. KIN17 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Smad2 Protein; Transforming Growth Factor beta

Squalene epoxidase (SQLE) is a key enzyme involved in cholesterol biosynthesis, but growing evidence also reveals that SQLE is abnormally expressed in some types of malignant tumours, even though the underlying mechanism remains poorly understood.. Bioinformatics analysis and RNA sequencing were applied to detect differentially expressed genes in clinical hepatocellular carcinoma (HCC). MTT, colony formation, AnnexinV-FITC/PI, EdU, wound healing, transwell, western blot, qRT-PCR, IHC, F-actin, RNA-sequencing, dual-luciferase reporters, and H&E staining were used to investigate the pharmacological effects and possible mechanisms of SQLE.. SQLE expression was specifically elevated in HCC, correlating with poor clinical outcomes. SQLE significantly promoted HCC growth, epithelial-mesenchymal transition, and metastasis both in vitro and in vivo. RNA sequencing and functional experiments revealed that the protumourigenic effect of SQLE on HCC was closely related to the activation of TGF-β/SMAD signalling, but the stimulatory effect of SQLE on TGF-β/SMAD signalling and HCC development is critically dependent on STRAP. SQLE expression is well correlated with STRAP in HCC, and further, to amplify TGF-β/SMAD signalling, SQLE even transcriptionally increased STRAP gene expression mediated by AP-2α. Finally, as a chemical inhibitor of SQLE, NB-598 markedly inhibited HCC cell growth and tumour development.. Taken together, SQLE serves as a novel oncogene in HCC development by activating TGF-β/SMAD signalling. Targeting SQLE could be useful in drug development and therapy for HCC.

Topics: Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Squalene Monooxygenase; Transforming Growth Factor beta

Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood.. We analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems.. We observed positive associations between L1 and activated TGFβ-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFβ-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p.. Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue.

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Long Interspersed Nucleotide Elements; NIMA-Interacting Peptidylprolyl Isomerase; Retroelements; Transforming Growth Factor beta; Up-Regulation

Tea domain transcription factor 4 (TEAD4) plays a pivotal role in tissue development and homeostasis by interacting with Yes-associated protein (YAP) in response to Hippo signaling inactivation. TEAD4 and YAP can also cooperate with transforming growth factor-β (TGF-β)-activated Smad proteins to regulate gene transcription. Yet, it remains unclear whether TEAD4 plays a YAP-independent role in TGF-β signaling. Here, we unveil a novel tumor suppressive function of TEAD4 in liver cancer via mitigating TGF-β signaling. Ectopic TEAD4 inhibited TGF-β-induced signal transduction, Smad transcriptional activity, and target gene transcription, consequently suppressing hepatocellular carcinoma cell proliferation and migration in vitro and xenograft tumor growth in mice. Consistently, depletion of endogenous TEAD4 by siRNAs enhanced TGF-β signaling in cancer cells. Mechanistically, TEAD4 associates with receptor-regulated Smads (Smad2/3) and Smad4 in the nucleus, thereby impairing the binding of Smad2/3 to the histone acetyltransferase p300. Intriguingly, these negative effects of TEAD4 on TGF-β/Smad signaling are independent of YAP, as impairing the TEAD4-YAP interaction through point mutagenesis or depletion of YAP and/or its paralog TAZ has little effect. Together, these results unravel a novel function of TEAD4 in fine tuning TGF-β signaling and liver cancer progression in a YAP-independent manner.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Mice; Signal Transduction; TEA Domain Transcription Factors; Transforming Growth Factor beta; YAP-Signaling Proteins

Targeting Nicotinamide adenine dinucleotide (NAD) metabolism has emerged as a promising anti-cancer strategy; we aimed to explore the health benefits of boosting NAD levels with nicotinamide riboside (NR) on hepatocellular carcinoma (HCC). We established three in vivo tumor models, including subcutaneous transplantation tumor model in both Balb/c nude mice (xenograft), C57BL/6J mice (allograft), and hematogenous metastatic neoplasm in nude mice. NR (400 mg/kg bw) was supplied daily in gavage. In-situ tumor growth or noninvasive bioluminescence were measured to evaluate the effect of NR on the HCC process. HepG2 cells were treated with transforming growth factor-β (TGF-β) in the absence/presence of NR in vitro. We found that NR supplementation alleviated malignancy-induced weight loss and metastasis to lung in nude mice in both subcutaneous xenograft and hematogenous metastasis models. NR supplementation decreased metastasis to the bone and liver in the hematogenous metastasis model. NR supplementation also significantly decreased the size of allografted tumors and extended the survival time in C57BL/6J mice. In vitro experiments showed that NR intervention inhibited the migration and invasion of HepG2 cells triggered by TGF-β. In summary, our results supply evidence that boosting NAD levels by supplementing NR alleviates HCC progression and metastasis, which may serve as an effective treatment for the suppression of HCC progression.

Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; NAD; Niacinamide; Transforming Growth Factor beta

Phytochemicals have been promising candidates for cancer therapy, affecting various cancer initiation and progression stages. Kaempferide is a mono methoxy flavone that shows potent anticancer effects on multiple cancers both in vitro and in vivo.. We evaluated the anticancer activity of kaempferide against HCC using an MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. HepG2, Huh7, and N1S1 were used for preliminary in vitro studies. This is followed by an apoptosis analysis assessed by caspase-3 and 9. The in vivo effects of the compound were studied in the N1S1 orthotopically injected SD (Sprague Dawley) rat model, where the animal was given kaempferide (25 mg/kg thrice a week) and vehicle (Cremophor:ethanol) iv. The expression of caspase-9 and a critical tumor marker, transforming growth factor beta 1 (TGF-β 1), were assessed in both control and treatment tumor samples.. Kaempferide-induced dose-dependent cytotoxicity in three HCC cell lines (HepG2: IC50 = 27.94 ± 2.199 µM; Huh7: IC50 = 25.65 ± 0.956 µM; and N1S1: IC50 = 15.18 ± 3.68 µM). Furthermore, caspase-dependent apoptosis was confirmed in vitro. Kaempferide showed a significant reduction in tumor size and tumor volume in vivo. Histopathological evaluation by hematoxylin and eosin (H&E) staining confirmed that altered cells were significantly demolished in the kaempferide-treated animals, which correlates with tumor reduction compared to the vehicle-treated group. Caspase-9 levels were also found to be increased in the treatment group. TGF-β 1, a crucial marker in invasion and metastasis of liver cancer, was also downregulated in the treatment group (control = 207.8 ± 22.9 pg/mL and kaempferide-treated = 157.3 ± 13.8 pg/mL).. We report for the first time the potential of kaempferide as a promising alternative against HCC, which further warrants its clinical validation.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 9; Cell Line, Tumor; Cell Proliferation; Liver Neoplasms; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Therapeutic targeting of the transforming growth factor beta (TGFβ) pathway in cancer represents a clinical challenge since TGFβ exhibits either tumor suppressive or tumor promoting properties, depending on the tumor stage. Thus, treatment with galunisertib, a small molecule inhibitor of TGFβ receptor type 1, demonstrated clinical benefits only in subsets of patients. Due to the functional duality of TGFβ in cancer, one can hypothesize that inhibiting this pathway could result in beneficial or adverse effects depending on tumor subtypes. Here, we report distinct gene expression signatures in response to galunisertib in PLC/PRF/5 and SNU-449, two cell lines that recapitulate human hepatocellular carcinoma (HCC) with good and poor prognosis, respectively. More importantly, integrative transcriptomics using independent cohorts of patients with HCC demonstrates that galunisertib-induced transcriptional reprogramming in SNU-449 is associated with human HCC with a better clinical outcome (i.e., increased overall survival), while galunisertib-induced transcriptional reprogramming in PLC/PRF/5 is associated with human HCC with a worse clinical outcome (i.e., reduced overall survival), demonstrating that galunisertib could indeed be beneficial or detrimental depending on HCC subtypes. Collectively, our study highlights the importance of patient selection to demonstrate a clinical benefit of TGFβ pathway inhibition and identifies Serpin Family F Member 2 (SERPINF2) as a putative companion biomarker for galunisertib in HCC.

Topics: Carcinoma, Hepatocellular; Gene Expression Profiling; Humans; Liver Neoplasms; Transcriptome; Transforming Growth Factor beta

TGF-β is related to the function of T cells in the tumor microenvironment. However, the characteristics of TGF-β affecting the function of CD8. In this study, flow cytometry, mass cytometry, immunohistochemistry, RNA-seq, single-cell RNA-seq, assay for transposase-accessible chromatin with high throughput sequencing, chromatin immunoprecipitation, and dual-luciferase reporter gene assay were used to study the regulatory effect and molecular mechanism of TGF-β on HCC infiltrating CD8. Here, we demonstrated that the overall effect of TGF-β on CD8. Our study describes a "self-rescue" mechanism of CD8

Topics: Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Humans; Liver Neoplasms; STAT1 Transcription Factor; Transforming Growth Factor beta; Tumor Microenvironment

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-β) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-β-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-β. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-β, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-β-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-β in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-β1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-β that may contribute to tumor progression, we found that NOX4 is also required for TGF-β-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-β-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-β-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-β signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-β pathway.

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; NADPH Oxidase 4; NADPH Oxidases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Recently, therapeutic antibodies against programmed cell death 1 (PD-1) and its ligand (PD-L1) have exerted potent anticancer effect in a variety of tumors. However, blocking the PD-1/PD-L1 axis alone is not sufficient to restore normal immune response. Other negative regulators of antitumor immunity, like TGF-β and VEGFA, are also involved in immune escape of tumor cells and induce immunotherapy resistance.. We developed a novel anti-TGF-β/VEGF bispecific antibody Y332D based on the Nano-YBODY™ technology platform. The CCK-8, flow cytometry, SBE4 luciferase reporter assay, western blotting and transwell assays were used to measure the biological activities of the anti-TGF-β moiety. The NFAT luciferase reporter assay, luminescent cell viability assay and tube formation assay were used to measure the biological activities of the anti-VEGF moiety. The in vivo anticancer efficacy of Y332D alone or in combination with PD-1 blockade was evaluated in H22, EMT-6, 4T1, and AKT/Ras-driven murine hepatocellular carcinoma tumor models. Immunofluorescent staining, flow cytometry, RNA-seq and quantitative RT-PCR were adopted to analyze the alterations in the tumor microenvironment.. Y332D could maintain specific binding affinities for TGF-β and VEGFA. Y332D almost entirely counteracted the in vitro biological functions of TGF-β and VEGFA, including immunosuppression, activated TGF-β signaling, epithelial-mesenchymal transition (EMT), activated VEGF/VEGFR signaling, HUVEC proliferation and tube formation. The in vivo experiment data demonstrated that Y332D was more effective in inhibiting tumor growth and metastasis than anti-TGF-β and anti-VEGF monotherapies. In combination therapies, Y332D plus PD-1 blockade exhibited the most potent and durable anticancer effect. Mechanistically, Y332D plus PD-1 blockade upregulated the density and function of tumor-infiltrating lymphocytes and exerted reinvigorated antitumor immunity.. Y332D could simultaneously block TGF-β and VEGF signalings. In comparison with the monotherapies, Y332D combined with PD-1 blockade exerts superior antitumor effect through improving immune microenvironment.

Topics: Animals; Antibodies, Bispecific; B7-H1 Antigen; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; Mice; Programmed Cell Death 1 Receptor; Transforming Growth Factor beta; Tumor Microenvironment

The class I- phosphatidylinositol-3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform-specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation.. Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT-qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ-overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8-based-PI3Kδ overexpression. These PI3Kδ-mediated cell morphogenesis and stroma remodelling were dependent on TGFβ/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression.. Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFβ/src/Notch-dependent mechanisms and open new directions for the classification and treatment of CCA patients.

Topics: Animals; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Cell Line, Tumor; Cholangiocarcinoma; Fibrosis; Humans; Liver Neoplasms; Mice; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Protein Isoforms; Transforming Growth Factor beta

Increased expression of TGFB regulatory factors DNMT3A and DNMT3B in non-neoplastic liver tissues of HCC patients is the goal of this study. Furthermore, we demonstrate that TGF- is capable of elevating the percentage of CD133+ cells present in liver cancer cell lines in a manner that is both consistent and long-lasting over several cell divisions. This process is linked to stable alterations in DNA methylation that occur over the whole of the genome and continue even after cell division. In addition, the silencing of de novo DNA methyl-transferases with siRNA is able to inhibit the phenotypic changes that are induced by TGF-. According to the findings of our research, there is a self-sustaining interaction between the DNA methylation machinery and the TGF- signaling pathway, which may be significant in the development of cellular phenotypes. CD133 positive and negative fractions expand within liver cancer cell lines in proportions that remain stable throughout time. In contrast to their CD133- counterparts, MACS-sorted CD133+ Huh7cells demonstrated the ability to shape themselves into spheres when grown under non-attachment circumstances. This study also found that the TGF- is responsible for the de novo induction of CD133, which is linked to an increase in the expression of DNMT3 genes and there is a correlation between the TGF-induced transition in the cell subpopulation and a distinct DNA methylome. TGF- has the potential to generate genome-wide alterations in DNA methylation, which ultimately leads to a persistent shift in the fraction of liver cancer cell subpopulations.

Topics: Carcinoma, Hepatocellular; DNA Methylation; DNA Methyltransferase 3B; Humans; Liver Neoplasms; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies worldwide. However, the molecular mechanism of HCC metastasis is largely unknown. Long non-coding RNA (lncRNA) plays a key role in gene regulation, and dysregulation of lncRNA is critical to cancer metastasis. LINC01980 has been reported in ESCC recently, but the mechanism underlying its function in HCC is still unknown. In this study, we found that LINC01980 was upregulated and associated with notably poor overall survival in HCC patients. Functionally, LINC01980 played a carcinogenic role and promoted HCC metastasis. Mechanically, LINC01980 enhanced the E2F5 expression via competitively binding miR-376b-5p, thereby inducing epithelial-mesenchymal transition and promoting HCC cells migration and invasion. In addition, LINC01980-mediated HCC cells metastasis was dependent on E2F5. What's more, TGF-β activated LINC01980 transcription through the canonical TGF-β/SMAD signaling pathway in HCC. In conclusion, LINC01980, activated by the canonical TGF-β/SMAD pathway, promoted HCC metastasis via miR-376b-5p/E2F5 axis. Therefore, LINC01980 might be a potential prognostic biomarker and therapeutic target of HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; E2F5 Transcription Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; RNA, Long Noncoding; Transforming Growth Factor beta

Krüppel-like factor 10 (KLF10) is involved in a positive feedback loop that regulates transforming growth factor β (TGFβ) signaling, and TGFβ plays an important role in the pathogenesis of liver disease. Here, we investigated whether KLF10 deletion affects the development of liver fibrosis and hepatocellular carcinoma (HCC).. We induced KLF10 deletion in C57BL/6 mice. Liver fibrosis was induced by feeding a diet high in fat and sucrose (high-fat diet [HFD]), whereas HCC was produced by intraperitoneal administration of N-diethylnitrosamine (DEN). An in vitro experiment was performed to evaluate the role of KLF10 in the cancer microenvironment using Hep3B and LX2 cells. An immunohistochemical study of KLF10 expression was performed using human HCC samples from 60 patients who had undergone liver resection.. KLF10 deletion resulted in an increased DEN-induced HCC burden with significant upregulation of SMAD2, although loss of KLF10 did not alter HFD-induced liver fibrosis. DEN-treated mice with KLF10 deletion exhibited increased levels of mesenchymal markers (N-cadherin and SNAI2) and tumor metastasis markers (matrix metalloproteinases 2 and 9). KLF10 depletion in Hep3B and LX2 cells using siRNA was associated with increased invasiveness. Compared with co-culture of KLF10-preserved Hep3B cells and KLF10-intact LX2 cells, co-culture of KLF10-preserved Hep3B cells and KLF10-depleted LX2 cells resulted in significantly enhanced invasion. Low KLF10 expression in resected human HCC specimens was associated with poor survival.. The results of this study suggest that loss of KLF10 facilitates liver cancer development with alteration in TGFβ signaling.

Topics: Animals; Carcinoma, Hepatocellular; Humans; Kruppel-Like Transcription Factors; Liver Cirrhosis; Liver Neoplasms; Mice; Mice, Inbred C57BL; Transforming Growth Factor beta; Tumor Microenvironment

Transforming growth factor beta (TGF-β) plays a key role in tumor progression, notably as a potent inducer of epithelial-mesenchymal transition (EMT). However, all of the molecular effectors driving TGFβ-induced EMT are not fully characterized. Here, we report that forkhead box S1 (FOXS1) is a SMAD (mothers against decapentaplegic)-dependent TGFβ-induced transcription factor, which regulates the expression of genes required for the initial steps of EMT (e.g., snail family transcription repressor 1) and to maintain a mesenchymal phenotype in hepatocellular carcinoma (HCC) cells. In human HCC, we report that FOXS1 is a biomarker of poorly differentiated and aggressive tumor subtypes. Importantly, FOXS1 expression level and activity are associated with a poor prognosis (e.g., reduced patient survival), not only in HCC but also in colon, stomach, and kidney cancers. Conclusion: FOXS1 constitutes a clinically relevant biomarker for tumors in which the pro-metastatic arm of TGF-β is active (i.e., patients who may benefit from targeted therapies using inhibitors of the TGF-β pathway).

Topics: Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Forkhead Transcription Factors; Humans; Liver Neoplasms; Prognosis; Transforming Growth Factor beta

Gemcitabine can alter the immunogenic microenvironments, and the effect of gemcitabine plus programmed death-1 (PD-1)/ programmed death-ligand 1 (PD-L1) blockade in hepatocellular carcinoma (HCC) is investigated.. Subcutaneous H22-green fluorescent protein (GFP) cells inoculation model was constructed and treated with gemcitabine, anti-PD-1 antibody (αPD-1), or the combination every four days when the tumor volumes reached about 50 mm. Gemcitabine treatment can effectively increase the total proportion of infiltrating immune cells, reduce the proportion of myeloid-derived suppressor cells (MDSCs) and macrophages, and increase T cells proportion without significant growth inhibition. While after gemcitabine treatment, PD-L1 expression on tumor cells and PD-1 on T cells were significantly up-regulated. Subcutaneous tumors volume were reduced considerably after gemcitabine plus αPD-1 treatment compared with gemcitabine (P<0.01) or αPD-1 monotherapy (P<0.001) with the increased proportion of IL-2

Topics: Animals; B7-H1 Antigen; Carcinoma, Hepatocellular; Cell Line, Tumor; Deoxycytidine; Gemcitabine; Humans; Liver Neoplasms; Mice; Neoplasm Recurrence, Local; Programmed Cell Death 1 Receptor; Transforming Growth Factor beta; Tumor Microenvironment

Dahuang Zhechong pill (DHZCP) improves the inhibitory immune status of mice with hepatocellular carcinoma (HCC) by regulating Treg/Th1 balance.. To study the multi-material basis and multi-mechanisms of DHZCP against HCC by regulating Treg/Th1 balance in vitro and in vivo.. Water-soluble (PW) and ethyl acetate (PE) polar parts of DHZCP affected the HCC immune system by inhibiting the differentiation of Tregs, reversing the balance of Treg/Th1, and significantly reduced the tumor volume and weight. However, petroleum ether and n-butanol polar parts had no above actions. The changes in emodin, chrysophanol, aloe vera emodin, emodin-8-O-β-D-glycoside, gallic acid, naringenin, baicalein, wogonin, norwogonin, apigenin, chrysin, glycyrrhizin, formononetin, and palmitic acid were closely related to the changes of Treg cells, which is the main material basis of DHZCP inhibition of Treg differentiation. Additionally, PW mainly inhibit the differentiation of Treg cells by affecting the metabolism of hepatoma cells, improving tumor microenvironment acidity, and glutamine depletion. However, PE inhibited the differentiation of Treg cells mainly by regulating the TGF-β/Smad pathway.. In this study, accurate analysis of multi-component was combined with pharmacodynamic evaluations to identify the pharmacodynamic substances of DHZCP in regulating Treg/Th1 balance, and clarified the multi-target mechanism of DHZCP to improve tumor immunity. The study style offers a novel approach for pharmacological research on TCM.

Topics: Animals; Carcinoma, Hepatocellular; Drugs, Chinese Herbal; Emodin; Glutamine; Liver Neoplasms; Mice; T-Lymphocytes, Regulatory; Tandem Mass Spectrometry; Transforming Growth Factor beta; Tumor Microenvironment

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Emodin; Humans; Liver Neoplasms; Macrophages; MicroRNAs; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta; Transforming Growth Factor beta1

Multidrug resistance is the main obstacle to curing hepatocellular carcinoma (HCC). Acid-sensing ion channel 1a (ASIC1a) has critical roles in all stages of cancer progression, especially invasion and metastasis, and in resistance to therapy. Epithelial to mesenchymal transition (EMT) transforms epithelial cells into mesenchymal cells after being stimulated by extracellular factors and is closely related to tumour infiltration and resistance. We used Western blotting, immunofluorescence, qRT-PCR, immunohistochemical staining, MTT, colony formation and scratch healing assay to determine ASIC1a levels and its relationship to cell proliferation, migration and invasion. ASIC1a is overexpressed in HCC tissues, and the amount increased in resistant HCC cells. EMT occurred more frequently in drug-resistant cells than in parental cells. Inactivation of ASIC1a inhibited cell migration and invasion and increased the chemosensitivity of cells through EMT. Overexpression of ASIC1a upregulated EMT and increased the cells' proliferation, migration and invasion and induced drug resistance; knocking down ASIC1a with shRNA had the opposite effects. ASIC1a increased cell migration and invasion through EMT by regulating α and β-catenin, vimentin and fibronectin expression via the AKT/GSK-3β/Snail pathway driven by TGFβ/Smad signals. ASIC1a mediates drug resistance of HCC through EMT via the AKT/GSK-3β/Snail pathway.

Topics: Acid Sensing Ion Channels; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3 beta; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

Long non-coding RNA (lncRNA) cancer susceptibility candidate 7 (CASC7) was reported to be participated in tumor development. This study was carried out to investigate the functions of CASC7 in hepatocellular carcinoma (HCC) progression. The expression of CASC7 and microRNA-30a-5p (miR-30a-5p) in HCC tissues and cells were detected by quantitative Real-time PCR (qRT-PCR). The expression of Krueppel-like factor 10 (KLF10), transforming growth factor-β (TGF-β), and SMAD3 were detected by Western Blot analysis. Transwell assay, flow cytometry, Cell Counting Kit-8 (CCK-8) assay and colony formation assay were performed to evaluate the effects of CASC7, KLF10 and miR-30a-5p on cell function. The relationship among CASC7, KLF10 and miR-30a-5p was evaluated by luciferase reporter assay and bioinformatics analyses. Tumor growth was detected in nude mice. The expression levels of CASC7 were increased and the expression levels of miR-30a-5p were reduced in HCC cells and tissues. Knockdown of CASC7 and overexpression of miR-30a-5p reduced tumor growth as well as HCC cell proliferation, invasion and migration. In HCC tumor tissues, the expression of miR-30a-5p was negatively correlated with the expression of CASC7. Moreover, as a target of miR-30a-5p, KLF10 was regulated by CASC7 and miR-30a-5p, and CASC7 regulated the KLF10/TGF-β/SMAD3 pathway via binding to miR-30a-5p, thereby promoting HCC cell progression.

Topics: Animals; Argonaute Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Liver Neoplasms; Mice; Mice, Nude; MicroRNAs; RNA, Long Noncoding; Transforming Growth Factor beta

Thermal ablation is a main curative therapy for early-stage hepatocellular carcinoma (HCC). However, insufficient ablation has been shown to promote HCC progression. E3 ligases have been approved to play important roles in malignant tumors. Whether E3 ligases are involved in HCC progression caused by insufficient ablation remains unclear. Herein, using RNA-sequencing coupled with an in vitro loss-of-function screen, we found that the E3 ligase Neuronal Precursor cell-expressed Developmentally Downregulated 4 (Nedd4) was upregulated in HCC insufficient ablation tissues and promoted HCC cells migration. The upregulation of Nedd4 was induced by METTL14-mediated N

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; Nedd4 Ubiquitin Protein Ligases; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Dysregulated transforming growth factor-beta (TGF-β) signaling contributes to fibrotic liver disease and hepatocellular cancer (HCC), both of which are associated with fatty liver disease. SIRT6 limits fibrosis by inhibiting TGF-β signaling through deacetylating SMAD2 and SMAD3 and limits lipogenesis by inhibiting SREBP1 and SREBP2 activity. Here, we showed that, compared to wild-type mice, high-fat diet-induced fatty liver is worse in TGF-β signaling-deficient mice (SPTBN1

Topics: Animals; Carcinoma, Hepatocellular; Fatty Liver; Fibrosis; Liver Neoplasms; Mice; Sirtuins; Sterol Regulatory Element Binding Protein 1; Transforming Growth Factor beta

Major HCV infections lead to chronic hepatitis, which results in progressive liver disease including fibrosis, cirrhosis and eventually hepatocellular carcinoma (HCC). P2X4 and P2X7 are most widely distributed receptors on hepatocytes.. Full length P2X4 (1.7kb) (Rattus norvegicus) was sub cloned in mammalian expression vector pcDNA3.1+. Two stable cell lines 293T/P2X4 (experimental) and 293T/ NV or null vector (control) were established. Both cell lines were inoculated with high viral titers human HCV sera and control human sera. Successfully infected cells harvested on day 5 and day 9 of post infection were used for further studies.. The results revealed a significant increase in gene expression of P2X4 on day 5 and day 9 Post -infection in cells infected with HCV sera compared with cells inoculated with control sera. Quantitative real time PCR analysis revealed that HO-1 was significantly upregulated in presence of P2X4 in HCV infected cells (P2X4/HCV) when compared with control NV/HCV cells. A significant decrease was observed in expression of Cu/ZnSOD in presence of P2X4 in HCV infected cells compared to control NV/HCV cells. However, expression of both antioxidants was observed unaltered in cells harvested on day 9 post infection. Gene expression of angiotensin II significantly increased in HCV infected cells in presence of P2X4 on day 5 and day 9 of post infection when compared with control NV/HCV cells. A significant increase in gene expression of TNF-α and TGF-β was observed in HCV infected cells in presence of P2X4 on day 9 post infection in comparison with control (NV/HCV cells). However, gene expression of adipokine leptin was not affected in both experimental (P2X4/HCV) and control (NV/HCV) groups on day 5 and day 9 of post infection. Extracellular matrix proteins, laminin and elastin genes expression also significantly increased in presence of P2X4 (HCV/P2X4) on day 9 of post-infection compared to control group NV/HCV cells.. In conclusion, these findings constitute the evidence that P2X4 receptors in the presence of HCV play a significant role in the regulation of key antioxidant enzymes (HO-1, Cu/ZnSOD), in the induction of proinflammatory. cytokine (TNF-α), profibrotic cytokine (TGF-β) vasoactive cytokine (angiotensin II). P2X4 also increases the expression of extracellular matrix proteins (laminin and elastin) in the presence of HCV.

Topics: Angiotensin II; Animals; Antioxidants; Carcinoma, Hepatocellular; Cytokines; Elastin; Fibrosis; Hepacivirus; Hepatitis C; Humans; Laminin; Liver Neoplasms; Rats; Receptors, Purinergic P2X4; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Hepatocellular carcinoma (HCC) accounts for the majority of liver cancer cases, while metastasis is considered the leading cause of HCC-related death. However, the currently available treatment strategies for efficient suppression of metastasis are limited. Therefore, novel therapeutic targets to inhibit metastasis and effectively treat HCC are urgently required.. Wound healing and Transwell assays were used to determine the migration and invasion abilities of HCC cells in vitro. Quantitative real-time PCR (qRT-PCR), protein array, immunofluorescence, and immunoprecipitation experiments were used to study the mechanism of DYRK1A-mediated metastasis. A tail vein metastasis model and H&E staining were utilized to assess metastatic potential in vivo.. The results of the current study demonstrated that dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) was upregulated in HCC tissues compared with normal liver tissues. Additionally, the level of DYRK1A was increased in primary HCC tissues of patients with metastasis compared with those of patients without metastasis, and DYRK1A overexpression correlated with worse outcomes in liver cancer patients. Gain- and loss-of-function studies suggested that DYRK1A enhanced the invasion and migration abilities of HCC cells by promoting epithelial-mesenchymal transition (EMT). Regarding the promoting effect of DYRK1A on cell invasion, the results showed that DYRK1A was coexpressed with TGF-β/SMAD and STAT3 signalling components in clinical tumour samples obtained from patients with HCC. DYRK1A also activated TGF-β/SMAD signalling by interacting with tuberous sclerosis 1 (TSC1) and enhanced metastasis of HCC cells by activating STAT3. Furthermore, DYRK1A promoted EMT by cooperatively activating STAT3/SMAD signalling.. Overall, the present study not only uncovered the promoting effect of DYRK1A on HCC metastasis and revealed the mechanism but also provided a new approach to predict and treat metastatic HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; STAT3 Transcription Factor; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC), the most common form of liver cancer, is a leading cause of tumor-associated mortality worldwide. Diagnosis based upon non-invasive criteria is currently challenged by the need for molecular information that requires tissue or liquid biopsies. The progression of HCC is often associated with chronic inflammation, expression levels of inflammatory mediators, chemokine, and cytokines. In this study, we try to evaluate the PI3K and pro-inflammatory cytokines, TGF-β, IL-1, and IL-6 expression level in patients with liver cancer.. The kupffer cells were isolated from patient's specimens. Real-time PCR was applied to evaluate the expression level of PI3K in cell lines or tumors. The concentrations of TGF-β, IL-1, and IL-6 were measured by the quantitative ELISA kit.. PI3K mRNA expression in cancer cells was increased markedly vs. normal cells. The ELISA results demonstrated over expression of TGF-β, IL-1, and IL-6 in patients and positive correlation between tumor size and stage.. This study suggests that targeting the expression level of PI3K and pro-inflammatory chemokine and cytokines, TGF-β, IL-1, and IL-6, may be a potential diagnostic strategy in HCC patients.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Cytokines; Humans; Interleukin-1; Interleukin-6; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Transforming Growth Factor beta

Regulatory T (Treg) cells play a vital role in maintaining the immunosuppressive tumor microenvironment. Lactate is a crucial metabolite in cancer and is related to tumor prognosis, metastasis, and overall survival. In this study, we focus on the effects of lactate on Treg cells. In vitro, lactate improves Treg cell stability and function, whereas lactate degradation reduces Treg cell induction, increases antitumor immunity, and decreases tumor growth in mice. Mechanistically, lactate modulates Treg cell generation through lactylation of Lys72 in MOESIN, which improves MOESIN interaction with transforming growth factor β (TGF-β) receptor I and downstream SMAD3 signaling. Cotreatment with anti-PD-1 and a lactate dehydrogenase inhibitor has a stronger antitumor effect than anti-PD-1 alone. Individuals with hepatocellular carcinoma who responded to anti-PD-1 treatment have lower levels of MOESIN lactylation in Treg cells than nonresponding individuals. Thus, we identify lactate as an essential small molecule that reinforces Treg cells in the tumor microenvironment through lactylation.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Lactic Acid; Liver Neoplasms; Mice; Mice, Knockout; Microfilament Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer in the world with high mortality due to its high potential of metastasis. Epithelial-mesenchymal transition (EMT) plays a key role in the pathogenesis of HCC occurrence and metastasis. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a novel tumor suppressor. There is little study about LHPP in human HCC development. In the present study, we aimed to investigate the role of LHPP in human HCC cell metastasis. We analyzed the LHPP expression level in human HCC tissues compared with normal tissues in the public database. We detected the mRNA level and protein level of LHPP in transformed liver cell line (LO2) and human HCC cell lines (MHCC-97 H, MHCC-97L, and HepG2). We performed genetic gain and loss of function experiments with LHPP using small interfering RNA (siRNA) and lentivirus infection. Then, we detected that LHPP suppressed proliferation and promoted apoptosis in hepatocellular carcinoma cell lines. Also, we investigated the role of LHPP in the EMT process. Finally, we examined the effect of LHPP on TGF-β-induced EMT. Interestingly, we also found that LHPP expression is positively regulated tumor suppressor p53. Our data showed that LHPP is significantly decreased in the human HCC tissues and human HCC cell lines compared with normal liver tissues and transformed liver cells. Knockdown of LHPP promotes HCC cell proliferation and metastasis, and LHPP expression levels negatively correlate with EMT-related genes. Furthermore, LHPP inhibits TGF-β-induced EMT in HCC cell lines. These studies validate LHPP as a tumor suppressor in liver cancer and provide a new genetic target for HCC diagnosis and treatment.

Topics: Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Inorganic Pyrophosphatase; Liver Neoplasms; RNA, Small Interfering; Transforming Growth Factor beta

The stem cells involved in formation of the complex human body are epithelial cells that undergo apicobasal polarization and form a hollow lumen. Epithelial plasticity manifests as epithelial to mesenchymal transition (EMT), a process by which epithelial cells switch their polarity and epithelial features to adopt a mesenchymal phenotype. The connection between the EMT program and acquisition of stemness is now supported by a substantial number of reports, although what discriminates these two processes remains largely elusive. In this study, based on 3D organoid culture of hepatocellular carcinoma (HCC)-derived cell lines and AAV8-based protein overexpression in the mouse liver, we show that activity modulation of isoform δ of phosphoinositide 3-kinase (PI3Kδ) controls differentiation and discriminates between stemness and EMT by regulating the transforming growth factor β (TGFβ) signaling. This study provides an important tool to control epithelial cell fate and represents a step forward in understanding the development of aggressive carcinoma.

Topics: Animals; Carcinoma, Hepatocellular; Class I Phosphatidylinositol 3-Kinases; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Transforming Growth Factor beta

Glucuronic acid metabolism participates in cellular detoxification, extracellular matrix remodeling and cell adhesion and migration. Here, we aimed to explore the crosstalk between dysregulated glucuronic acid metabolism and crucial metastatic signalling in glutathione S-transferase zeta 1 (GSTZ1)-deficient hepatocellular carcinoma (HCC).. Transwell, HCC xenograft and Gstz1. GSTZ1 was universally silenced in both human and murine HCC cells, and its deficiency contributed to HCC metastasis in vitro and in vivo. UDP-glucose 6-dehydrogenase (UGDH)-mediated UDP-glucuronic acid (UDP-GlcUA) accumulation promoted hepatoma cell migration upon GSTZ1 loss. UDP-GlcUA stabilized TGFβR1 mRNA by enhancing its binding to polypyrimidine tract binding protein 3, contributing to the activation of TGFβ/Smad signalling. UGDH or TGFβR1 blockade impaired HCC metastasis. In addition, UGDH up-regulation and UDP-GlcUA accumulation correlated with increased metastatic potential and decreased patient survival in GSTZ1-deficient HCC.. GSTZ1 deficiency and subsequent up-regulation of the glucuronic acid metabolic pathway promotes HCC metastasis by increasing the stability of TGFβR1 mRNA and activating TGFβ/Smad signalling. UGDH and a key metabolite, UDP-GlcUA, may serve as prognostic markers. Targeting UGDH might be a promising strategy for HCC therapy.

Topics: Animals; Carcinoma, Hepatocellular; Glucuronic Acid; Glutathione Transferase; Humans; Liver Neoplasms; Mice; RNA, Messenger; Transforming Growth Factor beta; Uridine Diphosphate; Uridine Diphosphate Glucose Dehydrogenase

Lnc-HUR1 is an HBV-related long non-coding RNA, which can promote the proliferation of hepatoma cells and the occurrence and development of liver cancer. In this study we explored the effect of lnc-HUR1 on the apoptosis of hepatocellular carcinoma cells by taking the approach of immunoblotting, quantitative real time PCR, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and flow cytometry. We found that overexpression of lnc-HUR1 significantly reduced the activity of caspase3/7 and the cleavage of PARP-1, while knocking down of lnc-HUR1 significantly increased the activity of caspase3/7 and promoted the cleavage of PARP-1 in HepG2 cells treated with TGF-β, pentafluorouracil or staurosporine. Consistently, the data from Annexin-V/PI staining showed that overexpression of lnc-HUR1 inhibited apoptosis, while knockdown of lnc-HUR1 promoted apoptosis. Moreover, overexpression of lnc-HUR1 up-regulated the apoptosis inhibitor Bcl-2 and down-regulated the pro-apoptotic factor BAX at both RNA and protein levels. In the CCL4-induced acute liver injury mice model, the expression of Bcl-2 in the liver tissue of lnc-HUR1 transgenic mice was higher than that of the control mice. The data from ChIP assay indicated that lnc-HUR1 reduced the enrichment of p53 on Bcl-2 and BAX promoters. All these results indicated that lnc-HUR1 inhibited the apoptosis by promoting the expression of apoptosis inhibitor Bcl-2 and inhibiting the expression of apoptosis promoting factor BAX. Further studies showed that lnc-HUR1 regulated the transcription of Bcl-2 and BAX in HCT116 cells, but had no effect on the expression of Bcl-2 and BAX in HCT116 p53

Topics: Animals; Annexins; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cell Proliferation; Hep G2 Cells; Hepatitis B virus; Humans; Liver Neoplasms; Mice; Poly(ADP-ribose) Polymerase Inhibitors; Proto-Oncogene Proteins c-bcl-2; RNA, Long Noncoding; Staurosporine; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Extracellular vesicles play crucial roles in intercellular communication in the tumor microenvironment. Here we demonstrate that in hepatic fibrosis, TGF-β stimulates the palmitoylation of hexokinase 1 (HK1) in hepatic stellate cells (HSCs), which facilitates the secretion of HK1 via large extracellular vesicles in a TSG101-dependent manner. The large extracellular vesicle HK1 is hijacked by hepatocellular carcinoma (HCC) cells, leading to accelerated glycolysis and HCC progression. In HSCs, the nuclear receptor Nur77 transcriptionally activates the expression of depalmitoylase ABHD17B to inhibit HK1 palmitoylation, consequently attenuating HK1 release. However, TGF-β-activated Akt functionally represses Nur77 by inducing Nur77 phosphorylation and degradation. We identify the small molecule PDNPA that binds Nur77 to generate steric hindrance to block Akt targeting, thereby disrupting Akt-mediated Nur77 degradation and preserving Nur77 inhibition of HK1 release. Together, this study demonstrates an overlooked function of HK1 in HCC upon its release from HSCs and highlights PDNPA as a candidate compound for inhibiting HCC progression.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Extracellular Vesicles; Hepatic Stellate Cells; Hexokinase; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Cytoplasmic and Nuclear; Transforming Growth Factor beta; Tumor Microenvironment

Hepatic fibrosis is a common pathologic stage in chronic liver disease development, which might ultimately lead to liver cirrhosis. Accumulating evidence suggests that adipose-derived stromal cells (ADSCs)-based therapies show excellent therapeutic potential in liver injury disease owing to its superior properties, including tissue repair ability and immunomodulation effect. However, cell-based therapy still limits to several problems, such as engraftment efficiency and immunoreaction, which impede the ADSCs-based therapeutics development. So, ADSCs-derived extracellular vesicles (EVs), especially for exosomes (ADSC-EXO), emerge as a promise cell-free therapeutics to ameliorate liver fibrosis. The effect and underlying mechanisms of ADSC-EXO in liver fibrosis remains blurred.. In this study, we obtained isolated ADSCs, collected and separated ADSCs-derived exosomes. We found that ADSC-EXO treatment could efficiently ameliorate DEN/CCl. These findings demonstrated that ADSC-derived exosomes could efficiently alleviate hepatic fibrosis by suppressing HSCs activation and remodeling glutamine and ammonia metabolism mediated by hepatocellular glutamine synthetase, which might be a novel and promising anti-fibrotic therapeutics for hepatic fibrosis disease.

Topics: Ammonia; Animals; Carbon Tetrachloride; Carcinoma, Hepatocellular; Diethylnitrosamine; Exosomes; Fibrosis; Glutamate-Ammonia Ligase; Glutamine; Hepatic Stellate Cells; Homeostasis; Liver Cirrhosis; Liver Neoplasms; Mice; Stromal Cells; Transforming Growth Factor beta

Topics: Animals; Antibodies, Neutralizing; Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Endothelial Cells; Glioma; Humans; Liver Neoplasms; Mice; Mice, Nude; Neovascularization, Pathologic; Nogo Proteins; Receptors, Transforming Growth Factor beta; Smad Proteins; Transforming Growth Factor beta; Tumor Microenvironment

STAT3 is involved in the progression of several cancers, and has been proposed as target for therapy. Indeed, the multitargeted tyrosine kinase inhibitor drug regorafenib, which indirectly inhibits STAT3, can significantly enhance the effects of anti-programmed death receptor (PD)-1 therapy in hepatocellular carcinoma (HCC) models. Here, we studied the impact of a direct STAT3 inhibitor on the tumor microenvironment and PD-1 blockade efficacy in HCC models.. Orthotopic mouse models of HCC (RIL-175 and HCA-1 grafts in syngeneic mice) were used to test the efficacy of the selective STAT3 inhibitor STX-0119 alone or combined with anti-PD-1 antibodies. We evaluated the effects of therapy on tumor vasculature and the immune microenvironment using immunofluorescence (IF), cell viability assay and quantitative real-time (qRT)-PCR in tumor tissues.. Combining anti-PD-1 antibodies with a STX-0119 failed to show a growth delay or survival benefit compared to each agent alone or control in any of the HCC models. Interestingly, evaluation of intratumoral CD8+ T cell infiltration by IF showed a significant increase after one-week treatment with STX-0119 (p=0.034). However, STX-0119 treatment simultaneously promoted increased immunosuppression in the tumor microenvironment by increasing the proportion of Tregs, tissue hypoxia and α-SMA activated cancer-associated fibroblasts (CAFs) measured by IF. Consistent with these findings, we found increased immature tumor vessels by IF and VEGF, Tgf-β and Vash2 expression by qPCR.. Pharmacologic STAT3 inhibition could significantly enhance CD8

Topics: Angiogenic Proteins; Animals; Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Immunosuppression Therapy; Liver Neoplasms; Mice; Protein Kinase Inhibitors; Receptors, Death Domain; STAT3 Transcription Factor; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A

Exosomes are deemed to be an important tool of intercellular communicators in cancer cells. Our study investigated the role of PRR34 long non-coding RNA antisense RNA 1 (PRR34-AS1) in regulating exosome secretion in hepatocellular carcinoma (HCC) cells.. Quantitative real-time polymerase chain reaction (RT-qPCR) analyzed the expression of PRR34-AS1. We assessed the function of PRR34-AS1 on the biological changes of THLE-3 cells and HCC cells. The downstream interaction between RNAS was assessed by mechanistic experiments.. PRR34-AS1 expression was upregulated in HCC cells in comparison to THLE-3 cells. PRR34-AS1 depletion repressed HCC cell proliferation, migration and invasion as well as EMT phenotype, while PRR34-AS1 up-regulation accelerated the malignant phenotypes of THLE-3 cells. PRR34-AS1 recruited DDX3X to stabilize the mRNA level of exosomal protein Rab27a. Moreover, PRR34-AS1 facilitated the malignant phenotypes of THLE-3 cells by elevating Rab27a expression to promote the exosome secretion of VEGF and TGF-β in HCC cells.. The current study revealed a novel function of PRR34-AS1 in accelerating exosome secretion in HCC cells and offered an insight into lncRNA function in the regulation of tumor cell biology.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; DEAD-box RNA Helicases; Exosomes; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; RNA, Long Noncoding; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines.. Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway.. We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes.. Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p15; Humans; Liver Neoplasms; RNA, Messenger; Smad Proteins; Transforming Growth Factor beta

Cancer cells are high in heterogeneity and versatility, which can easily adapt to the external stresses via both primary and secondary resistance. Targeting of tumour microenvironment (TME) is a new approach and an ideal therapeutic strategy especially for the multidrug resistant cancer. Recently, we invented AANG, a natural compound formula containing traditional Chinese medicine (TCM) derived Smad3 inhibitor Naringenin (NG) and Smad7 activator Asiatic Acid (AA), for rebalancing TGF-β/Smad signalling in the TME, and its implication on the multidrug resistance is still unexplored. Here, we observed that an equilibrium shift of the Smad signalling in patients with hepatocellular carcinoma (HCC), which was dramatically enhanced in the recurrent cases showing p-glycoprotein overexpression. We optimized the formula ratio and dosage of AANG that effectively inhibit the proliferation of our unique human multidrug resistant subclone R-HepG2. Mechanistically, we found that AANG not only inhibits Smad3 at post-transcriptional level, but also upregulates Smad7 at transcriptional level in a synergistic manner in vitro. More importantly, AANG markedly suppressed the growth and p-glycoprotein expression of R-HepG2 xenografts in vivo. Thus, AANG may represent a novel and safe TCM-derived natural compound formula for overcoming HCC with p-glycoprotein-mediated multidrug resistance.

Topics: Aged; Animals; Antineoplastic Agents; Biological Products; Carcinoma, Hepatocellular; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Synergism; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Middle Aged; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor beta (TGFβ) plays a key role in liver carcinogenesis. However, its action is complex, since TGFβ exhibits tumor-suppressive or oncogenic properties, depending on the tumor stage. At an early stage TGFβ exhibits cytostatic features, but at a later stage it promotes cell growth and metastasis, as a potent inducer of epithelial to mesenchymal transition (EMT). Here, we evaluated DNA methylation as a possible molecular mechanism switching TGFβ activity toward tumor progression in hepatocellular carcinoma (HCC). We report that decitabine, a demethylating agent already used in the clinic for the treatment of several cancers, greatly impairs the transcriptional response of SNU449 HCC cells to TGFβ. Importantly, decitabine was shown to induce the expression of EMT-related transcription factors (e.g.,

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Decitabine; DNA Methylation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Neoplasms; Neoplasm Recurrence, Local; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Aberrant activation of the TGF-β/SMAD signaling pathway is often observed in hepatocellular carcinoma (HCC). Whether lncRNA regulates the TGF-β/SMAD signaling remains largely unknown. Here, we identified an oncogenic lncRNA that was upregulated in HCC and was transcriptionally induced by TGF-β (named lnc-UTGF, lncRNA upregulated by TGF-β). Upon TGF-β stimulation, SMAD2/3 bound to the lnc-UTGF promoter and activated lnc-UTGF expression. In turn, the TGF-β/SMAD signaling was augmented by overexpressing lnc-UTGF, but was inhibited by silencing lnc-UTGF. Mechanism investigations revealed that lnc-UTGF interacted with the mRNAs of SMAD2 and SMAD4 via complementary base-pairing, resulting in enhanced stability of SMAD2/4 mRNAs. These data suggest a novel TGF-β/SMAD/lnc-UTGF positive feedback circuitry. Subsequent gain- and loss-of-function analyses disclosed that lnc-UTGF promoted the migration and invasion of hepatoma cells, and this effect of lnc-UTGF was attenuated by repressing SMAD2/4 expression or by mutating the SMAD2/4-binding sites in lnc-UTGF. Studies using mouse models further confirmed that in vivo metastasis of hepatoma xenografts was inhibited by silencing lnc-UTGF, but was enhanced by ectopic expression of lnc-UTGF. The lnc-UTGF level was positively correlated with the SMAD2/4 levels in xenografts. Consistently, we detected an association of lnc-UTGF upregulation with increase of SMAD2, SMAD4, and their metastasis effector SNAIL1 in human HCC. And high lnc-UTGF level was also significantly associated with enhanced metastasis potential, advanced TNM stages, and worse recurrence-free survival. Conclusion: there exists a lnc-UTGF-mediated positive feedback loop of the TGF-β signaling and its deregulation promotes hepatoma metastasis. These findings may provide a new therapeutic target for HCC metastasis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; HEK293 Cells; Humans; Liver Neoplasms; Mice; Neoplasm Metastasis; Neoplasm Proteins; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta

Patients with primary biliary cholangitis (PBC) are at increased risk for development of hepatocellular carcinoma (HCC), particularly in the presence of comorbidities such as excessive alcohol consumption. Although liver fibrosis is an important risk factor for HCC development, earlier predictors of future HCC development in livers with little fibrosis are needed but not well defined. The transforming growth factor (TGF)-β/Smad signaling pathway participates importantly in hepatic carcinogenesis. Phosphorylated forms (phospho-isoforms) in Smad-related pathways can transmit opposing signals: cytostatic C-terminally-phosphorylated Smad3 (pSmad3C) and carcinogenic linker-phosphorylated Smad3 (pSmad3L) signals.. To assess the balance between Smad signals as a biomarker of risk, we immunohistochemically compared Smad domain-specific Smad3 phosphorylation patterns among 52 PBC patients with various stages of fibrosis and 25 non-PBC patients with chronic hepatitis C virus infection. HCC developed in 7 of 11 PBC patients showing high pSmad3L immunoreactivity, but in only 2 of 41 PBC patients with low pSmad3L. In contrast, 9 of 20 PBC patients with minimal Smad3C phosphorylation developed HCC, while HCC did not occur during follow-up in 32 patients who retained hepatic tumor-suppressive pSmad3C. Further, PBC patients whose liver specimens showed high pSmad3L positivity were relatively likely to develop HCC even when little fibrosis was evident.. In this study, Smad phospho-isoform status showed promise as a biomarker predicting likelihood of HCC occurrence in PBC. Eventually, therapies to shift favorably Smad phospho-isoforms might decrease likelihood of PBC-related HCC.

Topics: Biomarkers; Carcinoma, Hepatocellular; Hepatitis C, Chronic; Humans; Liver Cirrhosis, Biliary; Liver Neoplasms; Risk Assessment; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor‑β (TGF-β) signaling is a crucial inducer of tissue fibrosis and extracellular matrix accumulation and a vital suppressor of epithelial cell proliferation and cancer metastasis. The nature of this multifunctional cytokine has prompted the development of TGF-β signaling inhibitors as therapeutic agents. Our research group has recently isolated the polyprenylated polycyclic acylphloroglucinol garcimultiflorone K (GMK) from the stems of Garcinia multiflora; GMK exhibits antiangiogenic activity in endothelial cells.. In the current study, we aimed to explore the antitumor effect and detailed mechanisms of Garcimultiflorone K in hepatocellular carcinoma cells.. Cell proliferation and viability were evaluated using the MTT assay. The migratory ability of HepG2 cells was measured using wound healing assays. The inhibitory effect of GMK against the nuclear translocation of Smad by TGF-β was assessed through immunofluorescence staining and Western blotting. To investigate TGF-β-dependent gene expression profiles upon GMK stimulation, RNA transcript levels were determined using reverse transcription polymerase chain reaction. The effects of GMK in Smad2-driven transcriptomic activities were studied using a reporter gene assay. Protein levels were detected using Western blotting.. Our data revealed that GMK inhibited TGF-β-induced cellular responses, including Smad protein phosphorylation, cell migration, and extracellular matrix production, during epithelial-mesenchymal transition (EMT). Mechanistic studies further demonstrated that GMK suppressed TGF-β signaling by downregulating TGF-β receptor II (TβRII).. These findings elucidate that TβRII expression in hepatic cells can be specifically suppressed by GMK to attenuate metastasis and the disease-promoting effects of EMT, representing a therapeutic approach.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Garcinia; Hep G2 Cells; Hepatic Stellate Cells; Humans; Liver Neoplasms; Mice; Phloroglucinol; Phosphorylation; Rats; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs) are key players in multicellular, stromal-dependent alterations leading to HCC pathogenesis. However, the intricate crosstalk between CAFs and other components in the tumor microenvironment (TME) remains unclear. This study aimed to investigate the cellular crosstalk among CAFs, tumor cells, and tumor-associated neutrophils (TANs) during different stages of HCC pathogenesis.. In the HCC-TME, CAF-derived cardiotrophin-like cytokine factor 1 (CLCF1) increased chemokine (C-X-C motif) ligand 6 (CXCL6) and TGF-β secretion in tumor cells, which subsequently promoted tumor cell stemness in an autocrine manner and TAN infiltration and polarization in a paracrine manner. Moreover, CXCL6 and TGF-β secreted by HCC cells activated extracellular signal-regulated kinase (ERK) 1/2 signaling of CAFs to produce more CLCF1, thus forming a positive feedback loop to accelerate HCC progression. Inhibition of ERK1/2 or CLCF1/ciliary neurotrophic factor receptor signaling efficiently impaired CLCF1-mediated crosstalk among CAFs, tumor cells, and TANs both in vitro and in vivo. In clinical samples, up-regulation of the CLCF1-CXCL6/TGF-β axis exhibited a marked correlation with increased cancer stem cells, "N2"-polarized TANs, tumor stage, and poor prognosis.. This study reveals a cytokine-mediated cellular crosstalk and clinical network involving the CLCF1-CXCL6/TGF-β axis, which regulates the positive feedback loop among CAFs, tumor stemness, and TANs, HCC progression, and patient prognosis. These results may support the CLCF1 cascade as a potential prognostic biomarker and suggest that selective blockade of CLCF1/ciliary neurotrophic factor receptor or ERK1/2 signaling could provide an effective therapeutic target for patients with HCC.

Topics: Cancer-Associated Fibroblasts; Carcinoma, Hepatocellular; Chemokine CXCL6; Cytokines; Disease Progression; Female; Humans; Liver Neoplasms; Male; MAP Kinase Signaling System; Middle Aged; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Metastasis remains the major obstacle of improving the survival of patients with hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is critical to cancer metastasis. Successful induction of EMT requires dramatic cytoskeleton rearrangement. However, the significance of microtubule (MT), one of the core components of cell cytoskeleton, in this process remains largely unknown. Here we revealed that STMN2, an important MT dynamics regulator, is barely expressed in normal live tissues but markedly up-regulated in HCCs, especially in those with early recurrence. High STMN2 expression correlates with aggressive clinicopathological features and predicts poor prognosis of HCC patients. STMN2 overexpression in HCC cells promotes EMT, invasion and metastasis in vitro and in vivo, whereas STMN2 knockdown has opposite results. Mechanistically, STMN2 modulates MTs disassembly, disrupts MT-Smad complex, and facilitates release from MT network, phosphorylation and nuclear translocation of Smad2/3 even independent of TGFβ stimulation, thereby enhancing TGFβ signaling. Collectively, STMN2 orchestrates MT disassembly to facilitate EMT via TGF-β signaling, providing a novel insight into the mechanisms underlying cancer metastasis. STMN2 is a promising prognostic biomarker and potential therapeutic target for HCC.

Topics: Active Transport, Cell Nucleus; Aged; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Signal Transduction; Smad2 Protein; Smad3 Protein; Stathmin; Transforming Growth Factor beta

The transforming growth factor β (TGF-β) activates JNK, phosphorylates Smad3 to linker-phosphorylated Smad3 (pSmad3L), resulting in liver tumorigenesis. However, the effect of pSmad3L on hepatocellular carcinoma (HCC) prognosis is obscure.. To detect the effect of pSmad3L on HCC prognosis and investigate the mechanism.. The expressions of pSmad3L, E-cadherin, vimentin and MicroRNA-140-5p (miR-140-5p) were detected by using immunohistochemistry, immunofluorescence and in situ hybridization. Next, the relationships of pSmad3L and HCC patients' prognoses, pSmad3L and EMT markers, pSmad3L and miR-140-5p were analyzed using Spearman's rank correlation test. JNK/pSmad3L specific inhibitor SP600125 or Smad3 mutant plasmid was used to suppress JNK/pSmad3L pathway, and QPCR assay was performed to investigate the effect of pSmad3L on miR-140-5p level. The proliferation and invasion of hepatoma cells were observed using colony formation assay and transwell assay.. We demonstrated that patient with high level of pSmad3L predicted poor prognosis. Next, we verified that pSmad3L promoted EMT of hepatoma cells in vivo and in vitro. In order to investigate the mechanism, we verified a negative correlation between pSmad3L and miR-140-5p, which was an EMT inhibitor, in the liver tissues of HCC patient and diethylnitrosamine (DEN)-induced rat HCC model. We further used SP600125 or pSmad3L mutant plasmid to decrease pSmad3L level of hepatoma cells, and inhibition of pSmad3L increased miR-140-5p level and suppressed EMT of hepatoma cells.. JNK/pSmad3L pathway induces EMT by inhibiting miR-140-5p in HCC progression.

Topics: Animals; Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; Rats; Smad3 Protein; Transforming Growth Factor beta

(1) Background: The transforming growth factor (TGF)-β plays a dual role in liver carcinogenesis. At early stages, it inhibits cell growth and induces apoptosis. However, TGF-β expression is high in advanced stages of hepatocellular carcinoma (HCC) and cells become resistant to TGF-β induced suppressor effects, responding to this cytokine undergoing epithelial-mesenchymal transition (EMT), which contributes to cell migration and invasion. Metabolic reprogramming has been established as a key hallmark of cancer. However, to consider metabolism as a therapeutic target in HCC, it is necessary to obtain a better understanding of how reprogramming occurs, which are the factors that regulate it, and how to identify the situation in a patient. Accordingly, in this work we aimed to analyze whether a process of full EMT induced by TGF-β in HCC cells induces metabolic reprogramming. (2) Methods: In vitro analysis in HCC cell lines, metabolomics and transcriptomics. (3) Results: Our findings indicate a differential metabolic switch in response to TGF-β when the HCC cells undergo a full EMT, which would favor lipolysis, increased transport and utilization of free fatty acids (FFA), decreased aerobic glycolysis and an increase in mitochondrial oxidative metabolism. (4) Conclusions: EMT induced by TGF-β in HCC cells reprograms lipid metabolism to facilitate the utilization of FFA and the entry of acetyl-CoA into the TCA cycle, to sustain the elevated requirements of energy linked to this process.

Topics: Carcinoma, Hepatocellular; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Silencing; Hep G2 Cells; Humans; Lipid Metabolism; Liver Neoplasms; Metabolome; Metabolomics; Mitochondria; Oxidative Phosphorylation; Receptors, Transforming Growth Factor beta; Transcriptome; Transforming Growth Factor beta

Liver fibrosis and fibrosis-related hepatocarcinogenesis are a rising cause for morbidity and death worldwide. Although transforming growth factor-β (TGF-β) is a critical mediator of chronic liver fibrosis, targeting TGF-β isoforms and receptors lead to unacceptable side effect. This study was designed to explore the antifibrotic effect of Compound kushen injection (CKI), an approved traditional Chinese medicine formula, via a therapeutic strategy of rebalancing TGF-β/Smad7 signaling.. Based on meta-analysis results, CKI improved the liver function and relieved liver fibrosis among patients. In our preclinical studies by using two mouse models, CKI treatment demonstrated promising antifibrotic effects and postponed hepatocarcinogenesis with improved liver function and histopathologic features. Mechanistically, we found that CKI inhibited HSCs activation by stabilizing the interaction of Smad7/TGF-βR1 to rebalance Smad2/Smad3 signaling, and subsequently decreased the extracellular matrix formation. Importantly, Smad7 depletion abolished the antifibrotic effect of CKI in vivo and in vitro. Moreover, matrine, oxymatrine, sophocarpine, and oxysophocarpine were identified as material basis responsible for the antifibrosis effect of CKI.. Our results unveil the approach of CKI in rebalancing TGF-β/Smad7 signaling in HSCs to protect against hepatic fibrosis and hepatocarcinogenesis in both preclinical and clinical studies. Our study suggests that CKI can be a candidate for treatment of hepatic fibrosis and related oncogenesis.

Topics: Animals; Carbon Tetrachloride; Carcinoma, Hepatocellular; Cell Line; Drugs, Chinese Herbal; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Medicine, Chinese Traditional; Meta-Analysis as Topic; Mice; Mice, Inbred C57BL; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) has emerged as one of the most prevalent life-threatening cancers, and the high mortality rate is largely due to the metastasis. The sustained activation of Smad4 and transforming growth factor-β (TGF-β) is closely associated with advanced HCC metastasis. However, the regulatory mechanism underlying the aberrant activation of Smad4 and TGF-β pathway remains elusive. In this study, using a functional screen of USPs siRNA library, we identified ubiquitin-specific proteases USP10 as a deubiquitinating enzyme (DUB) that sustains the protein level of Smad4 and activates TGF-β signaling. Further analysis showed that USP10 directly interacts with Smad4 and stabilizes it through the cleavage of its proteolytic ubiquitination, thus promoting HCC metastasis. The suppression of USP10 by either shRNAs or catalytic inhibitor Spautin-1 significantly inhibited the migration of HCC cells, whereas the reconstitution of Smad4 was able to efficiently rescue this defect. Overall, our study not only uncovers the regulatory effect of USP10 on the protein abundance of Smad4, but also indicates that USP10 could be regarded as a potential intervention target for the metastatic HCC in Smad4-positive patients.

Topics: Animals; Benzylamines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Protein Binding; Protein Stability; Quinazolines; RNA, Small Interfering; Smad4 Protein; Transforming Growth Factor beta; Ubiquitin Thiolesterase; Ubiquitination; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide and the majority of HCC patients occur with a background of hepatic fibrosis and cirrhosis. We have previously reported the hepatoprotective effects of steamed and freeze-dried mature silkworm larval powder (SMSP) in a chronic ethanol-treated rat model. Here, we assessed the anti-fibrotic and anti-carcinogenic effects of SMSP on diethylnitrosamine (DEN)-treated rats. Wistar rats were intraperitoneally injected with DEN once a week for 12 or 16 weeks with or without SMSP administration (0.1 and 1 g/kg). SMSP administration significantly attenuated tumor foci formation and proliferation in the livers of the rats treated with DEN for 16 weeks. SMSP administration also inhibited hepatic fibrosis by decreasing the levels of collagen fiber and the expression of pro-collagen I and alpha-smooth muscle actin (α-SMA). Moreover, SMSP supplementation improved the major parameters of fibrosis such as transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), tumor necrosis factor-alpha (TNF-α), plasminogen activator inhibitor-1 (PAI-1), and collagen type I (Col1A1) in the livers from the rats treated with DEN for 16 weeks. As s possible mechanisms, we investigated the effects of SMSP on the TGF-β and signal transducer and activator of transcription 3 (STAT3)-mediated signaling cascades, which are known to promote hepatic fibrosis. We found that SMSP treatment inhibited the activation of TGF-β and the phosphorylation of STAT3 pathway in DEN-treated rats. Moreover, SMSP administration suppressed the expressions of the target genes of TGF-β and STAT3 induced by DEN treatment. Our findings provide experimental evidences that SMSP administration has inhibitory effects of hepatic fibrosis and HCC induced by DEN in vivo and could be a promising strategy for the prevention or treatment of hepatic fibrosis and hepatocellular carcinogenesis.

Topics: Animals; Bombyx; Carcinogenesis; Carcinoma, Hepatocellular; Cell Proliferation; Diethylnitrosamine; Freeze Drying; Inflammation; Liver Cirrhosis; Liver Neoplasms; Male; Phosphorylation; Powders; Rats, Wistar; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFβ/SMAD signaling. SMAD3 and its adaptors, such as β2SP, are important mediators of TGFβ signaling and regulate gene expression to suppress stem cell-like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/β2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Exome Sequencing; Gene Deletion; Gene Expression Regulation; Gene Knockdown Techniques; Gene Silencing; Heterografts; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplastic Stem Cells; Oleanolic Acid; Phosphorylation; RNA, Small Interfering; Smad Proteins; Smad2 Protein; Smad3 Protein; Spectrin; Stem Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ubiquitin-Protein Ligases; Ubiquitination; Up-Regulation

Hepatocellular carcinoma (HCC) is the strongest independent predictor of mortality in non-alcoholic steatohepatitis (NASH)-related cirrhosis. The effects and mechanisms of combination of sodium-dependent glucose cotransporter inhibitor and canagliflozin (CA) and dipeptidyl peptidase-4 inhibitor and teneligliptin (TE) on non-diabetic NASH progression were examined. CA and TE suppressed choline-deficient, L-amino acid-defined diet-induced hepatic fibrogenesis and carcinogenesis. CA alone or with TE significantly decreased proinflammatory cytokine expression. CA and TE significantly attenuated hepatic lipid peroxidation. In vitro studies showed that TE alone or with CA inhibited cell proliferation and TGF-β1 and α1 (I)-procollagen mRNA expression in Ac-HSCs. CA+TE inhibited liver fibrogenesis by attenuating hepatic lipid peroxidation and inflammation and by inhibiting Ac-HSC proliferation with concomitant attenuation of hepatic lipid peroxidation. Moreover, CA+TE suppressed in vivo angiogenesis and oxidative DNA damage. CA or CA+TE inhibited HCC cells and human umbilical vein endothelial cell (HUVEC) proliferation. CA+TE suppressed vascular endothelial growth factor expression and promoted increased E-cadherin expression in HUVECs. CA+TE potentially exerts synergistic effects on hepatocarcinogenesis prevention by suppressing HCC cell proliferation and angiogenesis and concomitantly reducing oxidative stress and by inhibiting angiogenesis with attenuation of oxidative stress. CA+TE showed chemopreventive effects on NASH progression compared with single agent in non-diabetic rat model of NASH, concurrent with Ac-HSC and HCC cell proliferation, angiogenesis oxidative stress, and inflammation. Both agents are widely, safely used in clinical practice; combined treatment may represent a potential strategy against NASH.

Topics: Animals; Cadherins; Canagliflozin; Carcinogenesis; Carcinoma, Hepatocellular; Cell Proliferation; Collagen Type I; Collagen Type I, alpha 1 Chain; Cytokines; Disease Progression; DNA Damage; Drug Synergism; Hepatic Stellate Cells; Human Umbilical Vein Endothelial Cells; Humans; Lipid Peroxidation; Liver Cirrhosis; Liver Neoplasms; Male; Neovascularization, Pathologic; Non-alcoholic Fatty Liver Disease; Pyrazoles; Rats; Rats, Inbred Strains; Thiazolidines; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Immunological; B7-H1 Antigen; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Immunotherapy, Adoptive; Liver Neoplasms; Phenylurea Compounds; Programmed Cell Death 1 Receptor; Pyrazoles; Quinolines; Sunitinib; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor-Associated Macrophages

Functional rescue of NK-cells in solid tumors represents a central aim for new immunotherapeutic strategies. We have conducted a genomic, phenotypic and functional analysis of circulating NK-cells from patients with HCV-related liver cirrhosis and hepatocellular carcinoma. NK-cells were sorted from patients with HCC or liver cirrhosis and from healthy donors. Comparative mRNA gene expression profiling by whole-human-genome microarrays of sorted NK-cells was followed by phenotypic and functional characterization. To further identify possible mediators of NK-cell dysfunction, an in vitro model using media conditioned with patients' and controls' plasma was set up. Metabolic and cell motility defects were identified at the genomic level. Dysregulated gene expression profile has been translated into reduced cytokine production and degranulation despite a prevalent phenotype of terminally differentiated NK-cells. NKG2D-downregulation, high SMAD2 phosphorylation and other phenotypic and molecular alterations suggested TGF-β as possible mediator of this dysfunction. Blocking TGF-β could partially restore functional defects of NK-cells from healthy donors, exposed to TGF-β rich HCC patients' plasma, suggesting that TGF-β among other molecules may represent a suitable target for immunotherapeutic intervention aimed at NK-cell functional restoration. By an unbiased approach, we have identified energy metabolism and cell motility defects of circulating NK-cells as main mechanisms responsible for functional NK-cell impairment in patients with hepatocellular carcinoma. This opens the way to test different approaches to restore NK-cell response in these patients.

Topics: Aged; Carcinoma, Hepatocellular; Case-Control Studies; Cell Movement; Cytokines; Cytotoxicity, Immunologic; Energy Metabolism; Female; Follow-Up Studies; Hepacivirus; Hepatitis C; Humans; Killer Cells, Natural; Liver Cirrhosis; Liver Neoplasms; Male; Prognosis; Transforming Growth Factor beta; Tumor Cells, Cultured

Liver cancer is considered to be the sixth deadliest cancer worldwide. Hepatocellular carcinoma (HCC) is known to be the most prevalent type of liver cancer. The number of deaths due to HCC reported per year is on a constant rise especially in lesser developed countries. There are several contributing factors to this rise in number. Among other contributing factors is the late diagnosis of HCC. Patients are usually diagnosed when the disease reaches its advance stage. The present study was conducted with total 30 samples. It was designed for investigating the potential of TGF-β, NF-κβ, VEGF, AKT and PI3K as RNA based biomarkers in tumor educated platelets for early detection of HCC.. The results obtained from the transcriptional analysis revealed a significant high expression of TGF-β, NF-κβ, VEGF by 2.48, 2.35 and 2.78 folds respectively in comparison to the control. On the other hand, a decrease in expression by 0.6 and 0.65 folds was observed in AKT and PI3K respectively in comparison to controls. Although all selected RNA biomarkers showed promising potential to detect HCC however, AKT and PI3K were better able to detect early stage HCC.. The results obtained clearly indicate the increased expression of TGF-β, NF-κβ, VEGF in HCC patients. All these biomarkers are previously known for cancer initiation, progression and metastasis. The significant decrease in expression of AKT and PI3K in HCC patients needs further investigation. All the selected RNA biomarkers can be used for detection of HCC as they were able to distinguish HCC patients from controls successfully with AKT and PI3K showing better potential to detect early stage HCC. However, translational analysis for all these RNA biomarkers should be performed to gain further evidence for the ability of these biomarkers to be used for early HCC detection.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Blood Platelets; Carcinoma, Hepatocellular; DNA, Complementary; Early Detection of Cancer; Female; Humans; Liquid Biopsy; Liver Neoplasms; Male; Middle Aged; Neoplastic Cells, Circulating; NF-kappa B; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Young Adult

Feedback regulation plays a pivotal role in determining the intensity and duration of TGF-β signaling and subsequently affecting the pathophysiological roles of TGF-β, including those in liver malignancy. KLF2, a member of the Krüppel-like factor (KLF) family transcription factors, has been implicated in impeding hepatocellular carcinoma (HCC) development. However, the underlying molecular mechanisms are not fully understood. In the present study, we found that TGF-β stimulates the expression of KLF2 gene in several HCC cell lines. KLF2 protein is able to inhibit TGF-β/Smad signaling in HCC cells as assessed by luciferase reporter assay. Further studies indicated that KLF2 inhibits the transcriptional activity of Smad2/3 and Smad4 and ameliorates TGF-β-induced target gene expression, therefore creating a novel negative feedback loop in TGF-β signaling. Functionally, stably expression of KLF2 in HCCLM3 cells attenuated TGF-β-induced cancer cell motility in wound-healing and transwell assays by interfering with TGF-β-mediated upregulation of MMP2. Together, our results revealed that KLF2 protein has a tumor-suppressive function in HCC through a negative feedback loop over TGF-β signaling.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Liver Neoplasms; Neoplasm Proteins; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. MicroRNAs play a pivotal role in the progression of various cancers. To date, very little attention has been paid to miR-4458. Therefore, the aim of our study was to explore the function and underlying molecular mechanism of miR-4458 in HCC. We found that the expression of miR-4458 was reduced in HCC tissues and cell lines. Forced overexpression of miR-4458 inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, while downregulation of miR-4458 promoted the aggressive phenotype. Furthermore, transforming growth factor beta receptor 1 (TGFBR1), the modulator of the TGF-β signaling pathway, was verified to be a novel target gene of miR-4458 by dual-luciferase reporter gene assay. Upregulated miR-4458 dramatically abolished TGFBR1 and p-Smad2/3 expression, thus blocking the TGF-β signaling pathway. Moreover, restoration of TGFBR1 partially rescued the miR-4458-mediated suppressive effect on the migration, invasion, and EMT and reactivated the TGF-β signaling pathway in HCC cells. In summary, our findings first demonstrated a mechanism of miR-4458 in HCC cell migration, invasion, and EMT by regulating the TGF-β signaling pathway via directly targeting TGFBR1.

Topics: Aged; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Proteins; Receptor, Transforming Growth Factor-beta Type I; RNA, Neoplasm; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) metastasis is largely responsible for HCC-associated recurrence and mortality. We aimed to identify metastasis-related long non-coding RNAs (lncRNAs) to understand the molecular mechanism of HCC metastasis. We first identified that miR-1258 was downregulated in HCC tissues both in The Cancer Genome Atlas (TCGA) and Sun Yat-sen University Cancer Center (SYSUCC) dataset. MiR-1258 expression negatively correlated with recurrence-free survival and overall survival of HCC patients. MiR-1258 overexpression inhibited migration and invasion of HCC cells both in vitro and in vivo, whereas miR-1258 downregulation promoted cell metastasis. Luciferase assays verified direct binding of miR-1258 to Smad2 and Smad3, thereby attenuating TGF-β/Smad signaling. We further established that lncRNA LINC01278 was a negative regulator of miR-1258. In vivo and in vitro assays demonstrated that LINC01278-mediated HCC metastasis was dependent on miR-1258 expression. Furthermore, miR-1258 downregulation in turn increased LINC01278 expression. We also observed that TCF-4 could bind to the LINC01278 promoter site. In addition, LINC01278 downregulation decreased migration and invasion of HCC cells induced by β-catenin and TGF-β1 both in vitro and in vivo. We uncovered a novel mechanism for β-catenin/TCF-4-LINC01278-miR-1258-Smad2/3 feedback loop activation in HCC metastasis, and the study indicated that LINC01278 could serve as a therapeutic target for HCC metastasis.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Humans; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Neoplasm Transplantation; RNA, Long Noncoding; Smad2 Protein; Smad3 Protein; Transcription Factor 4; Transforming Growth Factor beta; Transplantation, Heterologous; Wnt Signaling Pathway; Wnt1 Protein

Integrin beta-like 1 (ITGBL1) is involved in the migration and invasion of several cancers; however, its roles in the development and progression of hepatocellular carcinoma (HCC) remain largely unknown.. Immunohistochemistry staining was used to investigate the expression pattern of ITGBL1 and its prognostic values in HCC patients. The transwell, wound-healing assays, xenograft and orthotopic mouse models were employed to determine the effects of ITGBL1 on HCC cell migration and invasion in vitro and in vivo. The biological mechanisms involved in cell migration and invasion caused by ITGBL1 were determined with Western blotting and RT-PCR methods.. ITGBL1 expression was significantly increased in HCC tissues compared to adjacent normal tissues. Patients with higher ITGBL1 expression were associated with more reduced overall survival. ITGBL1 overexpression promoted migration and invasion in SMMC-7721 and HepG2 cells in vitro and in vivo, whereas knockdown or knockout ITGBL1 in CSQT-2 cells significantly reduced cell migration and invasion abilities. In SMMC-7721 cells, ITGBL1 overexpression stimulated TGF-β/Smads signalling pathway, along with the KRT17 and genes involved in the epithelial-mesenchymal transition (EMT). In contrast, ITGBL1 knockout inhibited the TGF-β/Smads signalling pathway in CSQT-2 cells.. These findings suggested that ITGBL1 promoted migration and invasion in HCC cells by stimulating the TGF-β/Smads signalling pathway. ITGBL1 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Integrin beta1; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Middle Aged; Prognosis; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Topics: Animals; beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Movement; Cell Nucleus; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Homoharringtonine; Humans; Liver Neoplasms; Male; Mice, Nude; Phosphorylation; Protein Transport; Receptor, EphB4; Transforming Growth Factor beta

Deoxycholic acid (DCA), a secondary bile acid, is reportedly increased in the serum of patients with nonalcoholic steatohepatitis and animals with experimentally induced hepatocellular carcinoma (HCC), but its contribution to malignant behaviors of HCC has not been precisely clarified. This study aimed to examine the effect of DCA on hepatic stellate cells (HSCs), a major component of nonparenchymal cells in the liver, and its subsequent indirect effect on HCC cells.. LX2 cells, a human HSC line, were treated with DCA in vitro. Then, HuH7 cells, a human hepatoma cell line, were incubated in conditioned media of DCA-treated LX2 to investigate the subsequent effect focusing on malignant behaviors.. DCA resulted in cellular senescence in LX2 with the decreased cell proliferation via cell cycle arrest at G0/1 phase, together with the induction of senescence-associated secretory phenotype (SASP) factors. To investigate the influence of SASP factors secreted by HSCs in response to DCA, HCC cells were treated with conditioned media that promoted cell migration and invasion via induction of epithelial mesenchymal transition. These changes were attenuated in the presence of neutralizing antibody against IL8 or TGFβ. Pathological analysis of surgical specimens from HCC patients revealed that senescent HSCs were detected in the stroma surrounding HCC.. Our data suggest an important role of HSC senescence caused by DCA for the malignant biological behaviors of HCC via induction of SASP factors, particularly IL8 and TGFβ.

Topics: Antibodies, Neutralizing; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cellular Senescence; Deoxycholic Acid; Epithelial-Mesenchymal Transition; Hepatic Stellate Cells; Humans; Interleukin-8; Liver; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta

To observe the inhibitory effect of solanine on regulatory T cells (Treg) in transplanted hepatoma mice and to study the mechanism of solanine inhibiting tumor growth.. The levels of Treg cells and IL-2, IL-10, and TGF. Compared with healthy people, Treg cells and IL-2, IL-10, and TGF. Solanine may enhance the antitumor immune response by downregulating the proportion of CD4

Topics: Adult; Aged; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Mice, Nude; Middle Aged; Signal Transduction; Smad Proteins; Solanine; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Escape; Tumor Microenvironment; Xenograft Model Antitumor Assays

Nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) is a long noncoding RNA (lncRNA) that has been shown to be overexpressed in hepatocellular carcinoma (HCC). However, the molecular mechanism involving NNT-AS1 in HCC remains to be extensively investigated. The activation of TGF-

Topics: Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; Female; Hep G2 Cells; Humans; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; NADP Transhydrogenases; RNA, Long Noncoding; Transforming Growth Factor beta

Rab23 is a member of Ras-related small GTPase family, which plays a critical role in the progression of wide range of tumors. However, its biological function in hepatocellular carcinoma still remains unclear. Here, we investigated the effects of Rab23 on proliferation and migration in hepatocellular carcinoma cell and its potential mechanisms. We found over-expression of Rab23 promoted the Hep3B hepatocellular carcinoma cell migration, which could be reversed by Rab23 silencing. Rab23 induced Rac1 activation and followed progression of epithelial-mesenchymal transition (EMT) along with upregulation of N-cadherin, snail as well as vimentin and downregulation of E-cadherin via upregulating Transforming Growth Factor-β (TGF-β). Silencing Rac1 significantly attenuated Rab23-induced HepG2 migration and TGF-β. Moreover, knockdown of TGF-β effectively attenuated Rab23-induced EMT. Taken together, we demonstrated a mechanistic cascade of Rab23 enhangcing Rac1 activation and subsequent TGF-β expression, leading to hepatocellular carcinoma cell migration.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; rab GTP-Binding Proteins; rac1 GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta

Functional E-cadherin loss, a hallmark of epithelial-mesenchymal transition (EMT), is important for metastasis. However, the mechanism of Snail2 in hepatocellular carcinoma (HCC) EMT and metastasis remains unclear. Here, we showed that Snail2 was upregulated in primary HCC, and significantly increased during transforming growth factor-β-induced liver cell EMT. Snail2-overexpressing and knockdown cell lines have been established to determine its function in EMT in HCC. H3K9 methylation was upregulated and H3K4 and H3K56 acetylation were downregulated at the E-cadherin promoter in Snail2-overexpressing cancer cells. Furthermore, Snail2 interacted with G9a and histone deacetylases (HDACs) to form a complex to suppress E-cadherin transcription. Snail2 overexpression enhanced migration and invasion in HCC cells, whereas G9a and HDAC inhibition significantly reversed this effect. Moreover, Snail2 overexpression in cancer cells increased tumor metastasis and shortened survival time in mice, whereas G9a and HDAC inhibitors extended survival. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also suggests novel treatment strategies for HCC.

Topics: Acetylation; Animals; Azepines; Cadherins; Carcinoma, Hepatocellular; Cell Movement; Disease Progression; Down-Regulation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Histocompatibility Antigens; Histone Deacetylase Inhibitors; Histone Deacetylases; Histone-Lysine N-Methyltransferase; Humans; Hydroxamic Acids; Liver; Liver Neoplasms; Methylation; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Quinazolines; Snail Family Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

The Epidermal Growth Factor Receptor (EGFR) and the Transforming Growth Factor-beta (TGF-β) are key regulators of hepatocarcinogenesis. Targeting EGFR was proposed as a promising therapy; however, poor success was obtained in human hepatocellular carcinoma (HCC) clinical trials. Here, we describe how EGFR is frequently downregulated in HCC patients while TGF-β is upregulated. Using 2D/3D cellular models, we show that after EGFR loss, TGF-β is more efficient in its pro-migratory and invasive effects, inducing epithelial to amoeboid transition. EGFR knock-down promotes loss of cell-cell and cell-to-matrix adhesion, favouring TGF-β-induced actomyosin contractility and acquisition of an amoeboid migratory phenotype. Moreover, TGF-β upregulates RHOC and CDC42 after EGFR silencing, promoting Myosin II in amoeboid cells. Importantly, low EGFR combined with high TGFB1 or RHOC/CDC42 levels confer poor patient prognosis. In conclusion, this work reveals a new tumour suppressor function for EGFR counteracting TGF-β-mediated epithelial to amoeboid transitions in HCC, supporting a rational for targeting the TGF-β pathway in patients with low EGFR expression. Our work also highlights the relevance of epithelial to amoeboid transition in human tumours and the need to better target this process in the clinic.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Liver Neoplasms; Models, Biological; Prognosis; Signal Transduction; Transforming Growth Factor beta

Increasing evidence indicates that Six2 contributes to tumorigenesis in various tumor including hepatocellular carcinoma (HCC). This study aimed to determine the role of Six2 in HCC and to elucidate the association of Six2 with clinical pathological characteristics.. The expressions of Six2 in HCC tumor, para-tumor tissue and portal vein tumor thrombus (PVTT) were detected by tissue microarray technique, immunohistochemistry, real-time RT-PCR and Western blotting. Chi-square and Kaplan-Meier analysis were used to analyze the correlation between Six2 expression and prognosis of HCC patients. Lentivirus mediated Six2 knockdown, spheroid formation assay, proliferation assay and subcutaneous tumor implantation were performed to determine the function of Six2.. In 274 HCC samples, Six2 was strongly expressed. Kaplan-Meier analysis revealed that high expression of Six2 was correlated with a shorter overall survival (OS) and disease-free survival (DFS). Moreover, Six2 expression was associated with sex, alpha-fetoprotein, tumor size and portal vein invasion. Six2 was highly expressed in PVTT. Six2 knockdown inhibited HCC cell lines proliferation, migration, and self-renewal in vitro and in vivo. In addition, low-expression of Six2 weakened TGF-β induced Smad4 activation and epithelial-mesenchymal transition in HCC cell lines.. Elevated Six2 expression in HCC tumor patients was associated with negative prognosis. Upregulated Six2 promoted tumor growth and facilitated HCC metastasis via TGF-β/Smad signal pathway.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease-Free Survival; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Liver Neoplasms; Male; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Nerve Tissue Proteins; Smad4 Protein; Transforming Growth Factor beta; Tumor Burden; Up-Regulation

Sorafenib (Sor) is a multi-kinase inhibitor. It is recommended for the treatment of advanced hepatocellular carcinoma (HCC). However, Sor has severe and marked side effects. On the other hand, taurine (Tau) has been shown to enhance the therapeutic effects of cancer chemotherapy and also to enhance the function of leukocytes. Here, we aimed to investigate the enhancing efficacy of Sor as well as minimizing its marked side effects by using Tau in combination in an immunological aspect. We evaluated the influence of Sor and Tau combination on the expression pattern of FOXP3 gene in HepG2 cells compared to peripheral blood mononuclear leukocytes (PBMCs) as control normal cells. Also, the levels of TGF-β and IL-10 released in culture media of both cells were determined. Our results revealed that, Tau reduced cytotoxicity of Sor on PBMC indicated by lactic dehyrogenase (LDH) release assay. In addition, Sor-Tau combination led to FOXP3 down-regulation in hepatic cancer cells (HepG2). The results showed also that, TGF-β levels decreased significantly in their culture media. In contrary, the cytokine increased in PBMCs culture media. Moreover, IL-10 was significantly elevated in the culture media of both cells. This study could open new avenues for the improvement of therapeutic efficacy of Sorafenib treated HCC patients by using Tau in combination.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Cytokines; Drug Therapy, Combination; Forkhead Transcription Factors; Hep G2 Cells; Humans; L-Lactate Dehydrogenase; Leukocytes, Mononuclear; Liver Neoplasms; Protein Kinase Inhibitors; Sorafenib; Taurine; Transforming Growth Factor beta

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; Carcinoma, Hepatocellular; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Inactivation, Metabolic; Liver Neoplasms; MAP Kinase Signaling System; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Pregnane X Receptor; Proto-Oncogene Protein c-ets-1; Pyrazoles; Quinolines; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) plays a crucial role in hepatocellular carcinoma (HCC) progression. Hypoxia and excessive transforming growth factor-β (TGF-β) have been identified as inducers and target for EMT in HCC. Here, we show hypoxia inducible factor-1α (HIF-1α) and TGF-β form a feed-forward loop to induce EMT in HCC cells. Further mechanistic study indicates under both hypoxia and TGF-β stimulation, Smad and PI3K-AKT pathways are activated. We show sanguinarine, a natural benzophenanthridine alkaloid, impairs the proliferation of nine kinds of HCC cell lines and the colony formation of HCC cells. In hypoxic and TGF-β cell models, sanguinarine inhibits HIF-1α signaling and the expression of EMT markers, translocation of Snail and activation of both Smad and PI3K-AKT pathways. Sanguinarine could also inhibit TGF-β-induced cell migration in HCC cells. In vivo studies reveal that the administration of sanguinarine inhibits tumor growth and HIF-1α signaling, inhibits the expression changes of EMT markers as well as Smad and PI3K-AKT pathway proteins. Our findings suggest that sanguinarine is a promising candidate targeting HIF-1α/TGF-β signaling to improve the treatment for HCC patients.

Topics: Animals; Benzophenanthridines; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Hep G2 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Isoquinolines; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Signal Transduction; Transforming Growth Factor beta; Tumor Burden; Xenograft Model Antitumor Assays

Indoleamine-2,3-dioxygenase (IDO) is an enzyme that catalyzes tryptophan to kynurenine and studies have revealed that IDO play a vital role in regulation of liver immunity and inflammation activities. This study investigated the association between plasma IDO and disease severity and the possible marker role of IDO in the inflammatory process of hepatitis C. In this study, 80 individuals with HCV infection were retrospectively selected. Plasma levels of IDO, IL-10, and TGF-β were assayed by ELISA. Clinical characteristics of patients, including the levels of ALT, AST, and total bilirubin (TBil) were collected from clinical databases. HCV-related liver cirrhosis (HC-Cirr) and HCV-related Hepatocellular carcinoma (HCV-HCC) had significantly high plasma levels of IDO compared to other patient groups and healthy controls. Plasma IL-10 level were significantly greater in all chronic liver disease groups and with respect to TGF-β, the level was high in all the selected patients with HCV infection compare with controls. Moreover, HCV-HCC patients showed highest values for both IL-10 and TGF-β, with significant difference compared with other groups. In addition, plasma IDO was positively correlated with TGF-β among all patients with HCV infection (r = 0.4509, P < 0.0001), with IL-10 in CHC patients (r = 0.4787, P = 0.0047), with TBil in HCV-Cirr patients (r = 0.4671; P = 0.0093). High level of IDO and TGF-β is associated with hepatocyte necrosis and intrahepatic inflammation, and may be used as an index of disease progression for patients with chronic HCV infection.

Topics: Adult; Aged; Aged, 80 and over; Alanine Transaminase; Aspartate Aminotransferases; Bilirubin; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Female; Hepatitis C, Chronic; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-10; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Retrospective Studies; Transforming Growth Factor beta

Transforming growth factor (TGF)-β suppresses early hepatocellular carcinoma (HCC) development but triggers pro-oncogenic abilities at later stages. Recent data suggest that the receptor tyrosine kinase Axl causes a TGF-β switch toward dedifferentiation and invasion of HCC cells. Here, we analyzed two human cellular HCC models with opposing phenotypes in response to TGF-β. Both HCC models showed reduced proliferation and clonogenic growth behavior following TGF-β stimulation, although they exhibited differences in chemosensitivity and migratory abilities, suggesting that HCC cells evade traits of anti-oncogenic TGF-β. Transcriptome profiling revealed differential regulation of the chemokine CXCL5, which positively correlated with TGF-β expression in HCC patients. The expression and secretion of CXCL5 was dependent on Axl expression, suggesting that CXCL5 is a TGF-β target gene collaborating with Axl signaling. Loss of either TGF-β or Axl signaling abrogated CXCL5-dependent attraction of neutrophils. In mice, tumor formation of transplanted HCC cells relied on CXCL5 expression. In HCC patients, high levels of Axl and CXCL5 correlated with advanced tumor stages, recruitment of neutrophils into HCC tissue, and reduced survival. Conclusion: The synergy of TGF-β and Axl induces CXCL5 secretion, causing the infiltration of neutrophils into HCC tissue. Intervention with TGF-β/Axl/CXCL5 signaling may be an effective therapeutic strategy to combat HCC progression in TGF-β-positive patients.

Topics: Animals; Axl Receptor Tyrosine Kinase; Carcinoma, Hepatocellular; Chemokine CXCL5; Humans; Liver Neoplasms; Mice; Neutrophil Infiltration; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Transforming Growth Factor beta; Tumor Cells, Cultured

Hepatocellular carcinoma (HCC) is the leading cause of cancer mortality worldwide. Early growth response factor 1 (Egr1) plays a crucial role in cancer progression. However, its precise role in HCC has not been clear. Here, we identified the aggravating role of Egr1 in cell proliferation of HCC firstly. The expression of Egr1 was significantly increased in HCC tissues. Functionally, overexpression of Egr1 enhanced, whereas silenced Egr1 expression attenuated HCC cells proliferation in vitro. Mechanistically, up-regulated Egr1 induced cell proliferation through activating Transforming growth factor (TGF)-β1/Smad signaling pathway concomitantly with upregulation of p-Smad2 and p-Smad3. Secondly, miR-181a-5p was down-regulated in clinical HCC specimens and its expression was inversely correlated with Egr1 expression. Functionally, overexpression of miR-181a-5p inhibited, whereas decreased expression of miR-181a-5p promoted HCC cells proliferation in vitro. Furthermore, we demonstrated that miR-181a-5p overexpression directly suppressed Egr1, resulting in a down-regulated TGF-β1/Smad pathway. Besides, the silenced Egr1 expression could rescue the enhanced cell proliferation induced by miR-181a-5p inhibitor. Thus, we concluded that miR-181a-5p is a negative regulator of Egr1 that can suppress tumor proliferation in HCC through targeting Egr1/TGF-β1/Smad pathway, which may be a potential therapeutic approach of HCC.

Topics: Aged; Carcinoma, Hepatocellular; Cell Proliferation; Early Growth Response Protein 1; Female; Humans; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Proteins; RNA, Neoplasm; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most fatal cancers with common features of invasion and metastasis. Recent evidence indicate that the long noncoding RNA NORAD is a potential oncogene and is significantly upregulated in several cancers. However, the general biological role and clinical value of NORAD in HCC remains unknown. Here, NORAD expression was measured in 29 paired tumor and paratumor tissues via quantitative real-time polymerase chain reaction (qPCR). The effects of NORAD on HCC cell malignant potential were investigated via NORAD overexpression and knockdown both in vitro and in vivo. The mechanism of competitive endogenous RNAs (ceRNAs) was acquired and identified by bioinformatics analyses and luciferase assays. Moreover, the impact of NORAD level on the transforming growth factor β (TGF-β) pathway was further determined by qPCR. We found that HCC tissues had a high level of NORAD compared with the paratumor tissues, and NORAD upregulation was associated with the shorter overall survival of patients with HCC. Furthermore, NORAD overexpression was demonstrated to promote HCC cell migration and invasion. Mechanically, NORAD might function as a ceRNA to regulate miR-202-5p, which served as a tumor-suppressing microRNA via the TGF-β pathway. We address that NORAD has a tumor-promoting effect in HCC and describes a novel mechanism whereby NORAD regulates the TGF-β pathway as a ceRNA of Homo sapiens (hsa)-miR-202-5p.

Topics: Adult; Carcinoma, Hepatocellular; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

Hyper-activation of TGF-β signaling is critically involved in progression of hepatocellular carcinoma (HCC). However, the events that contribute to the dysregulation of TGF-β pathway in HCC, especially at the post-translational level, are not well understood.. Associations of deubiquitinase POH1 with TGF-β signaling activity and the outcomes of HCC patients were examined by data mining of online HCC datasets, immunohistochemistry analyses using human HCC specimens, spearman correlation and survival analyses. The effects of POH1 on the ubiquitination and stability of the TGF-β receptors (TGFBR1 and TGFBR2) and the activation of downstream effectors were tested by western blotting. Primary mouse liver tissues from polyinosinic:polycytidylic acid (poly I:C)- treated Mx-Cre+, poh1. Here we show that POH1 is a critical regulator of TGF-β signaling and promotes tumor metastasis. Integrative analyses of HCC subgroups classified with unsupervised transcriptome clustering of the TGF-β response, metastatic potential and outcomes, reveal that POH1 expression positively correlates with activities of TGF-β signaling in tumors and with malignant disease progression. Functionally, POH1 intensifies TGF-β signaling delivery and, as a consequence, promotes HCC cell metastatic properties both in vitro and in vivo. The expression of the TGF-β receptors was severely downregulated in POH1-deficient mouse hepatocytes. Mechanistically, POH1 deubiquitinates the TGF-β receptors and CAV1, therefore negatively regulates lysosome pathway-mediated turnover of TGF-β receptors.. Our study highlights the pathological significance of aberrantly expressed POH1 in TGF-β signaling hyperactivation and aggressive progression in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Caveolin 1; Cell Line, Tumor; Down-Regulation; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Prognosis; Proteasome Endopeptidase Complex; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Trans-Activators; Transforming Growth Factor beta; Ubiquitination

RING finger protein 38 (RNF38), a member of the RNF protein family, has just emerged as a vital driver of cancer progression. However, the oncogenic mechanisms of RNF38 remain unexplored.. Using frozen tumor tissue and tissue microarray from hepatocellular carcinoma (HCC) patients, we tried to probe the expression of RNF38 in HCC and its clinical value. Then the biological functions of RNF38 were analyzed in vivo and vitro. Stable isotope labeling with amino acids (SILAC) in cell culture and co-immunoprecipitation proteomic analyses were combined to reveal the potential mechanism of RNF38 in HCC progression.. We report that RNF38 expression was markedly higher in HCC tissues than in peritumor tissues. Correspondingly, RNF38 overexpression promoted the HCC cell migration and invasion and inhibited apoptosis both in vitro and in vivo. And elevated RNF38 expression induced HCC cell epithelial-mesenchymal transition by facilitating transforming growth factor-β (TGF-β) signaling via ubiquitinating and degrading neuroblast differentiation-associated protein (AHNAK), a well-established inhibitor of TGF-β signaling. Furthermore, AHNAK interference restored the HCC cell invasion and metastasis deprived by RNF38 downregulation. Clinically, elevated RNF38 and transforming growth factor beta receptor 1 (TGFBR1) expression was related to short overall survival (OS) and high cumulative recurrence rates in HCC patients.. High levels of RNF38 promote HCC by facilitating TGF-β signaling and are a novel marker for predicting the prognosis of HCC patients and a potential therapeutic target in HCC.

Topics: Adult; Animals; Carcinogenesis; Carcinoma, Hepatocellular; Carrier Proteins; Disease-Free Survival; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Isotope Labeling; Liver Neoplasms; Male; Membrane Proteins; Mice; Middle Aged; Neoplasm Proteins; Prognosis; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Combining chemotherapy with radiotherapy potentiates the outcome of cancer treatment for the more comprehensive attack. In the current study, we continued to assess the therapeutic efficaciousness of the newly synthesized gallium nanoparticles (GaNPs) combined with low level of gamma radiation (IR), on the incidence of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. Oral administration of GaNPs (1 mg/Kg b.wt.) 5 times per week for 6 weeks combined with IR to rats treated with DEN (20 mg/Kg b.wt. 5 times per week for 6 weeks) significantly reduced serum levels of alpha-fetoprotein (AFP), aspartate transferase (AST), alanine transferase (ALT), and gamma-glutamyltransferase (GGT). In addition, the immunoblotting results of matrix metalloproteinase-9 (MM-9) showed a marked downregulation of protein expression along with a significant decrease in the hepatic level of transforming growth factor β (TGF-β). Furthermore, GaNPs and/or low dose of radiation significantly elevated the level of caspase-3 gene transcript accompanied with evoked DNA fragmentation in rats treated with DEN. The ameliorative effect of GaNPs and IR well appreciated with the histopathological alteration finding in DEN groups. It can be concluded that the combination of GaNPs and/or IR can serve as a good therapeutic agent for the treatment of HCC, which ought to attract more studies.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Caspase 3; Diethylnitrosamine; Disease Models, Animal; DNA Fragmentation; Gallium; Gamma Rays; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; Metal Nanoparticles; Radiation Dosage; Rats; Transforming Growth Factor beta

EndMT has an important effect on metastasis and progression of tumor. This work will elucidate the effect of miR-494 on EndMT and development of HCC. Therefore, the differential miRNA expression among non-tumorous, para-tumorous and tumorous tissues was analyzed. Moreover, luciferase activities of SIRT3 3'UTR treated with miR-494 were determined. Then human hepatoma cell lines were dealt with mimics or inhibitors of miR-494, migration and proliferation ability were assessed. The expression of SIRT3 and markers of mesenchymal cell were analyzed. The influences of miR-494 on development of HCC through inducing EndMT by targeting SIRT3 and TGF-β/SMAD signaling pathways in hepatoma cell lines were investigated. Xenograft mice were used to explore the potential roles of miR-494 on EndMT and development of HCC in vivo. Our results showed that, compared with non-tumorous tissues, 17 miRNAs were upregulated and 3 miRNAs were down-regulated in tumor tissues. In tumor tissues, the miR-494 expression level was much more than the expression of para-tumorous and non-tumorous tissues. MiR-494 suppressed SIRT3 expression, additionally enhanced expression of mesenchymal cell markers, while exerted effects on cell proliferation and migration of hepatoma cell lines. Moreover, the antagomir of miR-494 could protect against development process in xenogarft murine model. In conclusions, our work demonstrated that miR-494 targeted to SIRT3, and was a crucial mediator of EndMT and development of HCC through regulating SIRT3/TGF-β/SMAD signaling pathway. It suggested that aim at SIRT3/TGF-β/SMAD signaling pathway through suppressing the miR-494 expression level, was a feasible therapy strategy for HCC.

Topics: 3' Untranslated Regions; Animals; Antagomirs; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Disease Progression; Down-Regulation; Epithelial-Mesenchymal Transition; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; MicroRNAs; Signal Transduction; Sirtuin 3; Smad Proteins; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Euphorbia factor L2 has potent effects on ascites, hydropsy and cancers.. We investigated the pharmacological effects of Euphorbia factor L2 (EFL2) on hepatocellular carcinoma (HCC).. MTT assay was conducted to determine the proliferative activity of EFL2 on Hep G2 and SMMC-7721 cells. Wound-healing assay, colony formation assay, western blotting and quantitative PCR were carried out to examine the cell migration, p-AKT and p-STAT3 signaling. Moreover, we used human tumor xenograft BALB/c nude mice to detect the effect of EFL2 on HCC in vivo.. EFL2 inhibited the proliferation of SMMC-7721 and Hep G2 cells in concentration- and time-dependent manners. EFL2 also suppressed the cell migration and colony formation of hepatocellular carcinoma cells. Using a transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) model, we provided evidences that EFL2 could also inhibit TGF-β induced cell growth, vimentin, N-cadherin expressions, activation of p-AKT and p-STAT3, whereas up-regulate E-cadherin expression. Furthermore, EFL2 inhibited tumor growth and STAT3 phosphorylation in vivo.. In conclusion, EFL2 has the potential to be explored as a candidate treatment agent for HCC by inhibiting cell growth and migration both in vitro and in vivo.

Topics: Animals; Antineoplastic Agents, Phytogenic; Benzoates; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Euphorbia; Fused-Ring Compounds; Hep G2 Cells; Humans; Liver Neoplasms; Macrocyclic Compounds; Male; Mice, Inbred BALB C; Polycyclic Compounds; Proto-Oncogene Proteins c-akt; STAT3 Transcription Factor; Transforming Growth Factor beta

Abnormal tumor microenvironment and the epithelial-mesenchymal transition (EMT) are important features of tumor metastasis. However, it remains unknown how signals can form complicated networks to regulate the sustainability of the EMT process. The aim of our study is to explore the possible interaction between tumor-associated macrophages and tumor cells in the EMT process mediated by microRNA (miR)-362-3p. In this study, we found that by releasing TGF-β, M2 macrophages mediate binding of Smad2/3 to miR-362-3p promoter, leading to overexpression of miR-362-3p. MicroRNA-362-3p maintains EMT by regulating CD82, one of the most important members of the family of tetraspanins. Our finding suggests that miR-362-3p can serve as a core factor mediating cross-talk between the TGF-β pathway in tumor-associated macrophages and tetraspanins in tumor cells, and thus facilitates the EMT process.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Kangai-1 Protein; Liver Neoplasms; Macrophages; Mice; Mice, Nude; MicroRNAs; Signal Transduction; Smad2 Protein; Smad3 Protein; Tetraspanins; Transforming Growth Factor beta

In spite of therapeutic approaches, liver cancer is still one of the deadliest type of tumor in which tumor microenvironment may play an active role in the outcome of the disease. Decorin, a small leucine-rich proteoglycan is not only responsible for assembly and maintenance of the integrity of the extracellular matrix, but a natural inhibitor of cell surface receptors, thus it exerts antitumorigenic effects. Here we addressed the question whether this effect of decorin is independent of the tumor phenotypes including differentiation, proliferation and invasion.. Four hepatoma cell lines HepG2, Hep3B, HuH7 and HLE, possessing different molecular backgrounds, were selected to investigate. After proliferation tests, pRTK arrays, WB analyses, and immunofluorescent examinations were performed on decorin treated and control cells for comparison.. Based on our results decorin may be a candidate therapeutic agent in the battle against liver cancer, but several questions need to be answered. It is certain that decorin is capable to exert its suppressor effect in hepatoma cells without respect to their phenotype and molecular background.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Decorin; Hep G2 Cells; Humans; Liver Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Phosphorylation; Proto-Oncogene Proteins c-myc; Receptor, IGF Type 1; Transforming Growth Factor beta; Tumor Microenvironment

TGFβ signaling plays an important role in orchestrating a favorable microenvironment for tumor cell growth and promoting epithelial-mesenchymal transition. As a conventional nonsteroidal anti-inflammation drugs, tolfenamic acid (TA) has been previously reported to exhibit anti-cancer activity. Herein, we investigated the effect of TA on TGFβ-mediated pro-metastatic activity and the underlying mechanisms in hepatocellular carcinoma (HCC). As a result, TA suppresses TGFβ-induced migration and glycolysis in HCC cells, which is accompanied with reduced Smad phosphorylation and subsequent nuclear transcription activity. Mechanistically, TA promotes lipid raft-caveolar internalization pathway of TGFβ receptor, therefore leading to its rapid turnover. Consistently, TA inhibits constitutively active TGFβ type I receptor induced Smad phosphorylation and EMT markers, whereas ectopic expression of TGFβ type II receptor could partially rescue TGFβ-mediated Smad2 phosphorylation and downstream genes expression in the presence of TA. Furthermore, TA inhibited HCC cells invasion in nude mice, associated with the alteration of characteristics related with EMT and glycolysis of cancer cells. Our study suggests TA could activate lipid raft pathway and modulate TGFβ mediated metastasis, implicating the potential application of TA as a modulator of tumor microenvironment in HCC.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Liver Neoplasms; Membrane Microdomains; Mice, Inbred BALB C; Mice, Nude; ortho-Aminobenzoates; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Development of hepatocellular carcinoma (HCC) is associated with alterations in the transforming growth factor-beta (TGF-β) signaling pathway, which regulates liver inflammation and can have tumor suppressor or promoter activities. Little is known about the roles of specific members of this pathway at specific of HCC development. We took an integrated approach to identify and validate the effects of changes in this pathway in HCC and identify therapeutic targets.. We performed transcriptome analyses for a total of 488 HCCs that include data from The Cancer Genome Atlas. We also screened 301 HCCs reported in the Catalogue of Somatic Mutations in Cancer and 202 from Cancer Genome Atlas for mutations in genome sequences. We expressed mutant forms of spectrin beta, non-erythrocytic 1 (SPTBN1) in HepG2, SNU398, and SNU475 cells and measured phosphorylation, nuclear translocation, and transcriptional activity of SMAD family member 3 (SMAD3).. We found somatic mutations in at least 1 gene whose product is a member of TGF-β signaling pathway in 38% of HCC samples. SPTBN1 was mutated in the largest proportion of samples (12 of 202, 6%). Unsupervised clustering of transcriptome data identified a group of HCCs with activation of the TGF-β signaling pathway (increased transcription of genes in the pathway) and a group of HCCs with inactivation of TGF-β signaling (reduced expression of genes in this pathway). Patients with tumors with inactivation of TGF-β signaling had shorter survival times than patients with tumors with activation of TGF-β signaling (P = .0129). Patterns of TGF-β signaling correlated with activation of the DNA damage response and sirtuin signaling pathways. HepG2, SNU398, and SNU475 cells that expressed the D1089Y mutant or with knockdown of SPTBN1 had increased sensitivity to DNA crosslinking agents and reduced survival compared with cells that expressed normal SPTBN1 (controls).. In genome and transcriptome analyses of HCC samples, we found mutations in genes in the TGF-β signaling pathway in almost 40% of samples. These correlated with changes in expression of genes in the pathways; up-regulation of genes in this pathway would contribute to inflammation and fibrosis, whereas down-regulation would indicate loss of TGF-β tumor suppressor activity. Our findings indicate that therapeutic agents for HCCs can be effective, based on genetic features of the TGF-β pathway; agents that block TGF-β should be used only in patients with specific types of HCCs.

Topics: Aged; Carcinoma, Hepatocellular; Case-Control Studies; Cluster Analysis; Female; Humans; Liver Neoplasms; Male; Middle Aged; Mutation; Signal Transduction; Transforming Growth Factor beta

Evading TGF-β-mediated growth inhibition is often associated with tumorigenesis in liver, including hepatocellular carcinoma (HCC). To better understand the functions and the underlying molecular mechanisms of TGF-β in HCC initiation and progression, we carried out transcriptome sequencing (RNA-Seq) to identify the target genes of TGF-β. CXXC5, a member of the CXXC-type zinc finger domain-containing protein family, was identified as a novel TGF-β target gene in Hep3B HCC cells. Knockdown of CXXC5 attenuated the expression of a substantial portion of TGF-β target genes and ameliorated TGF-β-induced growth inhibition or apoptosis of Hep3B cells, suggesting that CXXC5 is required for TGF-β-mediated inhibition of HCC progression. Analysis of the TCGA database indicated that CXXC5 expression is reduced in the majority of HCC tissue samples in comparison to that in normal tissues. Furthermore, CXXC5 associates with the histone deacetylase HDAC1 and competes its interaction with Smad2/3, thereby abolishing the inhibitory effect of HDAC1 on TGF-β signaling. These observations together suggest that CXXC5 may act as a tumor suppressor by promoting TGF-β signaling via a positive feedback loop, and reveal a strategy for HCC to bypass TGF-β-mediated cytostasis by disrupting the positive feedback regulation. Our findings shed new light on TGF-β signaling regulation and demonstrate the function of CXXC5 in HCC development.

Topics: Apoptosis; Carcinogenesis; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Progression; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Histone Deacetylase 1; Humans; Liver Neoplasms; Protein Interaction Maps; Signal Transduction; Smad Proteins; Transcription Factors; Transforming Growth Factor beta

Late-stage hepatocellular carcinoma (HCC) usually has a low survival rate because of the high risk of metastases and the lack of an effective cure. Disulfiram (DSF) has copper (Cu)-dependent anticancer properties in vitro and in vivo. The present work aims to explore the anti-metastasis effects and molecular mechanisms of DSF/Cu on HCC cells both in vitro and in vivo. The results showed that DSF inhibited the proliferation, migration and invasion of HCC cells. Cu improved the anti-metastatic activity of DSF, while Cu alone had no effect. Furthermore, DSF/Cu inhibited both NF-κB and TGF-β signalling, including the nuclear translocation of NF-κB subunits and the expression of Smad4, leading to down-regulation of Snail and Slug, which contributed to phenotype epithelial-mesenchymal transition (EMT). Finally, DSF/Cu inhibited the lung metastasis of Hep3B cells not only in a subcutaneous tumour model but also in an orthotopic liver metastasis assay. These results indicated that DSF/Cu suppressed the metastasis and EMT of hepatic carcinoma through NF-κB and TGF-β signalling. Our study indicates the potential of DSF/Cu for therapeutic use.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Copper; Disulfiram; Down-Regulation; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; NF-kappa B; Phenotype; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Xenograft Model Antitumor Assays

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factors

Understanding the complexity of changes in differentiation and cell survival in hepatocellular carcinoma (HCC) is essential for the design of new diagnostic tools and therapeutic modalities. In this context, we have analyzed the crosstalk between transforming growth factor β (TGFβ) and liver X receptor α (LXRα) pathways. TGFβ is known to promote cytostatic and pro-apoptotic responses in HCC, and to facilitate mesenchymal differentiation. We here demonstrate that stimulation of the nuclear LXRα receptor system by physiological and clinically useful agonists controls the HCC response to TGFβ. Specifically, LXRα activation antagonizes the mesenchymal, reactive oxygen species and pro-apoptotic responses to TGFβ and the mesenchymal transcription factor Snail mediates this crosstalk. In contrast, LXRα activation and TGFβ cooperate in enforcing cytostasis in HCC, which preserves their epithelial features. LXRα influences Snail expression transcriptionally, acting on the Snail promoter. These findings propose that clinically used LXR agonists may find further application to the treatment of aggressive, mesenchymal HCCs, whose progression is chronically dependent on autocrine or paracrine TGFβ.

Topics: Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Liver X Receptors; Neoplasm Proteins; Paracrine Communication; Snail Family Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

Upon stimulation of cells with transforming growth factor β (TGF-β), Smad proteins form trimeric complexes and activate a broad spectrum of target genes. It remains unresolved which of the possible Smad complexes are formed in cellular contexts and how these contribute to gene expression. By combining quantitative mass spectrometry with a computational selection strategy, we predict and provide experimental evidence for the three most relevant Smad complexes in the mouse hepatoma cell line Hepa1-6. Utilizing dynamic pathway modeling, we specify the contribution of each Smad complex to the expression of representative Smad target genes, and show that these contributions are conserved in human hepatoma cell lines and primary hepatocytes. We predict, based on gene expression data of patient samples, increased amounts of Smad2/3/4 proteins and Smad2 phosphorylation as hallmarks of hepatocellular carcinoma and experimentally verify this prediction. Our findings demonstrate that modeling approaches can disentangle the complexity of transcription factor complex formation and its impact on gene expression.

Topics: Aged; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA-Binding Proteins; Female; Hep G2 Cells; Hepatocytes; Humans; Liver; Liver Neoplasms; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Middle Aged; Phosphorylation; Signal Transduction; Smad Proteins; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta

Hepatocytes are epithelial cells with highly specialized polarity. The disorder and loss of hepatocyte polarity leads to a weakness of cell adhesion and connection, the induction of epithelial-mesenchymal transition, and eventually the occurrence of hepatocellular carcinoma (HCC). Cluster of differentiation 147 (CD147), a tumor-related glycoprotein, promotes epithelial-mesenchymal transition and the invasion of HCC. However, the function of CD147 in hepatocyte depolarization is unknown. Here we identified that CD147 was basolaterally polarized in hepatocyte membrane of liver tissues and HepG2 cells. CD147 not only promoted transforming growth factor-β1-mediated hepatocyte polarity loss but also directly induced endocytosis and down-regulation of E-cadherin which contributed to hepatocyte depolarization. Overexpression of CD147 induced Src activation and subsequently recruited ubiquitin ligase Hakai for E-cadherin ubiquitination and lysosomal degradation, leading to decreases of partitioning defective 3 expression and β-catenin nuclear translocation. This signal transduction was initiated by competitive binding of CD147 with integrin β1 that interrupted the interaction between the Arg-Gly-Asp motif of fibronectin and integrin β1. The specific antibodies targeting integrin α5 and β1 reversed the decrease of E-cadherin and partitioning defective 3 levels induced by CD147 overexpression. In human liver tissues, CD147 polarity rates significantly declined from liver cirrhosis (71.4%) to HCC (10.4%). CD147-polarized localization negatively correlated with Child-Pugh scores in human liver cirrhosis (r = -0.6092, P < 0.0001) and positively correlated with differentiation grades in HCC (r = 0.2060, P = 0.004). HCC patients with CD147-polarized localization had significantly better overall survival than patients with CD147 nonpolarity (P = 0.021).. The ectopic CD147-polarized distribution on basolateral membrane promotes hepatocyte depolarization by activation of the CD147-integrin α5β1-E-cadherin ubiquitination-partitioning defective 3 decrease and β-catenin translocation signaling cascade, replenishing a molecular pathway in hepatic carcinogenesis. (Hepatology 2018;68:317-332).

Topics: Adaptor Proteins, Signal Transducing; Animals; Basigin; Cadherins; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Polarity; Disease Progression; Hep G2 Cells; Hepatocytes; Humans; Integrin alpha5beta1; Liver Neoplasms; Lysosomes; Membrane Proteins; Mice; Mice, Knockout; src-Family Kinases; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Ubiquitination

Tunicamycin (TM) is an N-linked glycosylation (NLG) inhibitor with strong antitumor activity, the exact underlying molecular mechanism of which remains to be elucidated. In our previous studies, we found that TM reversed drug resistance and improved the efficacy of combination treatments for hepatocellular carcinomas (HCC). Here, we investigated the effects of TM on HCC cell proliferation and migration as well as the mechanism of those effects. Our results showed that TM inhibited cell proliferation and migration as well as induced apoptosis of hepatocellular carcinoma cells. TM inhibited proliferation of HCC cells by inducing cell apoptosis and cell cycle arrest at the G2/M phase. Meanwhile, TM inhibited migration of HCC cells by suppressing CD44s-mediated epithelial-mesenchymal transition (EMT). TM inhibited migration and invasion of HCC cells by decreasing CD44 expression and altering its glycosylation. In addition, CD44s is involved in promoting EMT and is associated with a poor prognosis in HCC patients. Overexpression of CD44s promoted tumor migration and activated phosphorylation of ERK1/2 in HCC cells, whereas TM inhibited CD44s overexpression-associated cell migration. The ability of TM to inhibit cell migration and invasion was enhanced or reversed in CD44s knockdown cells and cells overexpressing CD44s, respectively. The MEK/ERK inhibitor U0126 and TM inhibited hyaluronic acid-induced cell migration in HCC cells. Furthermore, TM inhibited exogenous transforming growth factor beta (TGF-β)-mediated EMT by an ERK1/2-dependent mechanism and restored the TGF-β-mediated loss of E-cadherin. In summary, our study provides evidence that TM inhibits proliferation and migration of HCC cells through inhibition of CD44s and the ERK1/2 signaling pathway.

Topics: Antineoplastic Agents; Apoptosis; Cadherins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Signal Transduction; Transforming Growth Factor beta; Tunicamycin

Resistance to TGF-β-induced growth repression is prevalent in various cancer cells, but the underlying mechanisms remain unclear. In this study, we showed that activation of TGF-β signaling caused Sma- and Mad-related family (Smad) 2 and Smad3 to bind directly to the promoter region of miR-195, and then activated miR-195 transcription in normal hepatocytes. Conversely, miR-195 inhibited the expression of Smad7 by binding to its 3'-UTR, thereby strengthening TGF-β-Smad signaling. These data identify a novel TGF-β-miR-195 positive regulatory circuitry in normal hepatocytes. Further investigation revealed that HDAC1, a histone deacetylase that was abnormally overexpressed in hepatocellular carcinoma, could bind to the miR-195 promoter via Smad3 and cause hypoacetylation in the histones associated with the miR-195 promoter in hepatoma cells. This resulted in transcriptional repression of miR-195 and, subsequently, disruption of the TGF-β-miR-195 regulatory loop and evasion of TGF-β-mediated growth inhibition. Moreover, silencing HDAC1 in hepatoma cells restored TGF-β-mediated growth suppression, but this effect was attenuated if miR-195 expression decreased. These findings suggest that HDAC1-induced miR-195 down-regulation is an important mechanism for tumor cells to resist the cytostatic activity of TGF-β, and highlight the importance of TGF-β-Smad2/3-miR-195-Smad7 circuitry in preventing uncontrolled cell proliferation.-Wang, R., Fu, T., You, K., Li, S., Zhao, N., Yang, J., Zhuang, S.-M. Identification of a TGF-β-miR-195 positive feedback loop in hepatocytes and its deregulation in hepatoma cells.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Histone Deacetylase 1; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; MicroRNAs; Smad Proteins; Transforming Growth Factor beta

AGO1 is a major component of RNA-induced silencing complexes and plays a crucial role in solid tumors. The aim of our study was to investigate AGO1 functions in hepatocellular carcinoma (HCC). Using small interfering RNA, AGO1 functions were investigated in HCCLM3 cell lines. Cell proliferation, immigration, and invasion significantly decreased after AGO1 depletion using MTT, wound-healing, and transwell assay. The associated proteins in the epithelial-mesenchymal transition (EMT) and the activation of its signal pathways were measured using western blot. After AGO1 depleted, increased E-cadherin and decreased N-cadherin, Vimentin, Snail, and Zeb1 were founded. In its upstream pathway, the phosphorylation of ERK1/2(Thr202/Tyr204), Smad2(S425/250/255), and Smad4 were significantly inhibited. Meanwhile, inhibitor of ERK1/2(LY3214996) significantly inhibited the growth and migration of the AGO1 cells. The nuclear importing of Smad4 was blocked and furthermore, the transcription of Snail was also influenced for the decrease of combination between Smad4 and the promotor region of Snail. After Snail was overexpressed, the invasion of HCCLM3 cells was significantly rescued. Immunohistochemistry in tissue microarrays consisting of 200 HCC patients was used to analyze the associations between AGO1 expression and prognosis. Intratumoral AGO1 expression was an independent risk factor for overall survival (P = 0.008) and recurrence-free survival (P < 0.001). In conclusion, AGO1 may promote HCC metastasis through TGF-β pathway, and AGO1 may be a reliable prognostic factor in HCC.

Topics: Argonaute Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Disease-Free Survival; Down-Regulation; Epithelial-Mesenchymal Transition; Eukaryotic Initiation Factors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Phosphorylation; Prognosis; Risk Factors; Signal Transduction; Smad2 Protein; Smad4 Protein; Snail Family Transcription Factors; Transforming Growth Factor beta

Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Erythroblasts; Gene Expression Regulation, Neoplastic; Glial Cell Line-Derived Neurotrophic Factor Receptors; Hep G2 Cells; Humans; Leukocyte Common Antigens; Leukocytes; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Invasiveness; Nerve Tissue Proteins; Signal Transduction; Spleen; Transforming Growth Factor beta

Sorafenib is the only FDA approved drug for the treatment of advanced hepatocellular carcinoma (HCC) and other malignancies. Studies indicate that TGF-β signalling is associated with tumour progression in HCC. Autocrine and paracrine TGF-β promotes tumour growth and malignancy by inducing epithelial-mesenchymal transition (EMT). Sorafenib is believed to antagonize tumour progression by inhibiting TGF-β-induced EMT. It improves survival of patients but HCC later develops resistance and relapses. The underlying mechanism of resistance is unknown. Understanding of the molecular mechanism of sorafenib inhibition of TGF-β-induced signalling or responses in HCC may lead to development of adjunctive effective therapy for HCC. In this study, we demonstrate that sorafenib suppresses TGF-β responsiveness in hepatoma cells, hepatocytes, and animal liver, mainly by downregulating cell-surface type II TGF-β receptors (TβRII) localized in caveolae/lipid rafts and non-lipid raft microdomains via caveolae/lipid rafts-mediated internalization and degradation. Furthermore, sorafenib-induced downregulation and degradation of cell-surface TβRII is prevented by simultaneous treatment with a caveolae disruptor or lysosomal inhibitors. On the other hand, sorafenib only downregulates cell-surface TβRII localized in caveolae/lipid rafts but not localized in non-lipid raft microdomains in hepatic stellate cells. These results suggest that sorafenib inhibits TGF-β signalling mainly by inducing caveolae/lipid raft-mediated internalization and degradation of cell-surface TβR-II in target cells. They may also imply that treatment with agents which promote formation of caveolae/lipid rafts, TGF-β receptor kinase inhibitors (e.g., LY2157299) or TGF-β peptide antagonists (by liver-targeting delivery) may be considered as effective adjunct therapy with sorafenib for HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Caveolae; Cell Line, Transformed; Cell Membrane; Dose-Response Relationship, Drug; Endocytosis; HEK293 Cells; Hep G2 Cells; Humans; Liver Neoplasms; Male; Membrane Microdomains; Mice; Mice, Inbred C57BL; Mink; Rats; Receptor, Transforming Growth Factor-beta Type II; Sorafenib; Transforming Growth Factor beta; Treatment Outcome

Background: Transforming growth factor-beta (TGF-β) signaling is recognized as being critical for carcinogenesis.\ Vitamin D has proved to exert numerous tumor suppressive effects. Effects of bone marrow derived mesenchymal stem\ cells (BM-MSCs) on tumor progression are still controversial. The present study was conducted to evaluate the effects\ of BM-MSCs and vitamin D on TGF-β signaling in an experimental hepatocellular carcinoma (HCC) model in rats.\ Materials and Methods: The study was conducted on fifty female white albino rats divided equally into 5 groups:\ controls, HCC induced by diethyl-nitrosamine (DENA) and carbon tetrachloride (CCl4), HCC plus MSCs, HCC plus\ vitamin D and HCC plus both MSCs and vitamin D. The following parameters were assessed in rat liver tissues: TGF-β\ and Smad2 protein levels by ELISA and western blotting, respectively, gene expression of Smad3, Smad7, Snail,\ HNF4α and MMP-2 and histopathological lesions. Serum levels of alpha fetoprotein (AFP), ALT and albumin were\ also assessed. Results: TGF-β protein levels and gene expression of its downstream effectors (Smad3 and Snail), in\ addition to Smad2 protein levels were significantly higher in the HCC group than in the control group. On the other\ hand, they were significantly down-regulated in all treated groups with most significant amelioration with both MSCs\ and vitamin D. Also, the serum levels of AFP were significantly increased in the untreated HCC group, and this was\ again reversed in all treated groups. Histopathological examination of liver tissue revealed that administration of\ MSCs or vitamin D into HCC rat group improved the histopathological picture with residual tumor pathology, while\ administration of both MSCs and vitamin D showed better restoration of liver parenchyma. These data suggest that\ the TGF-β signaling pathway could be used as a therapeutic target in HCC.

Topics: Alkylating Agents; Animals; Carcinoma, Hepatocellular; Cells, Cultured; Diethylnitrosamine; Female; Gene Expression Regulation, Neoplastic; Liver Neoplasms, Experimental; Mesenchymal Stem Cells; Rats; Signal Transduction; Transforming Growth Factor beta; Vitamin D; Vitamins

To understand the cellular and molecular changes in peripheral blood that can lead to the development of hepatocellular carcinoma (HCC) and provide new methods for its diagnosis and treatment.. Peripheral blood mononuclear cells were isolated from the peripheral blood of HCC patients and normal controls and then analyzed by flow cytometry. The percentage of transforming growth factor-β (TGF-β)+ regulatory cells (Tregs) in the peripheral blood was measured, and the expression of TGF-β was also determined. Then, the relationship between the changes and the 5-year survival of patients was analyzed. In addition, recombinant human TGF-β (rhTGF-β) and recombinant human interleukin-6 were added to stimulate the cultured cells, and their effects on HCC were evaluated.. The expression of TGF-β and the percentage of TGF-β+ Tregs in the peripheral blood of HCC patients increased significantly compared with normal controls. Compared with the low TGF-β expression group, the high TGF-β expression group had a significantly lower 5-year survival rate, and the same result was found in the two TGF-β+ Treg groups, suggesting that TGF-β and TGF-β+ Tregs were negatively correlated with the overall survival of the patients. In addition, rhTGF-β promoted the growth of tumor cells and induced high expression levels of IL-6, which further promoted tumor proliferation.. The results showed that TGF-β may promote tumor growth and proliferation by inducing the production of IL-6, and TGF-β and TGF-β+ Tregs may serve as new markers for predicting a poor prognosis in HCC.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Female; Flow Cytometry; Forkhead Transcription Factors; Humans; Interleukin-6; Liver Neoplasms; Male; Middle Aged; Primary Cell Culture; Prognosis; Recombinant Proteins; Survival Rate; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC), one of the most common maligant cancers in the world, is difficult to diagnose in the early time. MicroRNAs (miRNAs), small non-coding RNAs, perform vital functions in cellular differentiation, metabolism and physiological processes. MiR-133a acts as a tumor suppressor in breast, lung and gastric cancer, while the molecular circadian mechanism has not been clear in HCC. In the present study, we certified that the expression of miR-133a decreased in HCC tissues and cell lines and that miR-133a inhibited proliferation, migration and invasion of hepatocellular carcinoma cells. Fos-related antigen 2 (FOSL2), also named FRA-2, was predicted to be a downstream target of miR-133a based on bioinformatic analysis and the prediction was verified by Western Blot, qRT-PCR and luciferase reporter assay. In addition, there was a negative correlation between miR-133a and FOSL2 expression in HCC samples. Furthermore, we verified that overexpression of miR-133a suppressed biological behaviour of HCC through TGF-β/Smad3 signaling pathway. In brief, miR-133a may be a potential prognostic biomarker and may thus be a new therapy in HCC.

Topics: Animals; Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Fos-Related Antigen-2; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Proteins; RNA, Messenger; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Topics: Animals; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Transforming Growth Factor beta; TRPC6 Cation Channel

To explore the biological roles of alpha-fetoprotein (AFP), a tumor-associated antigen in human hepatocellular carcinoma (HCC).. After knockdown of AFP in HepG2 cells by transfection of specific Stealth™ RNAi, the expression of AFP were detected by reverse transcription polymerase chain reaction at mRNA level and by enzyme-linked immunosorbent assay at the protein level. Then, the effect of silenced AFP on cell proliferation was assessed by dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay, and apoptosis assessment with Hoechst33258 and flow cytometry (double stain with fluorescein isothiocyanate/propidium iodide), the roles of AFP in the cell cycle regulation were assessed by flow cytometry. We also detected the expression of some key proteins related to apoptosis pathway by Western immunoblot analysis.. After the transfection for 48 h, the expression of AFP gene was almost abolished, the cell proliferation was inhibited by 47.61%, the number of cells undergoing early apoptosis was significantly increased to 59.47%; cell cycle was arrested with the increase of G0/G1 phase cells from 45.3% to 58.4%. Inhibition of AFP expression also results in decreasing of transforming growth factor-β (TGF-β), mutant P53 expression, and increasing of Bax/Bcl-2 ratio, activation of caspase-3.. The results suggest that AFP may positively regulate cell proliferation by enhancing the apoptosis resistance via effect on TGF-β and p53/Bax/caspase-3 signaling pathway in HepG2 cells. As such, the knockdown of AFP gene should be further investigated in vivo as a novel approach to HCC treatment.

Topics: alpha-Fetoproteins; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hep G2 Cells; Humans; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53

CHI3L1 (YKL40) is a secreted glycoprotein and elevated serum CHI3L1 level has been proved to be associated with poor prognosis in many human cancers. However, the mechanism of how CHI3L1 causes poor prognosis in cancers is still unknown. Here, considering that CHI3L1 is a liver specific/enriched protein, we use hepatocellular carcinoma as a model to study the function of CHI3L1. We showed that, both in vivo and in vitro, overexpression of CHI3L1 could promote liver cancer cells growth, migration and invasion. We then used RNA-seq to analyze the expression profiles of CHI3L1 overexpressed in two HCC cell lines and found that CHI3L1 overexpression affected genes that were involved in cell-cell adhesion, extracellular exosome and adherens junction. Western blot analysis further revealed that CHI3L1 could activate TGF-β signal pathways. Our data added new understanding of the mechanism of CHI3L1's action. 1) CHI3L1 promoted cancer cell proliferation by regulating cell cycles; 2) CHI3L1 promoted cancer cell invasion and metastasis; 3) CHI3L1 regulate liver cancer potentially by regulating the TGF-β signaling pathways; 4) CHI3L1 has direct kinase activities or activate kinase to phosphorylate SMAD2, SMAD3.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Proliferation; Chitinase-3-Like Protein 1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Neoplasm Invasiveness; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Invasion and metastasis are the main cause of recurrence and death in advanced hepatocellular carcinoma (HCC). Revealing the mechanisms of HCC metastasis is important for developing new therapeutic approaches and reducing patient mortality. Ubiquitin specific protease 4 (USP4), is involved in tumorigenesis by deubiquitinating some important oncogenic proteins and impacting their degradation. In the present study, we found that USP4 was significantly upregulated in HCC tumor tissues and the high expression of USP4 was associated with distant metastasis and poor survival in patients. Using gene interference, we demonstrated that USP4 knockdown significantly inhibited HCC cell migration and invasion

Topics: Animals; Carcinoma, Hepatocellular; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Neoplasm Invasiveness; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Transforming Growth Factor beta; Ubiquitin-Specific Proteases; Ubiquitination

LncRNA has been shown to associates with the initiation and progression of hepatocellular carcinoma (HCC). Recently, some studies showed that HANR function as an oncogene in HCC; however, the detailed mechanism of HANR-regulated HCC tumorigenesis and progression needs to be elucidated.. We used RT-qPCR method to probe genes expression. MTT assay, wound healing assay and transwell invasion assay were utilized to examine proliferation and migration and invasion abilities of HepG2 cells. Xenograft tumor experiment was used to show the growth of tumors in vivo.. HANR was evidently upregulated in HCC tumors and cells compared to normal tissues and cells. Besides, HANR knockdown induces attenuated cell proliferation, migration, invasion of HCC cells. By bioinformatic analysis and dual luciferase reporter assay, we found that miR-214 was the downstream target of HANR. Furthermore, miR-214 inhibitor largely enhanced tumor phenotypes of HCC cells regulated by HANR knockdown. HANR and miR-214 regulated the EZH2, then affecting TGFBR2 level. Finally, we demonstrated that EZH2 overexpression could greatly rescue HANR knockdown or miR-214 mimic-induced HCC tumorigenesis and progression.. In this study, we report a newly identified regulatory mechanism HANR/miR-214/EZH2/TGF-β axis, which is implicated in tumorigenesis and progression of HCC. Our findings suggest that HANR facilitates the development of therapeutical strategies or diagnostic markers by targeting HANR.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Enhancer of Zeste Homolog 2 Protein; Hep G2 Cells; Heterografts; Humans; Liver Neoplasms; Mice, Nude; MicroRNAs; RNA, Long Noncoding; Transforming Growth Factor beta

The epithelial mesenchymal transition (EMT) is a physiological multistep process involving epithelial cells acquiring a mesenchymal-like phenotype. It is widely demonstrated that EMT is linked to tumor progression and metastasis. The transforming growth factor (TGF)-β pathways have been widely investigated, but its role in the hepatocarcinoma EMT is still unclear. While the biochemical pathways have been extensively studied, the alteration of biomechanical behavior correlated to cellular phenotype and motility is not yet fully understood. To better define the involvement of TGF-β1 in the metastatic progression process in different hepatocarcinoma cell lines (HepG2, PLC/PRF/5, HLE), we applied a systematic morphomechanical approach in order to investigate the physical and the structural characteristics. In addition, we evaluated the antitumor effect of LY2157299, a TGF-βR1 kinase inhibitor, from a biomechanical point of view, using Atomic Force and Confocal Microscopy. Our approach allows for validation of biological data, therefore it may be used in the future as a diagnostic tool to be combined with conventional biomolecular techniques.

Topics: Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytoskeleton; Elastic Modulus; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Mechanotransduction, Cellular; Microscopy, Atomic Force; Neoplasm Grading; Transforming Growth Factor beta

It is widely understood that transforming growth factor β (TGF-β) has dual functions in tumors-tumor promoter or tumor suppressor. As a tumor promoter, TGF-β drives tumor initiation and progression partially by suppressing the antitumor responses of CD8 cytotoxic T lymphocytes (CTLs) in the tumor microenvironment. Here, we investigated the prognostic value of measuring TGF-β and CD8 CTLs levels and their relationship in human hepatocellular carcinoma (HCC). Immunohistochemical staining was conducted to analyze the prognostic value of TGF-β expression and/or CD8 CTLs levels in 407 HCC patients. The relationship between TGF-β and CD8 T-cell was also evaluated using HCC cell lines and patients' peripheral blood. Lower TGF-β expression or a higher CD8 CTL density was associated with better overall survival and recurrence-free survival, and the patients with low TGF-β expression and more CD8 CTLs had the best prognosis. Although there was no correlation between TGF-β expression and the density of CD8 CTLs, the survival of patients with more CD8 CTL cells was only significantly improved when the tumor expressed low levels of TGF-β. Furthermore, the TGF-β levels was not associated with the proportion of CD8 T cells, but negatively related to interferon γ secretion by CD8 T cells in peripheral blood of HCC patients. Higher TGF-β also resulted in decreased interferon γ secreted by CD8 T cells in vitro. In conclusion, our study suggests that TGF-β is a poor prognostic factor for patients and negatively affect the prognostic value of CD8 CTLs through suppressing antitumor activity of CD8 T-cell in HCC.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Female; Humans; Interferon-gamma; Liver Neoplasms; Lymphocyte Count; Male; Middle Aged; Predictive Value of Tests; Prognosis; Survival Analysis; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Young Adult

Understanding cancer stem cell (CSC) maintenance pathways is critical for the development of CSC-targeting therapy. Here, we investigated the functional role of the cyclin D1-dependent activation of Smad2/3 and Smad4 in hepatocellular carcinoma (HCC) CSCs and in HCC primary tumors. Cyclin D1 sphere-derived xenograft tumor models were employed to evaluate the therapeutic effects of a Smad inhibitor in combination with chemotherapy. Cyclin D1 overexpression confers stemness properties by enhancing single sphere formation, enhancing the CD90+ and EpCAM+ population, increasing stemness gene expression, and increasing chemoresistance. Cyclin D1 interacts with and activates Smad2/3 and Smad4 to result in cyclin D1-Smad2/3-Smad4 signaling-regulated liver CSC self-renewal. The cyclin D1-dependent activation of Smad2/3 and Smad4 is also found in HCC patients and predicts disease progression. A Smad inhibitor impaired cyclin D1-Smad-mediated self-renewal, resulting in the chemosensitization. Thus, pretreatment with a Smad inhibitor followed by chemotherapy not only successfully suppressed tumor growth but also eliminated 57% of the tumors in a cyclin D1 sphere-derived xenograft model. Together, The cyclin D1-mediated activation of Smad2/3 and Smad4 is an important regulatory mechanism in liver CSC self-renewal and stemness. Accordingly, a Smad inhibitor induced CSC differentiation and consequently significant chemosensitization, which could be an effective strategy to target CSCs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Differentiation; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; Male; Mice; Mice, SCID; Neoplastic Stem Cells; Prognosis; Signal Transduction; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Here, we demonstrate a mechanism of cell adhesion-mediated drug resistance using a variety of HCC spheroid models to overcome environment-mediated drug resistance in HCC. We classified spheroids into two groups, tightly compacted and loosely compacted aggregates, based on investigation of dynamics of spheroid formation. Our results show that compactness of HCC spheroids correlated with fibroblast-like characteristics, collagen 1A1 (COL1A1) content, and capacity for chemoresistance. We also showed that ablation of COL1A1 attenuated not only the capacity for compact-spheroid formation, but also chemoresistance. Generally, connective tissue growth factor (CTGF) acts downstream of transforming growth factor (TGF)-β and promotes collagen I fiber deposition in the tumor microenvironment. Importantly, we found that TGF-β-independent CTGF is upregulated and regulates cell adhesion-mediated drug resistance by inducing COL1A1 in tightly compacted HCC spheroids. Furthermore, losartan, which inhibits collagen I synthesis, impaired the compactness of spheroids via disruption of cell-cell contacts and increased the efficacy of anticancer therapeutics in HCC cell line- and HCC patient-derived tumor spheroids. These results strongly suggest functional roles for CTGF-induced collagen I expression in formation of compact spheroids and in evading anticancer therapies in HCC, and suggest that losartan, administered in combination with conventional chemotherapy, might be an effective treatment for liver cancer.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Liver Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor beta

In the present study, the role of microRNA‑15a (miR‑15a) was investigated in hepatitis B virus (HBV)‑associated liver cancer. The results revealed that the expression levels of miR-15a were increased in HBV-associated liver cancer tissues compared with the levels in normal tumor‑adjacent tissues. Moreover, Smad-7 protein expression in patients with HBV-associated liver cancer was higher than that in normal tumor-adjacent tissues. In addition, miR-15a expression and Smad-7 protein expression were increased in HepG2 hepatocellular carcinoma cells compared with that noted in L-02 normal hepatocytes. In HepG2 cells, miR-15a inhibition suppressed cell proliferation and increased Smad-7 protein expression. The inhibition of miR-15a was also demonstrated to decrease transforming growth factor (TGF)-β1 protein expression and Smad-2, p-Smad-2 and Smad-4 expression levels in HepG2 cells. Furthermore, FSP1 protein expression and caspase-3/-7 activities were enhanced by miR-15a inhibition in HepG2 cells compared with the control group. Treatment with recombinant TGF-β1 was demonstrated to activate Smad‑2/-4 and FSP1 protein expression and increase caspase-3/-7 activity in HepG2 cells. Collectively, these findings demonstrate that the miR-15a/Smad-7/TGF-β pathway is important in HBV-associated liver cancer.

Topics: Apoptosis; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatitis B virus; Humans; Liver Neoplasms; Male; MicroRNAs; Recombinant Proteins; S100 Calcium-Binding Protein A4; Signal Transduction; Smad2 Protein; Smad7 Protein; Transforming Growth Factor beta

Platelets in the primary tumor microenvironment play crucial roles in the regulation of tumor progression, but the mechanisms underlying are poorly understood. Here, we report that platelet releasates exerted a proliferative effect on hepatocellular carcinoma (HCC) cells both in vitro and in vivo. This effect depended on a reduction of KLF6 expression in HCC cells. After incubation with either platelets or platelet granule contents, SMMC.7721 and HepG2 cells exhibited significant increases in proliferation and decreases in apoptosis. However, no effect was observed when incubating cancer cells with resuspended activated platelet pellet which exhausted of releasates. Platelet releasates also increased the population of HCC cells in the S and G2/M phases of the cell cycle and reduced the cell population in the G0/G1 phase. Moreover, knocking down KLF6 expression significantly diminished the platelet-mediated enhancement of HCC growth. In addition, blocking TGF-β signaling with the TGF-β receptor inhibitor SB431542 counteracted the effect of platelets on KLF6 expression and proliferation of HCC cells. Based on these findings, we conclude that platelet releasates, especially TGF-β, promote the proliferation of SMMC.7721 and HepG2 cells by decreasing expression of KLF6. This discovery identifies a potential new therapeutic target for the prevention and treatment of hepatocellular carcinoma.

Topics: Animals; Apoptosis; Benzamides; Blood Platelets; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Dioxoles; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hep G2 Cells; Humans; Kruppel-Like Factor 6; Liver Neoplasms; Mice; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

TGFβ1 pathway antagonists have been considered promising therapies to attenuate TGFβ downstream signals in cancer cells. Inhibiting peptides, as P-17 in this study, are bound to either TGFβ1 or its receptors, blocking signal transduction. However, for efficient use of these TGFβ1antagonist as target therapeutic tools, improvement in their delivery is required. Here, a plasmid carrying specific shDNA (SHT-DNA), small interfering RNA (siRNA), and the peptide (P-17) were loaded separately into folic acid (FA)-functionalized nano-carriers made of Bovine Serum Albumin (BSA). The two building blocks of the carrier, (BSA and FA) were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane of hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. Finally, cellular studies were performed to assess the targeting efficiency of the hybrid carriers. These vectors were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. A novel fabrication of Hybrid Polymeric-Protein Nano-Carriers (HPPNC) for delivering TGF β1 inhibitors to HCC cells has been developed. SHT-DNA, siRNA and P-17 have been successfully encapsulated. TGF β1 inhibitors-loaded HPPNC were efficiently uptaken by HLF cells.

Topics: Animals; Carcinoma, Hepatocellular; Cattle; Colloids; Drug Carriers; Drug Delivery Systems; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Liver Neoplasms; Microscopy, Atomic Force; Microscopy, Fluorescence; Peptides; Polymers; RNA, Small Interfering; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Transforming Growth Factor beta

Hook1 is a member of the hook family of coiled-coil proteins, which is recently found to be associated with malignant tumors. However, its biological function in hepatocellular carcinoma is yet unknown. Here, we evaluated the Hook1 levels in human hepatocellular carcinoma samples and matched peritumoral tissues by real-time polymerase chain reaction. Small interfering RNA knockdown and a transforming growth factor-β-induced epithelial-mesenchymal transition model were employed to investigate the biological effects of Hook1 in hepatocellular carcinoma. Our results indicated that Hook1 levels were significantly lower in hepatocellular carcinoma tissues than in the peritumoral tissues. In addition, Hook1 expression was significantly associated with hepatocellular carcinoma malignancy. Hook1 was downregulated after transforming growth factor-β-induced epithelial-mesenchymal transition. Moreover, Hook1 knockdown promoted epithelial-mesenchymal transition and attenuated the sensitivity of hepatocellular carcinoma cells to doxorubicin. In summary, our results indicate that downregulation of Hook1 plays a pivotal role in hepatocellular carcinoma progression via epithelial-mesenchymal transition. Hook1 may be used as a novel marker and therapeutic molecular target in hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Doxorubicin; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Liver Neoplasms; Microtubule-Associated Proteins; RNA, Small Interfering; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) suppresses early stages of tumorigenesis, but also contributes to migration and metastasis of cancer cells. A large number of human tumors contain mutations that inactivate its receptors, or downstream proteins such as Smad transcription factors, indicating that the TGF-β signaling pathway prevents tumor growth. We investigated the effects of TGF-β inhibition on liver tumorigenesis in mice.. C57BL/6 mice received hydrodynamic tail-vein injections of transposons encoding HRAS. TGF-β inhibition via overexpression of SMAD7 (or knockdown of SMAD2, SMAD3, or SMAD4) consistently reduced formation and growth of liver tumors in mice that expressed activated RAS plus shRNA against p53, or in mice that expressed activated RAS and TAZ. TGF-β signaling activated transcription of the Snail gene in liver tumors induced by HRAS. In analyses of transgenic mice, we found TGF-β signaling to be required for formation of liver tumors upon expression of activated RAS and shRNA down-regulating p53, and upon expression of activated RAS and TAZ. Snail is the TGF-β target that is required for hepatic tumorigenesis in these models.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, ras; Genetic Predisposition to Disease; Hep G2 Cells; Humans; Liver; Liver Neoplasms, Experimental; Male; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Smad Proteins; Snail Family Transcription Factors; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Up-Regulation

Sorafenib, a multikinase inhibitor, is the standard therapy for patients with advanced-stage hepatocellular carcinoma (HCC). However, resistance develops to the treatment, therefore, we tried to unravel the underlying mechanism in the resistance of HCC cells to sorafenib via the development of more effective therapeutic strategies.. Various liver cancer cell lines were treated with either sorafenib only or with sorafenib after infection of adenovirus expressing short hairpin RNA (shRNA) against transforming growth factor-β (TGF-β) and p38 activity was examined using western blotting.. p38 MAP kinase activity was inhibited by low concentrations of sorafenib, which could potentially lead to sorafenib resistance in HCC cell lines. Subsequently, we used constitutive form of MKK3/6 (MKK3/6E) to confirm that massive cell death was induced by the activation of p38, and demonstrated the ability to activate p38 without any stimulation. In addition, sorafenib resistance was reduced by the activation of p38. Subsequently, we confirmed that TGF-β shRNA effectively recovered the phosphorylation of p38 inhibited by sorafenib, and increased the sensitivity of HCC cells to sorafenib, thereby inducing cell death and overcoming the resistance of HCC cells to sorafenib.. Our study provides a new therapeutic strategy for HCC that overcomes the resistance of HCC to sorafenib by down-regulation of TGF-β.

Topics: Adenoviridae; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; Niacinamide; p38 Mitogen-Activated Protein Kinases; Phenylurea Compounds; Phosphorylation; RNA, Small Interfering; Signal Transduction; Sorafenib; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide owing to its high rates of metastasis and recurrence. The oncogene IQ motif-containing GTPase activating protein 3 (IQGAP3) is ubiquitously overexpressed in several human cancers, including liver, ovary, lung, large intestine, gastric, bone marrow, and breast malignancies and is involved in the invasion and metastasis of cancer cells. Therefore, we aimed to determine the biological role and molecular mechanism of IQGAP3 in HCC.. We used 120 archived clinical HCC samples, 9 snap-frozen HCC tumor tissues, and 4 normal liver tissues. Expression of IQGAP3 mRNA and protein in HCC cell lines (Hep3B, SMMC-7721, HCCC-9810, HepG2, BEL-7404, HCCLM3, QGY-7701, Huh7, and MHCC97H) and normal liver epithelial cells LO2 was examined by western blot, quantitative polymerase chain reaction, and immunohistochemistry. In addition, wound-healing and transwell matrix penetration assays were used to assess the migratory and invasive abilities of HCC cells, respectively.. Expression of the IQGAP3 was robustly upregulated in HCC cells and tissues. High expression of IQGAP3 in HCC correlated with aggressive clinicopathological features and was an independent poor prognostic factor for overall survival. Furthermore, ectopic expression of IQGAP3 markedly enhanced HCC cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) in vitro and promoted metastasis of orthotopic hepatic tumors in nude mice. Conversely, silencing endogenous IQGAP3 showed an opposite effect. Mechanistically, IQGAP3 promoted EMT and metastasis by activating TGF-β signaling.. IQGAP3 functions as an important regulator of metastasis and EMT by constitutively activating the TGF-β signaling pathway in HCC. Our findings present new evidence of the role of IQGAP3 in EMT and metastasis, indicating its potential as a prognostic biomarker candidate and a therapeutic target against HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; GTPase-Activating Proteins; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; RNA, Messenger; Signal Transduction; Statistics, Nonparametric; Transforming Growth Factor beta; Up-Regulation

FOXP3 has been discovered to be expressed in tumor cells and participate in the regulation of tumor behavior. Herein, we investigated the clinical relevance and biological significance of FOXP3 expression in human hepatocellular carcinoma (HCC).. Expression profile of FOXP3 was analyzed using real-time RT-PCR, western blotting and immunofluorescence on HCC cell lines, and immunostaing of a tissue microarray containing of 240 primary HCC samples. The potential regulatory roles of FOXP3 were dissected by an integrated approach, combining biochemical assays, analysis of patient survival, genetic manipulation of HCC cell lines, mouse xenograft tumor models and chromatin immunoprecipitation (ChIP) sequencing.. FOXP3 was constitutively expressed in HCC cells with the existence of splice variants (especially exon 3 and 4 deleted, Δ3,4-FOXP3). High expression of FOXP3 significantly correlated with low serum α-fetoprotein (AFP) level, absence of vascular invasion and early TNM stage. Survival analyses revealed that increased FOXP3 expression was significantly associated with better survival and reduced recurrence, and served as an independent prognosticator for HCC patients. Furthermore, FOXP3 could potently suppress the proliferation and invasion of HCC cells in vitro and reduce tumor growth in vivo. However, Δ3,4-FOXP3 showed a significant reduction in the tumor-inhibiting effect. The inhibition of FOXP3 on HCC aggressiveness was acted probably by enhancing the TGF-β/Smad2/3 signaling pathway.. Our findings suggest that FOXP3 suppresses tumor progression in HCC via TGF-β/Smad2/3 signaling pathway, highlighting the role of FOXP3 as a prognostic factor and novel target for an optimal therapy against this fatal malignancy.

Topics: Aged; Animals; Carcinoma, Hepatocellular; Cell Proliferation; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Middle Aged; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins

Hypermethylation-induced epigenetic silencing of tumour suppressor genes (TSGs) are frequent events during carcinogenesis. MicroRNA-142 (miR-142) is found to be dysregulated in cancer patients to participate into tumour growth, metastasis and angiogenesis. However, the tumour suppressive role of miR-142 and the status of methylation are not fully understood in hepatocellular carcinoma (HCC).. Hepatocellular carcinoma tissues and corresponding non-neoplastic tissues were collected. The expression and function of miR-142 and TGF-β in two HCC cell lines were determined. The miRNA-mRNA network of miR-142 was analysed in HCC cell lines.. We found that the miR-142 expression was reduced in tumour tissues and two HCC cell lines HepG2 and SMMC7721, which correlated to higher TNM stage, metastasis and differentiation. Moreover, miR-142 was identified to directly target and inhibit transforming growth factor β (TGF-β), leading to decreased cell vitality, proliferation, EMT and the ability of pro-angiogenesis in TGF-β-dependent manner. Interestingly, the status of methylation of miR-142 was analysed and the results found the hypermethylated miR-142 in tumour patients and cell lines. The treatment of methylation inhibitor 5-Aza could restore the expression of miR-142 to suppress the TGF-β expression, which impaired TGF-β-induced tumour growth.. These findings implicated that miR-142 was a tumour suppressor gene in HCC and often hyermethylated to increase TGF-β-induced development of hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; Neoplasm Metastasis; Neovascularization, Pathologic; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Carcinoma, Hepatocellular; Caveolin 1; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hepatocytes; Humans; Liver Neoplasms; NADPH Oxidase 4; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins

Whole-cell tumor vaccines have shown much promise; however, only limited success has been achieved for the goal of eliciting robust tumor-specific T-cell responses.. Hepatocellular carcinoma (HCC) cells, H22 and Hepa1-6, were modified by blocking the STAT3 signaling pathway with a STAT3 decoy oligodeoxynucleotide, and the immunogenicity and possibility of using these cell lysates as a vaccine were evaluated.. STAT3-blocked whole HCC cell lysates inhibited tumor growth and tumorigenesis, and prolonged the survival of tumor-bearing mice. In addition, STAT3-blocked whole HCC cell lysates stimulated the activation of T cells and natural killer (NK) cells, and enhanced the infiltration of cytotoxic CD8. The newly generated STAT3-blocked whole-cell HCC vaccine has potential for cancer cell vaccination.

Topics: Animals; Cancer Vaccines; Carcinoma, Hepatocellular; Cell Line, Tumor; Female; Humans; Immunity, Cellular; Immunity, Humoral; Interleukin-10; Killer Cells, Natural; Liver Neoplasms; Lymphocyte Activation; Mice; Oligonucleotides; Signal Transduction; STAT3 Transcription Factor; T-Lymphocytes; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The sympathetic nervous system (SNS) is known to play a significant role in tumor initiation and metastasis. Hepatocellular carcinoma (HCC) frequently occurs in cirrhotic livers after chronic inflammation, and the SNS is hyperactive in advanced liver cirrhosis. However, it remains unclear whether the SNS promotes hepatocarcinogenesis by modulating chronic liver inflammation. In this study, a retrospective pathological analysis and quantification of sympathetic nerve fiber densities (tyrosine hydroxylase, TH

Topics: Adult; Aged; Animals; Carcinogenesis; Carcinoma, Hepatocellular; Female; Humans; Inflammation; Interleukin-6; Kupffer Cells; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Middle Aged; Nerve Fibers; Prognosis; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-1; Retrospective Studies; Sympathetic Nervous System; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) is a multifunctional cytokine which is importantly implicated in hepatocarcinogenesis. The current study provides novel evidence that TGFβ upregulates the expression of multiple receptor tyrosine kinases (RTKs), including IGF1R, EGFR, PDGFβR, and FGFR1 in human hepatocellular carcinoma (HCC) cells. This, in turn, sensitized HCC cells to individual cognate RTK ligands, leading to cell survival. Our data showed that the TGFβ-mediated increase in growth factor sensitivity led to evasion of apoptosis induced by the mutikinase inhibitor, sorafenib. Conversely, we observed that inhibition of the TGFβ signaling pathway by LY2157299, a TGFβRI kinase inhibitor, enhanced sorafenib-induced apoptosis, in vitro. Our findings disclose an important interplay between TGFβ and RTK signaling pathways, which is critical for hepatocellular cancer cell survival and resistance to therapy. © 2016 Wiley Periodicals, Inc.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Liver; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Pyrazoles; Quinolines; Receptor Protein-Tyrosine Kinases; Signal Transduction; Sorafenib; Transforming Growth Factor beta

Mucin 1 (MUC1), as an oncogene, is overexpressed in hepatocellular carcinoma (HCC) cells and promotes the progression and tumorigenesis of HCC through JNK/TGF-β signaling pathway. In the present study, RNA interference (RNAi) and JNK inhibitor SP600125, which target MUC1 and/or JNK, were used to treat HCC cells in vitro, and the results showed that both silencing the expression of MUC1 and blocking the activity of JNK inhibited the proliferation of HCC cells. In addition, MUC1-stable-knockdown and SP600125 significantly inhibited the growth of tumors in the subcutaneous transplant tumor models that established in BALB/c nude mice rather than MUC1 or JNK siRNAs transiently transfection. Furthermore, the results from immunohistochemical staining assays showed that the inhibitory effects of MUC1 gene silencing and SP600125 on the proliferation of HCC cells in vivo were through the JNK/TGF-β signaling pathway. These results indicate that MUC1 and JNK are attractive targets for HCC therapy and may provide new therapeutic strategies for the treatment of HCC.

Topics: Animals; Anthracenes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mucin-1; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta

Modulation of microRNA expression is a potential treatment for hepatocellular carcinoma (HCC). Therefore, the epigenetically regulated microRNA-449 family (miR-449a, miR-449b, miR-449c) was characterized with regards to its functional effects and target genes in HCC.. After transfection of miR-449a, miR-449b, and/or miR-449c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. Binding specificities, target genes, and regulated pathways of each miRNA were identified by microarray analyses. Target genes were validated by luciferase reporter assays and expression analyses in vitro. Furthermore, target gene expression was analyzed in 61 primary human HCCs compared to normal liver tissue.. Tumor suppressive effects, binding specificities, target genes, and regulated pathways of miR-449a and miR-449b differed from those of miR-449c. Transfection of miR-449a, miR-449b, and/or miR-449c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. Importantly, miR-449a, miR-449b, and, to a lesser degree, miR-449c directly targeted SOX4, which codes for a transcription factor involved in epithelial-mesenchymal transition and HCC metastasis, and thereby inhibited TGF-β-mediated cell migration.. This study provides detailed insights into the regulatory network of the epigenetically regulated miRNA-449 family and, for the first time, describes distinct tumor suppressive effects and target specificities of miR-449a, miR-449b, and miR-449c. Our results indicate that particularly miR-449a and miR-449b may be considered for miRNA replacement therapy to prevent HCC progression and metastasis.. In this study, we demonstrated that the microRNA-449 family acts as a tumor suppressor in liver cancer by causing cell death and inhibiting cell migration. These effects are caused by downregulation of the oncogene SOX4, which is frequently overexpressed in liver cancer. We conclude that the microRNA-449 family may be a target for liver cancer therapy.

Topics: Acetylation; Animals; Carcinoma, Hepatocellular; Cell Movement; Genes, Tumor Suppressor; Histones; Humans; Liver Neoplasms; Mice; MicroRNAs; SOXC Transcription Factors; Transforming Growth Factor beta

Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy.. Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n=164) and intrahepatic cholangiocarcinoma (iCCA) (n=149).. Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliary-derived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p<0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p=0.008), showed significant upregulation of transforming growth factor (TGF)-β signaling and enrichment of inflammation-related and immune response signatures (p<0.001). Stem-cell tumors were characterized by spalt-like transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p<0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs.. Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-β signaling.. Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bile Duct Neoplasms; Carcinoma, Hepatocellular; Cholangiocarcinoma; Chromosomal Instability; Female; Humans; Liver Neoplasms; Male; Middle Aged; Mutation; Signal Transduction; Transforming Growth Factor beta; Young Adult

As part of its potential pro-tumorigenic actions, Transforming Growth Factor-(TGF)-β induces epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells. Whether EMT induces changes in tumor cell plasticity has not been fully explored yet. Here, we analyze the effects of TGF-β on the EMT and stem-related properties of HCC cells and the potential correlation among those processes. The translational aim of the study was to propose a TGF-β/EMT/stem gene signature that would help in recognizing HCC patients as good candidates for anti-TGF-β therapy. Results indicate that when TGF-β induces EMT in HCC cells, a switch in the expression of stem genes is observed and their stemness potential and migratory/invasive capacity are enhanced. However, TGF-β may induce a partial EMT in some epithelial HCC cells, increasing the expression of mesenchymal genes and CD44, but maintaining epithelial gene expression. Epithelial cells show higher stemness potential than the mesenchymal ones, but respond to TGF-β increasing their migratory and invasive capacity. In HCC patient samples, TGFB1 expression most frequently correlates with a partial EMT, increase in mesenchymal genes and CD44 expression, as well as maintenance or over-expression of epithelial-related genes.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Plasticity; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Liver Neoplasms; Mesenchymal Stem Cells; Neoplasm Invasiveness; Neoplastic Stem Cells; Phenotype; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Time Factors; Transcriptome; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

The capacity of natural killer (NK) cells to kill tumor cells without specific antigen recognition provides an advantage over T cells and makes them potential effectors for tumor immunotherapy. However, the efficacy of NK cell adoptive therapy can be limited by the immunosuppressive tumor microenvironment. Transforming growth factor-β (TGF-β) is a potent immunosuppressive cytokine that can suppress NK cell function. To convert the suppressive signal induced by TGF-β to an activating signal, we genetically modified NK-92 cells to express a chimeric receptor with TGF-β type II receptor extracellular and transmembrane domains and the intracellular domain of NK cell-activating receptor NKG2D (TN chimeric receptor). NK-92 cells expressing TN receptors were resistant to TGF-β-induced suppressive signaling and did not down-regulate NKG2D. These modified NK-92 cells had higher killing capacity and interferon γ (IFN-γ) production against tumor cells compared with the control cells and their cytotoxicity could be further enhanced by TGF-β. More interestingly, the NK-92 cells expressing TN receptors were better chemo-attracted to the tumor cells expressing TGF-β. The presence of these modified NK-92 cells significantly inhibited the differentiation of human naïve CD4

Topics: Animals; Cancer Vaccines; Carcinoma, Hepatocellular; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Cytotoxicity, Immunologic; Humans; Immunotherapy, Adoptive; Interferon-gamma; Killer Cells, Natural; Liver Neoplasms; Mice; Mice, Nude; Neoplasms, Experimental; NK Cell Lectin-Like Receptor Subfamily K; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment; Xenograft Model Antitumor Assays

Hyperactivation of transforming growth factor-β (TGF-β) signaling pathway is a common feature of hepatocellular carcinoma (HCC) progression. However, the driver factors leading to enhanced TGF-β activity are not well characterized. Here, we explore the mechanisms that loss of Krüppel-like factor 4 (KLF4) exacerbates oncogenic TGF-β signaling in human HCC. The expression of KLF4 and TGF-β signaling components in primary HCC and their clinicopathologic relevance and significance was evaluated by using tissue microarray and immunohistochemistry. Cellular and molecular impacts of altered KLF4 expression and TGF-β signaling were determined using immunofluorescence, western blot, reverse-transcriptase PCR, chromatin immunoprecipitation and promoter reporter assays. Loss of KLF4 expression in primary HCC closely correlated with decreased Smad7 expression, increased p-Smad2/3 expression and independently predicts reduced overall and relapse-free survival after surgery. TGF-β signaling components were expressed in most HCC cells, and activation of TGF-β signaling promoted cell migration and invasion. Enforced KLF4 expression blocked TGF-β signal transduction and inhibited cell migration and invasion via activation of Smad7 transcription, whereas deletion of its C-terminal zinc-finger domain diminished this effect. KLF4 protein physically interacts with the Smad7 promoter. Promoter deletion and point mutation analyses revealed that a region between nucleotides -15 bp and -9 bp of the Smad7 promoter was required for the induction of Smad7 promoter activity by KLF4. Our data indicate that KLF4 suppresses oncogenic TGF-β signaling by activation of Smad7 transcription, and that loss of KLF4 expression in primary HCC may contribute to activation of oncogenic TGF-β signaling and subsequent tumor progression.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Disease Progression; Down-Regulation; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Liver Neoplasms; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

TGFβ is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.

Topics: Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytokines; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Hep G2 Cells; Humans; Liver Neoplasms; Microtubule-Associated Proteins; p300-CBP Transcription Factors; Phagosomes; Promoter Regions, Genetic; Retinoblastoma Protein; Signal Transduction; Transforming Growth Factor beta

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFβ signaling pathway was necessary for fibrogenesis. PDGF-C transgenic (PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFβ/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFβ signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cells, Cultured; Female; Hepatic Stellate Cells; Hepatocytes; Liver; Liver Cirrhosis; Liver Neoplasms; Lymphokines; Male; Mice; Mice, Knockout; Mice, Transgenic; Platelet-Derived Growth Factor; Rats; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor (TGF)-β induces cell growth arrest in well-differentiated hepatocellular carcinoma (HCC) while hepatitis B virus X protein (HBx) minimizes the tumor suppression of TGF-β signaling in early chronic hepatitis B. However, how to reverse the oncogenic effect of HBx and sustain the tumor-suppressive action of TGF-β has yet to be investigated. The present study examined the effect of TGF-β and a c-Jun N-terminal kinase (JNK) inhibitor on cell growth in HCC cells with forced expression of HBx. It was found that HBx promoted cell growth via activation of the JNK/pSMAD3L pathway and inhibition of the transforming growth factor-beta type I receptor (TβRI)/pSMAD3C pathway. pSMAD3L/SMAD4 and pSMAD3C/SMAD4 complexes antagonized each other to regulate c-Myc expression. In the absence of HBx, TGF-β induced cell growth arrest through activation of the TβRI/pSMAD3C pathway in well-differentiated HCC cells. In the presence of HBx, TGF-β had no effect on cell growth. JNK inhibitor SP600125 significantly reversed the oncogenic action of HBx and favored TGF-β to regain the ability to inhibit the cell growth in HBx-expressing well-differentiated HCC cells. In conclusion, targeting JNK signaling favors TGF-β to block HBx-induced cell growth promotion in well-differentiated HCC cells. As an adjunct to anti-viral therapy, the combination of TGF-β and inhibition of JNK signaling is a potential therapy for HBV-infected HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Hepatitis B virus; Hepatitis B, Chronic; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Signal Transduction; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Viral Regulatory and Accessory Proteins

The immune status of the tumor microenvironment influences tumor progression, and hepatocellular carcinoma (HCC) with an immunosuppressive signature often is associated with a poor prognosis. This study examined the impact of a bone marrow-derived dendritic cell (DC) vaccine loaded with autologous tumor cell lysate on tumor progression and the tumor microenvironment using an orthotopic murine HCC model. An orthotopic murine HCC was established by implantation of Hepa1-6 cells in the liver. The impact of DC vaccine loaded with Hepa1-6 cell lysate on tumor progression, survival, and tumor-infiltrating lymphocytes and cytokines was examined. Treating mice with DC vaccine loaded with Hepa1-6 cell lysate inhibited the progression of murine HCC generated through orthotopic implantation of Hepa1-6 cells and resulted in a 90 % survival rate by day 60 compared with a survival rate lower than 5 % for untreated mice. This anti-tumor response was associated with inhibition of STAT3 phosphorylation within the tumor. The DC vaccine reduced accumulation of Foxp3

Topics: Animals; Cancer Vaccines; Carcinoma, Hepatocellular; CD4 Antigens; Cell Line, Tumor; Dendritic Cells; Disease Models, Animal; Forkhead Transcription Factors; Immunotherapy, Active; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred Strains; Phosphorylation; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Precancerous lesion, a well-established histopathologically premalignant tissue with the highest risk for tumourigenesis, develops preferentially from activation of DNA damage checkpoint and persistent inflammation. However, little is known about the mechanisms by which precancerous lesions are initiated and their physiological significance.. Laser capture microdissection was used to acquire matched normal liver, precancerous lesion and tumour tissues. miR-484(-/-), Ifnar1(-/-) and Tgfbr2(△hep) mice were employed to determine the critical role of the interferon (IFN)-microRNA pathway in precancerous lesion formation and tumourigenesis. RNA immunoprecipitation (RIP), pull-down and chromatin immunoprecipitation (ChIP) assays were applied to explore the underlying mechanisms.. miR-484 is highly expressed in over 88% liver samples clinically. DEN-induced precancerous lesions and hepatocellular carcinoma were dramatically impaired in miR-484(-/-) mice. Mechanistically, ectopic expression of miR-484 initiates tumourigenesis and cell malignant transformation through synergistic activation of the transforming growth factor-β/Gli and nuclear factor-κB/type I IFN pathways. Specific acetylation of H3K27 is indispensable for basal IFN-induced continuous transcription of miR-484 and cell transformation. Convincingly, formation of precancerous lesions were significantly attenuated in both Tgfbr2(△hep) and Ifnar1(-/-) mice.. These findings demonstrate a new protumourigenic axis involving type I IFN-microRNA signalling, providing a potential therapeutic strategy to manipulate or reverse liver precancerous lesions and tumourigenesis.

Topics: Acetylation; Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Hepatocytes; Humans; Interferon Type I; Liver; Liver Neoplasms; Mice; Mice, Knockout; MicroRNAs; NF-kappa B; NIH 3T3 Cells; Pentanones; Precancerous Conditions; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Interferon alpha-beta; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Zinc Finger Protein GLI1

Sulforaphane is recognized as a safe antitumor agent derived from various cruciferous vegetables, including broccoli. It has been demonstrated that sulforaphase is a potent antitumor agent in diverse cancers. However, its effect on hepatocellular carcinoma remains largely unknown. Here, we show that sulforaphane inhibits TGF-β-induced epithelial-mesenchymal transition of hepatocellular carcinoma cell via the reactive oxygen species-dependent pathway. We found sulforaphane inhibited hepatocellular carcinoma cell proliferation in a dose- and time-dependent manner. Sulforaphane induced G0/G1 phase cell cycle arrest and promoted cell apoptosis. A set of experiments showed that sulforaphase inhibited hepatocellular carcinoma cell migration and invasion, inhibited the formation of fibroblast like mesenchymal cells and the expression of Vimentin, but increased the expression of E-cadherin, suggesting sulforaphane suppresses epithelial-mesenchymal transition (EMT) process. Cotreatment with N-acetyl-L-cysteine inhibited sulforaphane-inhibited invasion and upregulation of E-cadherin and almost completely abolished the sulforaphane-induced expression of Vimentin. The effect of sulforaphane on the growth of hepatocellular carcinoma cells was confirmed by a xenograft tumor growth model. All our finding indicated that sulforaphane is a promising and safe strategy for treating hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Movement; Cell Shape; Epithelial-Mesenchymal Transition; Female; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Inhibitory Concentration 50; Isothiocyanates; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Reactive Oxygen Species; Sulfoxides; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

We reported that poor prognoses of hepatocellular carcinoma (HCC) patients after radiofrequency ablation (RFA) are owing to up-regulation of expression of hypoxia-inducible factor-1 and epithelial cell adhesion molecule. We investigated aggressive progression in residual liver tumors (RLTs) after RFA to focus on expression of epithelial-mesenchymal transition (EMT)-related genes and miRNAs. Ten patients with recurrent HCC post-RFA who underwent hepatectomy (RFA group) and 78 patients with HCC without prior RFA (non-RFA group) were enrolled. We examined expression of transforming growth factor (TGF)-β, Twist, vimentin, and Snail-1 mRNAs in tumor tissues, and expression of miR-34a and miR-200c. Expression of TGF-β, Twist and Snail-1 in the RFA group was significantly higher than that in the non-RFA group (P < 0.05); vimentin expression in the RFA group was higher than that in the non-RFA group (P = 0.07). Expression of miR-200c and miR-34a in the RFA group was significantly lower than that in the non-RFA group (miR-200c: P = 0.04; miR-34a: P < 0.01). Increased expression of EMT markers through down-regulation of miRNA expression in RLTs after RFA may be related to poor prognoses of HCC patients with aggressive local recurrence after RFA.

Topics: Carcinoma, Hepatocellular; Catheter Ablation; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Liver Neoplasms; MicroRNAs; Neoplasm Recurrence, Local; Nuclear Proteins; Transforming Growth Factor beta; Twist-Related Protein 1; Vimentin

Hepatocellular carcinoma progression is thought to be driven by cancer stem cells (CSCs). No clinical trial has, as yet, shown convincing long-term disease free survival results for the majority of patients in HCC. So it is important to discover new anti-cancer agents. In our study, we chose sophocarpine, which is derived from the foxtail-like sophora herb, for its efficacy to inhibit HCC including CSCs and potential mechanism study. Our results show that sophocarpine could not only reduce HCC cell viability, eliminate HCC and reverse hepatoma cells malignant phenotype, but also reduce the ratio of CSCs and inhibit the sphere formation of CSCs in vitro. In vivo, sophocarpine significantly displayed antitumor effects in subcutaneous xenograft HCC models and orthotopic transplantation tumor models. Further studies showed that sophocarpine could exert anti-tumor effects partly via downregulating the activity of the cancer stem cell related pathways and inhibiting EMT induced by TGF-β.

Topics: Alkaloids; Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Catenins; Cell Differentiation; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3 beta; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Phenotype; Proto-Oncogene Proteins c-akt; Signal Transduction; Spheroids, Cellular; Time Factors; Transforming Growth Factor beta; Tumor Burden; Xenograft Model Antitumor Assays

The epithelial-mesenchymal transition (EMT) is the key process that drives tumor metastasis. Accumulating evidence suggests that the deregulation of some microRNAs (miRNAs), is implicated in this process. Here, we highlight the function and molecular mechanism of miR-630 and its potential clinical application in hepatocellular carcinoma (HCC). First, we identified the clinical relevance of miR-630 expression in a screen of 97 HCC patient tissues. Patients with low miR-630 expression had higher recurrence rates and shorter overall survival than those with high miR-630 expression. Functional studies demonstrated the overexpression of miR-630 in HCC cells attenuated the EMT phenotype in vitro. Conversely, inhibition of miR-630 promoted EMT in HCC cells. Mechanistically, our data revealed that miR-630 suppressed EMT by targeting Slug. Knockdown of Slug expression reversed miR-630 inhibitor-mediated EMT progression. Furthermore, we found that the TGF-β-Erk/SP1 and JNK/c-Jun signaling pathways repressed miR-630 transcription through occupying transcription factor binding sites. Ectopic expression of miR-630 restored the TGF-β-activated EMT process. Taken together, these findings demonstrate, in HCC cells, miR-630 exerts its tumor-suppressor functions through the TGF-β-miR-630-Slug axis and provides a potential prognostic predictor for HCC patients.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Mice; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Prognosis; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) accounts for 75-80 % of primary liver cancer, and usually arises after years of liver disease. Thus it is important to understand the molecular mechanisms which drive or mediate the development of HCC.. In this work, we examined whether CD109 was associated with a poor prognosis in HCC and explored possible underlying mechanisms.. We examined the CD109 and Ki67 expression levels in 97 patients with HCC using immunohistochemistry. CD109 levels in HCC cells were down-regulated by shRNA transfection. The cycle progression and cell proliferation status of HCC cells were evaluated by flow cytometry and CCK-8 assay. The effect of CD109 on proliferation and apoptosis was investigated by western blot and TUNEL activity assays.. The CD109 protein was up-regulated in HCC tissue compared with adjacent noncancerous tissue. CD109 expression levels in the 97 patients with HCC were positively correlated with histological grade. Univariate and multivariate survival analysis revealed that CD109 was a significant predictor of overall survival among HCC patients. CD109 shRNA knockdown delayed the G1-S phase transition, abrogated cell proliferation, and increased cell apoptosis. Furthermore, CD109 impaired TGF-β/Smad signaling through control of p-smad2.. CD109 promoted HCC proliferation and predicted poor prognosis. In addition, CD109 expression was associated with anti-apoptosis in HCC cells.

Topics: Adult; Antigens, CD; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 8; Cell Cycle; Cell Proliferation; Cell Survival; Female; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; GPI-Linked Proteins; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Proteins; Phosphorylation; Prognosis; Proliferating Cell Nuclear Antigen; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; Signal Transduction; Smad Proteins; Smad2 Protein; Transforming Growth Factor beta

Over-activation of transforming growth factor-β (TGF-β) signaling pathway promotes cell migration and invasion in hepatocellular carcinoma (HCC). The Hepatitis B virus X protein (HBx) is involved in the enhancement of TGF-β signaling pathway in HCC while the mechanism remains unclear. Protein phosphatase magnesium dependent 1A (PPM1a) functions as a phosphatase essential for terminating the TGF-β signaling pathway by dephosphorylating p-Smad2/3. In this study, we found that HBx dose-dependently downregulated PPM1a protein level in the presence of TGF-β, while having no effect on its mRNA level. Further study showed that HBx increased the ubiquitination of PPM1a and accelerated its proteasomal degradation. Restoration of PPM1a almost completely abrogated HBx mediated promotion on HCC migration and invasion. This involvement of PPM1a in HBx-related HCC was further confirmed with immunohistochemical analysis in HCC tissue. Compared with paired pericarcinous tissue, HCC tissue showed decreased PPM1a level. Besides, PPM1a level is negatively correlated with HBx expression. Taken together, our present study suggests that HBx-induced degradation of PPM1a is a novel mechanism for over-activation of TGF-β pathway in HCC development, which might provide potential candidates for clinical diagnosis and treatment.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Down-Regulation; Hep G2 Cells; Humans; Liver Neoplasms; Protein Phosphatase 2C; Trans-Activators; Transfection; Transforming Growth Factor beta; Viral Regulatory and Accessory Proteins

Macrophages are a major component of most solid tumours and can exert both anti- and pro-tumourigenic functions. Although the immunosuppressive/pro-tumour roles of macrophages have been widely examined, significantly less is known about macrophage subpopulations that have potential anti-tumour properties in humans. In the present study, a population of CD169(+) macrophages with relatively high expression levels of HLA-DR and CD86 was identified in human hepatocellular carcinoma tissues. The frequency of CD169-expressing macrophages within cancer nests was significantly lower than that found in paired non-tumour areas. In vitro experiments revealed that in the presence of anti-CD3 stimulation, CD169(+) macrophages could significantly enhance the proliferation, cytotoxicity, and cytokine production capacity of CD8(+) T cells in a CD169 molecule-dependent manner. Autocrine TGF-β produced by tumour-stimulated macrophages was involved in the down-regulation of CD169 expression on these cells. Moreover, the accumulation of CD169(+) macrophages in tumour tissues was negatively associated with disease progression and predicted favourable survival in hepatocellular carcinoma patients, which was in contrast to the trend observed for total CD68(+) macrophages. Therefore, CD169 might act as a co-stimulatory molecule for cytotoxic T-cell activation, and could define a population of tumour-infiltrating macrophages with potential anti-tumour properties in human hepatocellular carcinoma tissues. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Adult; Aged; Aged, 80 and over; B7-2 Antigen; Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Down-Regulation; Female; HLA-DR Antigens; Humans; Liver Neoplasms; Lymphocyte Activation; Macrophages; Male; Middle Aged; Sialic Acid Binding Ig-like Lectin 1; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Microenvironment; Young Adult

Diosmetin (Dio) is a major active component of flavonoid compounds. A previous study demonstrated that Dio exhibited anticancer activity and induced apoptosis in HepG2 human hepatoma cells via cytochrome P450, family 1-catalyzed metabolism. The present study observed that cell proliferation of HepG2 cells was inhibited by Dio treatment and tumor protein p53 was significantly increased following Dio treatment. Following addition of recombinant transforming growth factor‑β (TGF‑β) protein to Dio‑treated HepG2 cells, cell growth inhibition and cell apoptosis was partially reversed. These findings suggest a novel function for the TGF‑β/TGF‑β receptor signaling pathway and that it may be a key target of Dio‑induced cell apoptosis in HepG2 cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Proliferation; Flavonoids; Gene Expression; Hep G2 Cells; Humans; Liver Neoplasms; Phosphorylation; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Intrahepatic and extrahepatic metastases are frequently detected in hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is believed to drive metastasis. There are not many well-established model systems to study EMT in HCC. Here we identified an atypical EMT while characterizing a population of mesenchymal cells in Huh7.5 hepatoma cell cultures. Cells with distinct morphology appeared during geneticin treatment of Huh7.5 cultures. Molecular characterization of geneticin resistant Huh7.5M cells confirmed EMT. Huh7.5M cells expressed cancer stem cell markers. p38MAPK and ERK1/2 were substantially activated in Huh7.5M cells. Their Inhibition elevated E-Cadherin expression with concerted suppression of Vimentin and anchorage independent growth in Huh7.5M cells. TGFβ could not induce EMT in Huh7.5 cultures, but enriched mesenchymal populations, similar to geneticin. Huh7.5M cells formed more aggressive solid tumors, primarily comprising cells with epithelial morphology, in nude mice. Canonical EMT-TFs did not participate in this atypical EMT, indicating that the established canonical EMT-TFs do not drive every EMT and there is a dire need to identify additional factors. The system that we characterized is a unique model to study EMT, MET and biphasic TGFβ signaling in HCC and offers considerable potential to facilitate more insightful studies on deeper questions in tumor metastasis.

Topics: Animals; Antigens, CD; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cellular Reprogramming; Epithelial-Mesenchymal Transition; Gentamicins; Heterografts; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mice, Nude; Neoplastic Stem Cells; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Vimentin

Fibroblasts are rich in the surrounding microenvironment of hepatocellular carcinoma (HCC) because most HCCs occur in fibrotic or cirrhotic livers. However, the role of cancer-associated fibroblasts (CAFs) in HCC metastasis remains obscure. Here, we reported that CAFs promote the migration and invasion of HCC cells in vitro and facilitate the HCC metastasis to the bone, brain and lung in NOD/SCID mice. The RayBio human chemokine antibody array revealed that CAFs secret higher levels of CCL2, CCL5, CCL7 and CXCL16 than peri-tumor fibroblasts. CCL2 and CCL5 increase the migration but not the invasion of HCC cells, while CCL7 and CXCL16 promote both migration and invasion of HCC cells. Moreover, CCL2 and CCL5 stimulate the activation of the hedgehog (Hh) pathway, while CCL7 and CXCL16 enhance the activity of the transforming growth factor-β (TGF-β) pathway in HCC cells. The neutralizing antibodies of chemokines notably attenuate the effect of CAFs on HCC metastasis and compromised the activation of Hh and TGF-β pathways in HCC cells. In summary, CAF-secreted CCL2, CCL5, CCL7 and CXCL16 promote HCC metastasis through the coordinate activation of Hh and TGF-β pathways in HCC cells.

Topics: Animals; Bone Neoplasms; Brain Neoplasms; Cancer-Associated Fibroblasts; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Chemokine CCL2; Chemokine CCL5; Chemokine CCL7; Chemokine CXCL16; Chemokines; Chemokines, CXC; Hedgehog Proteins; Humans; Liver Neoplasms; Lung Neoplasms; Male; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Neoplasm Invasiveness; Paracrine Communication; Receptors, Scavenger; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

Combretastatin A-1 phosphate (CA1P) is a microtubule polymerization inhibitor that binds to the colchicine-binding site of tubulin. We demonstrated that CA1P has outstanding anti-cancer activity against hepatocellular carcinoma (HCC) in vitro and in vivo. As determined by fluorescence staining and western blots (WBs), CA1P induced reactive oxygen species (ROS) accumulation and apoptosis in HepG2 cells with a down-regulation of Mcl-1. Additional studies indicated that CA1P induced microtubule depolymerization-mediated AKT inactivation, which resulted in GSK-3β activation, Wnt/β-Catenin pathway inhibition, and Mcl-1 down-regulation. The induction of HepG2 cell apoptosis by CA1P was prevented by a GSK-3β-specific inhibitor. Furthermore, immunohistochemistry studies on hepatocellular carcinoma mouse models showed that CA1P had activity against tumor-associated macrophages (TAMs). CA1P induced TAM apoptosis in vitro through the same mechanism observed with HepG2 cells, and it eliminated TAMs in the tumor microenvironment (TME) in vivo. In TME, the expression of TGF-β and TNF-α was also altered. The adoptive transfer of macrophages partly rescued the growth of tumor inhibited by CA1P. These findings indicate that CA1P has great potential to impact both cancer cells and the microenvironment, and our results should accelerate the application of CA1P for HCC therapy in clinic.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Dose-Response Relationship, Drug; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Inhibitory Concentration 50; Liver Neoplasms; Macrophages; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Stilbenes; Transforming Growth Factor beta; Tubulin Modulators; Tumor Burden; Tumor Microenvironment; Tumor Necrosis Factor-alpha; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

The current lack of cancer stem cell (CSC) markers that are easily evaluated by blood samples prevents the establishment of new therapeutic strategies in hepatocellular carcinoma (HCC). Herein, we examined whether sex determining region Y-box 9 (SOX9) represents a new CSC marker, and whether osteopontin (OPN) can be used as a surrogate marker of SOX9 in HCC. In HCC cell lines transfected with a SOX9 promoter-driven enhanced green fluorescence protein gene, FACS-isolated SOX9(+) cells were capable of self-renewal and differentiation into SOX9(-) cells, and displayed high proliferation capacity in vitro. Xenotransplantation experiments revealed that SOX9(+) cells reproduced, differentiated into SOX9(-) cells, and generated tumors at a high frequency in vivo. Moreover, SOX9(+) cells were found to be involved in epithelial-mesenchymal transition (EMT) and activation of TGFb/Smad signaling. Gain/loss of function experiments showed that SOX9 regulates Wnt/beta-catenin signaling, including cyclin D1 and OPN. Immunohistochemistry of 166 HCC surgical specimens and serum OPN measurements showed that compared to SOX9(-) patients, SOX9(+) patients had significantly poorer recurrence-free survival, stronger venous invasion, and higher serum OPN levels. In conclusion, SOX9 is a novel HCC-CSC marker regulating the Wnt/beta-catenin pathway and its downstream target, OPN. OPN is a useful surrogate marker of SOX9 in HCC.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Green Fluorescent Proteins; Humans; Liver Neoplasms; Mice, SCID; Multivariate Analysis; Neoplastic Stem Cells; Osteopontin; Phenotype; Prognosis; Smad Proteins; SOX9 Transcription Factor; Transforming Growth Factor beta; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Accumulating evidences have demonstrated that mesenchymal stem cells (MSC) could be recruited to the tumor microenvironment. Umbilical cord mesenchymal stem cells (UCMSC) were attractive vehicles for delivering therapeutic agents against cancer. Nevertheless, the safety of UCMSC in the treatment of tumors including hepatocellular carcinoma (HCC) was still undetermined.. In this study, an in vitro co-culture system was established to evaluate the effect of UCMSC on the cell growth, cancer stem cell (CSC) characteristics, drug resistance, metastasis of 3D-cultured HCC cells, and the underlying mechanism was also investigated.. It was found that after co-cultured with UCMSC, the metastatic ability of 3D-cultured HCC cells was significantly enhanced as indicated by up-regulation of matrix metalloproteinase (MMP), epithelial-mesenchymal transition (EMT)-related genes, and migration ability. However, cell growth, drug resistance and CSC-related gene expression of HCC cells were not affected by UCMSC. Moreover, EMT was reversed, MMP-2 expression was down-regulated, and migration ability of HCC cell was significantly inhibited when TGF-β receptor inhibitor SB431542 was added into the co-culture system.. Therefore, these data indicated that UCMSC could significantly enhance the tumor cell metastasis, which was due to the EMT of HCC cells induced by TGF-β.

Topics: Benzamides; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Cisplatin; Coculture Techniques; Dioxoles; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinases; Mesenchymal Stem Cells; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Microenvironment

We recently described that the anti-apoptotic AMPK-related kinase, SNARK, promotes transforming growth factor (TGF)-β signaling in hepatocellular carcinoma (HCC) cells, as a potentially new therapeutic target. Here we explored FDA-approved drugs inhibiting the enzymatic activity of SNARK, using an in vitro luminescence kinase assay system. Interestingly, the long-used anti-alcoholism drug disulfiram (DSF), also known as Antabuse, emerged as the top hit. Enzymatic kinetics analyses revealed that DSF inhibited SNARK kinase activity in a noncompetitive manner to ATP or phosphosubstrates. Comparative in vitro analyses of DSF analogs indicated the significance of the disulfide bond-based molecular integrity for the kinase inhibition. DSF suppressed SNARK-promoted TGF-β signaling and demonstrated anti-HCC effects. The chemical and enzymatic findings herein reveal novel pharmacological effects of and use for DSF and its derivatives, and could be conducive to prevention and inhibition of liver fibrosis and HCC.

Topics: Alcohol Deterrents; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Disulfiram; Drug Screening Assays, Antitumor; Enzyme Activation; Humans; Kinetics; Liver Neoplasms; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Signal Transduction; Transforming Growth Factor beta

miR122 is the prevalent miRNA in adult healthy liver and it is responsible for liver stem cell differentiation towards hepatocyte lineage. Its expression is frequently lost in hepatocellular carcinoma (HCC). We studied the effects of restoring miR122 expression in a distinctive cell line derived from human HCC-BCLC9 cells-with a solid stem-like cell profile, high tumor initiating ability and undetectable miR122 expression. We generated a stable BCLC9 cell line that expresses miR122 (BCLC9-miR122). Restitution of miR122 in BCLC9 cells, decreases cell proliferation rate and reduces significantly tumor size in vivo. BCLC9-miR122 cells down-regulate expression of MYC, KLF4, FOXM1, AKT2 and AKT3 genes and up-regulate FOXO1 and FOXO3A gene expression. In addition, miR122 transfected cells decreased AKT2 kinase activation while decreased FOXO1 and FOXO3A protein inactivation. Reduction in tumor size in BCLC9-miR122 associated with an increase in p38MAPK protein expression and activation leading to a low phospho-ERK1/2 to phospho-p38 ratio. Treatment of miR122 positive cells with an inhibitor of TGFBR1 activation, abolished tumor dormancy program and recovered cell proliferation rate through a Smad-independent TGF-β response.HCC stem-like cells can be directed towards cell differentiation and tumor dormancy by restoring miR122 expression. We demonstrate, for the first time, that dormancy program is achieved through a Smad-independent TGF-β pathway. Restablishing miR122 expression is a promising therapeutic strategy that would work concurrently reducing tumor aggressiveness and decreasing disease recurrence.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Disease Progression; Humans; Kruppel-Like Factor 4; Liver Neoplasms; Mice; MicroRNAs; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Spheroids, Cellular; Transforming Growth Factor beta

The biological role of BMP-9 signaling in liver cancer remains dubious. To explore the potential use of BMP-9 signaling for anti-cancer therapy, we used recombinant human BMP-9, which we referred to as MB109, to study the effect on growth of fifteen hepatocellular carcinoma (HCC) cell lines. MB109 effectively inhibits the proliferation of nine HCC cells in vitro. The anti-proliferative effect was found to be induced by turning on p21 signaling, which caused survivin suppression and G0/G1 cell cycle arrest. ID3 was identified to be the mediator of the MB109-induced p21 expression. Blocking the activity of p38 MAPK diminished ID3 and p21 expression, indicating that MB109 signals through a p38 MAPK/ID3/p21 pathway to arrest cell cycle progression. Moreover, prolonged MB109 treatment suppressed the expression of five prominent liver cancer stem cell (LCSC) markers, including CD44, CD90, AFP, GPC3 and ANPEP. Xenograft model confirmed the anti-tumor and LCSC-suppression capability of MB109 in vivo. Contrary to ongoing efforts of suppressing BMP-9 signaling to inhibit angiogenesis of cancer tissue, these results demonstrate an unexpected therapeutic potential of MB109 to stimulate BMP-9 signaling for anti-cancer therapies.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 2; Humans; Inhibitor of Differentiation Proteins; Liver Neoplasms; Mice; Models, Biological; Neoplasm Proteins; Neoplastic Stem Cells; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Emerging evidence suggests that small nucleolar RNAs (snoRNAs) and their host genes (SNHGs) have malfunctioning roles in the development of human cancers. We globally investigated the molecular mechanisms by which snoRNA host gene 6 (SNHG6) promotes hepatocellular carcinoma (HCC) progression using human tissues and cell lines. We found that SNHG6 is overexpressed in HCC tissues and in hepatoma cell lines and is closely associated with histologic grade, hepatitis B virus DNA, Barcelona Clinic Liver Cancer stage and portal vein tumor thrombus in patients with HCC. Knockdown of SNHG6 induced apoptosis and repressed cell cycle progression in hepatoma cell lines, whereas transgenic expression of SNHG6 in the immortalized human hepatic cell line L02 had opposite effects. Xenograft tumors grown from SNHG6-knockdown cells had smaller mean volumes than did tumors grown from control cells. SNHG6 may act as a competing endogenous RNA, effectively becoming a sink for miR-101-3p and thereby modulating the derepression of zinc finger E-box binding homeobox 1, imposing an additional level of post-transcriptional regulation. Functionally, SNHG6 promotes tumor growth and metastasis by inducing epithelial to mesenchymal transition. Further investigations showed that SNHG6 could affect HCC tumorigenesis by binding to up-frameshift protein 1 and regulating Smad7 expression.

Topics: Aged; Animals; Apoptosis; Binding, Competitive; Carcinoma, Hepatocellular; Cell Cycle; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Invasiveness; RNA Helicases; RNA Interference; RNA, Small Nucleolar; Signal Transduction; Smad7 Protein; Time Factors; Trans-Activators; Transfection; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Mesenchymal stem cells (MSCs) display differential migration ability toward different tumor-released factors. Migration of MSCs is highly important in induction of proliferation and invasiveness of hepatocellular carcinoma (HepG2) cells. In this study, the role of CXCR4/CXCL12 axis and TGF-βR signaling were evaluated in the migration of MSCs toward HepG2 cells. The MSCs were incubated with SDF-1α (CXCL12), antagonists of CXCR4, TGF-βR, and co-receptor of TGF-β, (endoglin) for 48h. Then, the migration of these cells toward HepG2 cells was analyzed using in vitro migration assay. SDF-1α at a concentration of 100nM MSCs revealed the highest migration rate toward the conditioned medium (1.62 fold compared to the migration of un-treated MSCs; p<0.05). Applying combination of the antagonists against CXCR4, TGF- βR, and co-receptor of TGF-β decreased the migration rate significantly (4.51 fold; p<001). Western blot analysis confirmed that RhoA activity is a core modulator in migration pathway. This study demonstrated that CXCR4 and TGF-βR signaling are important for migration of MSCs toward HepG2 cells. Identifying the key mediators in the migration of MSCs toward hepatocellular carcinoma cells and then development of the therapeutic inhibitors against these factors can be considered as an essential strategy in suppression of tumor progression and metastasis.

Topics: Benzamides; Benzylamines; Blotting, Western; Bone Marrow Cells; Carcinoma, Hepatocellular; Cell Movement; Chemokine CXCL12; Culture Media, Conditioned; Cyclams; Endoglin; Hep G2 Cells; Heterocyclic Compounds; Humans; Liver Neoplasms; Mesenchymal Stem Cells; Pyrazoles; Real-Time Polymerase Chain Reaction; Receptors, CXCR4; rhoA GTP-Binding Protein; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

To investigate the potential oncogene promoting recurrence of hepatocellular carcinoma (HCC) following liver transplantation (LT), throughput RNA sequencing was performed in a subgroup of HCC patients. The up-regulated FAM83D in HCC tissues was found and further verified in 150 patients by real-time PCR and immunohistochemistry. FAM83D overexpression significantly correlated with high HCC recurrence rate following LT and poor HCC characteristics such as high AFP, poor differentiation. Of cancer stem cells (CSCs) markers, CD44 expression was effectively suppressed when FAM83D was knocked down by siRNA. Meanwhile, the siRNA transfected cells suppressed formation of sphere and ability of self-renew. In a xenograft tumorigenesis model, FAM83D knockdown apparently inhibited tumor growth and metastasis. Microarray assays revealed that FAM83D promotes CD44 expression via activating the MAPK, TGF-β and Hippo signaling pathways. Furthermore, CD44 knockdown presented reverse effect on above signaling pathways, which suggested that FAM83D was a key activator of loop between CD44 and above signaling pathways. In conclusion, FAM83D promotes HCC recurrence by promoting CD44 expression and CD44+ CSCs malignancy. FAM83D provides a novel therapeutic approach against HCC recurrence after LT.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Disease Models, Animal; Gene Expression; Gene Knockdown Techniques; Heterografts; Hippo Signaling Pathway; Humans; Hyaluronan Receptors; Kaplan-Meier Estimate; Liver Neoplasms; Liver Transplantation; Mice; Microtubule-Associated Proteins; Neoplastic Stem Cells; Prognosis; Protein Serine-Threonine Kinases; Recurrence; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) has been implicated in the processes of embryogenesis, tissue fibrosis and carcinogenesis. Transforming growth factor-β (TGF-β) has been identified as a key driver of EMT and plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). The aim was to identify microRNA (miR) expression in TGF-β-induced hepatocyte EMT.. We treated a human hepatocyte cell line PH5CH8 with TGF-β to induce an EMT-like change in phenotype and then identified dysregulated miRs using TaqMan Low Density Arrays. MiR expression was altered using miR-181a mimic and inhibitor in the same system and gene changes were identified using TaqMan gene arrays. MiR-181a gene expression was measured in human and mouse cirrhotic or HCC liver tissue samples. Gene changes were identified in rAAV-miR-181a-expressing mouse livers using TaqMan gene arrays.. We identified miR-181a as a miR that was significantly up-regulated in response to TGF-β treatment. Over-expression of a miR-181a mimic induced an in vitro EMT-like change with a phenotype similar to that seen with TGF-β treatment alone and was reversed using a miR-181a inhibitor. MiR-181a was shown to be up-regulated in experimental and human cirrhotic and HCC tissue. Mouse livers expressing rAAV-miR-181a showed genetic changes associated with TGF-β signalling and EMT.. MiR-181a had a direct effect in inducing hepatocyte EMT and was able to replace TGF-β-induced effects in vitro. MiR-181a was over-expressed in cirrhosis and HCC and is likely to play a role in disease pathogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Hepatocytes; Humans; In Vitro Techniques; Liver Cirrhosis; Liver Neoplasms; Mice; MicroRNAs; Transforming Growth Factor beta

The poor prognosis of hepatocellular carcinoma (HCC) is mainly due to tumor recurrence and metastases. Recently, epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis. However, the underlying molecular mechanisms are yet to be elucidated. Here, we show that 30-kDa Tat-interacting protein (TIP30), also called CC3, is significantly downregulated during transforming growth factor-β-induced EMT. In our in vitro and in vivo studies, we show that decreased TIP30 expression leads to EMT, as well as enhanced motility and invasion of HCC cells. Also, increased self-renewal ability and chemotherapeutic resistance are observed with TIP30 depletion. Moreover, Snail is one of the key transcription factors promoting EMT, and overexpression of TIP30 greatly decreased nucleic accumulation in Snail through the regulation of intracellular localization. Small interfering RNAs targeting Snail attenuated EMT and tumor-initiating properties induced by TIP30 deficiency. We further confirmed that TIP30 competitively interrupted the interaction of Snail with importin-β2 to block the nuclear import of Snail. Consistently, TIP30 expression significantly correlates with E-cadherin expression in HCC patients. TIP30 or combination of E-cadherin is a powerful marker in predicting the prognosis of HCC. Taken together, our results suggest a novel and critical role of TIP30 involved in HCC progression and aggressiveness.

Topics: Acetyltransferases; Animals; beta Karyopherins; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Prognosis; RNA Interference; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

In hepatocellular carcinoma (HCC), intrahepatic metastasis frequently correlates with epithelial to mesenchymal transition (EMT) of malignant hepatocytes. Several mechanisms have been identified to be essentially involved in hepatocellular EMT, among them transforming growth factor (TGF)-β signaling. Here we show the up-regulation and activation of the receptor tyrosine kinase Axl in EMT-transformed hepatoma cells. Knockdown of Axl expression resulted in abrogation of invasive and transendothelial migratory abilities of mesenchymal HCC cells in vitro and Axl overexpression-induced metastatic colonization of epithelial hepatoma cells in vivo. Importantly, Axl knockdown severely impaired resistance to TGF-β-mediated growth inhibition. Analysis of the Axl interactome revealed binding of Axl to 14-3-3ζ, which is essentially required for Axl-mediated cell invasion, transendothelial migration, and resistance against TGF-β. Axl/14-3-3ζ signaling caused phosphorylation of Smad3 linker region (Smad3L) at Ser213, resulting in the up-regulation of tumor-progressive TGF-β target genes such as PAI1, MMP9, and Snail as well as augmented TGF-β1 secretion in mesenchymal HCC cells. Accordingly, high Axl expression in HCC patient samples correlated with elevated vessel invasion of HCC cells, higher risk of tumor recurrence after liver transplantation, strong phosphorylation of Smad3L, and lower survival. In addition, elevated expression of both Axl and 14-3-3ζ showed strongly reduced survival of HCC patients.. Our data suggest that Axl/14-3-3ζ signaling is central for TGF-β-mediated HCC progression and a promising target for HCC therapy.

Topics: 14-3-3 Proteins; Autocrine Communication; Axl Receptor Tyrosine Kinase; Carcinoma, Hepatocellular; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Liver Neoplasms; Male; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta

The elevation of cancer stem cells (CSCs)-like properties is involved in the initiation and progression of various human cancers. Current standard practices for treatment of cancers are less than satisfactory because of CSCs-mediated recurrence. For this reason, targeting the CSCs or the cancer cells with CSCs-like properties has become the new approach for the cancer treatments. In addition to treating leukemia, arsenic trioxide (As₂O₃) also suppresses other solid tumors. However, the roles of As₂O₃ in the regulation of CSCs-like properties remain largely uninvestigated. Here by using sphere formation assay, luciferase reporter assay, and some other molecular biology approaches, we found that As₂O₃ attenuated the CSCs-like properties in human hepatocellular carcinoma (HCC). Briefly, in HCC cells and mice xenograft models, As₂O₃ improved the expression of miR-491 by DNA-demethylation. MiR-491, which targeted the SMAD3-3'-UTR, decreased the expressions of SMAD3, and inhibited the CSCs-like properties in HCC cells. Knockdown of either miR-491 or SMAD3 attenuated the As₂O₃-induced inhibition of endogenous transforming growth factor beta signal and the CSCs-like properties. Further, in HCC patients, miR-491 is inversely correlated with the expressions of SMAD3, CD133, and the metastasis/recurrence outcome. By understanding a novel mechanism whereby As₂O₃ inhibits the CSCs-like properties in HCC, our study would help in the design of future strategies of developing As₂O₃ as a potential HCC chemopreventive agent when used alone or in combination with other current drugs.

Topics: 3' Untranslated Regions; AC133 Antigen; Animals; Antigens, CD; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplastic Stem Cells; Oxides; Peptides; RNA Interference; Signal Transduction; Smad3 Protein; Time Factors; Transfection; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Dihydromyricetin (DHM) is a flavonoid compound which possesses potent antitumor activity. In the present study, it was demonstrated that DHM significantly inhibited proliferation and induced apoptosis in mouse hepatocellular carcinoma Hepal‑6 cells. Transforming growth factor β (TGF‑β) is recognized as a major profibrogenic cytokine and is therefore a common target for drugs in the treatment of liver disease. The present study aimed to investigate whether TGF‑β was involved in DHM‑triggered cell‑viability inhibition and apoptosis induction. An MTT assay was used to evaluate the viability of Hepal‑6 cells following DHM treatment. TGF‑β signalling is mediated by Smads and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is a crucial regulator of reactive oxygen species ROS production. TGF‑β, Smad3, phosphorylated (p)‑Smad2/3 and NOX4 protein expression levels were evaluated by western blot analysis. TGF‑β and NOX4 gene expression levels were determined by quantitative polymerase chain reaction. The results indicated that DHM downregulated TGF‑β, Smad3, p‑Smad2/3 and NOX4 in a concentration‑dependent manner. A cell counting assay indicated that DHM also inhibited Hepal‑6 cell growth in a concentration‑dependent manner. TGF‑β expression was significantly decreased following DHM treatment. In conclusion, the results of the present study defined and supported a novel function for DHM, indicating that it induced cell apoptosis by downregulating ROS production via the TGF‑β/Smad3 signaling pathway in mouse hepatocellular carcinoma Hepal‑6 cells.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Flavonols; Liver Neoplasms; Mice; Models, Biological; NADPH Oxidase 4; NADPH Oxidases; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Male; RNA, Long Noncoding; Transforming Growth Factor beta

Topics: Autocrine Communication; Axl Receptor Tyrosine Kinase; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Male; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta

In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1-Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1-Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild-type mice. Lung metastases were found in 75% of Sulf1-Tg mice but not in wild-type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor-β (TGF-β)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo. This effect of SULF1 on the TGF-β/SMAD pathway is functional; overexpression of SULF1 promotes TGF-β-induced gene expression and epithelial-mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF-β from the cell surface. We also show that SULF1 expression decreases the interaction between TGF-β1 and its heparan sulfate proteoglycan sequestration receptor, TGFβR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence-free survival (hazard ratio 4.1, 95% confidence interval 1.9-8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF-β expression and with several TGF-β-related epithelial-mesenchymal transition genes in human HCC.. Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF-β pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC.

Topics: Animals; Carcinoma, Hepatocellular; Liver Neoplasms; Male; Mice; Smad Proteins; Sulfotransferases; Transcriptional Activation; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) exerts apoptotic effects on various types of malignant cells, including liver cancer cells. However, the precise mechanisms by which TGF-β induces apoptosis remain poorly known. In the present study, we have showed that threonine 32 (Thr32) residue of FoxO3 is critical for TGF-β to induce apoptosis via Bim in hepatocarcinoma Hep3B cells. Our data demonstrated that TGF-β induced FoxO3 activation through specific de-phosphorylation at Thr32. TGF-β-activated FoxO3 cooperated with Smad2/3 to mediate Bim up-regulation and apoptosis. FoxO3 (de)phosphorylation at Thr32 was regulated by casein kinase I-ε (CKI-ε). CKI inhibition by small molecule D4476 could abrogate TGF-β-induced FoxO/Smad activation, reverse Bim up-regulation, and block the sequential apoptosis. More importantly, the deregulated levels of CKI-ε and p32FoxO3 were found in human malignant liver tissues. Taken together, our findings suggest that there might be a CKI-FoxO/Smad-Bim engine in which Thr32 of FoxO3 is pivotal for TGF-β-induced apoptosis, making it a potential therapeutic target for liver cancer treatment.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carcinoma, Hepatocellular; Cell Line, Tumor; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Membrane Proteins; Proto-Oncogene Proteins; Threonine; Transforming Growth Factor beta

The NEK6 (NIMA-related kinases 6) is reported to play potential roles in tumorigenesis. Although it is suggested to function in several cellular pathways, the underlying mechanism in tumorigenesis is still largely unknown. In the present study, we discovered interaction of NEK6 with Smad4, a key member of transforming growth factor beta (TGFβ) pathway. Over-expression of NEK6 in hepatocellular carcinoma (HCC) cell lines suppresses TGFβ-mediated transcription activity in a kinase activity-dependent manner. In addition, NEK6 suppresses the cell growth arrest induced by TGFβ. Mechanically, NEK6 blocks nuclear translocation of Smad4, which is essential for TGFβ function. Moreover, we identified that NEK6 could be regulated by TGFβ and hypoxia. Our study sheds new light on the roles of NEK6 in canonical TGFβ/Smad pathway and tumorigenesis.

Topics: Carcinoma, Hepatocellular; Cell Growth Processes; Cell Hypoxia; Cell Line, Tumor; Cell Nucleus; Humans; Liver Neoplasms; NIMA-Related Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Signal Transduction; Smad4 Protein; Transcription, Genetic; Transforming Growth Factor beta

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy.

Topics: Autocrine Communication; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Enzyme Activation; Gene Knockdown Techniques; Gene Silencing; HEK293 Cells; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase 8; Mucin-1; Phosphorylation; Protein Binding; Smad3 Protein; Transcription Factor AP-1; Transforming Growth Factor beta

The aim of this study was to investigate the mitogen-activated protein kinase (MAPK) pathway, which crosstalk with TGF-β/Smad signaling via linker phosphorylation of Smad2/3 to promote hepatocarcinogenesis. After DEN-induced hepatocellular carcinoma (HCC) in rats showed increased phosphorylation of JNK1/2, p38, and ERK1/2, we next antagonized TGF-β1-induced phosphorylation of JNK1/2, p38, ERK1/2, Smad2/3 signaling in HepG2 cells using SP600125, SB203580, and PD98059, respectively. Cell proliferation and invasion were assessed by MTT assay and transwell invasion chambers, respectively. Smad2/3, Smad4, and Smad7 expressions and PAI-1 messenger RNA (mRNA) transcription were measured by using immuno-precipitation/immuno-blotting and real-time RT-PCR, respectively. All the MAPK-specific inhibitors suppressed cell invasion, while all but PD98059 suppressed cell proliferation. Both SP600125 and SB203580 blocked pSmad2C/L and oncogenic pSmad3L. PD98059 blocked pSmad2L but had no effect on elevated pSmad2C and oncogenic pSmad3L. All but PD98059 blocked Smad2/3/4 complex formation and restored Smad7 expression, while all the three MAPK-Specific inhibitors repressed PAI-1 mRNA transcription. Both SP600125 and SB203580 inhibited HepG2 cells' proliferation and invasion by blocking oncogenic pSmad3L and Smad2/3/4 complex formation. PD98059 repressed PAI-1 mRNA by an unknown mechanism.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Proliferation; Diethylnitrosamine; Flavonoids; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Plasminogen Activator Inhibitor 1; Rats; Smad Proteins; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-β role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-β inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-β effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-β-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-β actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatocytes; Humans; Liver Diseases; Liver Neoplasms; Male; Mice, 129 Strain; Mice, Knockout; Mice, Transgenic; Microscopy, Confocal; Middle Aged; Multivariate Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Smad7 Protein; Survival Analysis; Transforming Growth Factor beta; Y-Box-Binding Protein 1

MicroRNAs (miRNAs) play an important role in tumorigenesis, but their role in tumor-induced immune suppression is largely unknown. STAT3 signaling, a key pathway mediating immune suppression in the tumor microenvironment, is responsible for the transcription of several important miRNAs. In this study, we observed that miR-146a, a known important regulator of immune responses, was downregulated by blocking activated STAT3 in hepatocellular carcinoma (HCC) cells. Furthermore, miR-146a inhibition in HCC cells not only altered the STAT3 activation-associated cytokine profile but also reversed HCC-induced NK cell dysfunction in vitro and improved the anti-tumor effect of lymphocytes in vivo. Importantly, ChIP and luciferase reporter assays confirmed that STAT3 directly bound to the miR-146a promoter and induced miR-146a expression. These findings indicated that miR-146a expression was regulated by aberrantly activated STAT3 in HCC cells and exerted negative effects on anti-tumor immune response, which resulted in the upregulation of cytokines such as TGF-β, IL-17, VEGF and downregulation of type I IFN to create an immunosuppressive microenvironment. This further insight into understanding the mechanism responsible for tumor-induced immune suppression highlights the potential application of miR-146a as a novel immunotherapeutic target for HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Hep G2 Cells; Humans; Interferon Type I; Interleukin-17; Liver Neoplasms; Lymphocytes; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Oligonucleotides, Antisense; STAT3 Transcription Factor; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Tumour-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Tim-3 is highly expressed on macrophages and regulates macrophage functions in several conditions. However, whether Tim-3 is involved in the activation and the function of TAMs has not been reported.. Tim-3 expression in HCC samples was evaluated by flow cytometry, immunohistochemistry and confocal analysis. We analysed the effects of Tim-3 knockdown on macrophages in growth of H22 tumour homografts in BALB/c mice. Tim-3 interference was performed by neutralising antibody, small interfering RNA or short hairpin RNA-expressing lentivirus. Cytokine production was evaluated by reverse transcription PCR, ELISA or Cytometric Bead Array. The effects of Tim-3 interference in macrophages were examined with regard to alternative activation of macrophages and proliferation and migration of Hepa1-6 cells. Cell growth curve, colony formation and transwell assays were involved to estimate cell proliferation and migration.. Tim-3 expression was significantly increased in both peripheral blood monocytes and TAMs in patients with HCC. The Tim-3 expression in monocytes/TAMs strongly correlated with higher tumour grades and the poor survival of patients with HCC. Consistently, HCC conditioned medium or transforming growth factor-β fostered Tim-3 expression and the alternative activation of macrophages. Moreover, Tim-3 interference in macrophages significantly inhibited the alternative activation of macrophages and suppressed HCC cell growth both in vitro and in vivo. Blocking interleukin 6 reversed the Tim-3-mediated effects on HCC cell growth in vitro.. Tim-3 displays critical roles in microenvironment-induced activation and protumoral effects of TAMs in HCC. Interference of Tim-3 might be great potential in HCC therapy.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Hepatitis A Virus Cellular Receptor 2; Humans; Immunohistochemistry; Liver Neoplasms, Experimental; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Receptors, Virus; RNA, Neoplasm; Transforming Growth Factor beta

Previously isolated pathways screened from individual genes were investigated at either the transcriptional or translational level; however, the consistency between the pathways screened at the gene expression levels was obscure in metastatic human hepatocellular carcinoma (HCC). To elucidate this question, we performed a transcriptomic (16,353 genes) and proteomic (7861 proteins) analysis simultaneously on six metastatic HCC cell lines against two nonmetastatic HCC cell lines, with all HBV traceable and close genetic-backgrounds for a comparative study. The quantitative and integrated results showed that significant genes were screened differentially with 351 transcripts from the transcriptome and 304 proteins from the proteome, with limited overlapping genes (7%). However, we discovered that these discrete 351 transcripts and 304 proteins screened share extrusive significant-pathways/networks with a 77% overlap, including active TGF-β, RAS, NFκB, and Wnt, and inactive HNF4A, which are responsible for HCC metastasis. We conclude that the discrete, but significant genes predicted by either ome play intrinsically important roles in the linkage of responsible pathways shared by both omes in HCC metastasis.

Topics: Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Liver Neoplasms; Proteome; ras Proteins; Transforming Growth Factor beta; Wnt Signaling Pathway

Topics: Animals; Carcinoma, Hepatocellular; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms, Experimental; Macrophage Activation; Male; Receptors, Virus; RNA, Neoplasm; Transforming Growth Factor beta

Keratin 19 (K19) is a known marker of poor prognosis and invasion in human hepatocellular carcinoma (HCC). However, the relationship between K19 and cancer stem cells (CSCs) is unclear. Here, we determined whether K19 can be used as a new CSC marker and therapeutic target in HCC.. HCC cell lines were transfected with a K19 promoter-driven enhanced green fluorescence protein gene. CSC characteristics, epithelial-mesenchymal transition (EMT), and TGFb/Smad signaling were examined in FACS-isolated K19(+)/K19(-) cells. K19 and TGFb receptor 1 (TGFbR1) expression in 166 consecutive human HCC surgical specimens was examined immunohistochemically.. FACS-isolated single K19(+) cells showed self-renewal and differentiation into K19(-) cells, whereas single K19(-) cells did not produce K19(+) cells. K19(+) cells displayed high proliferation capacity and 5-fluorouracil resistance in vitro. Xenotransplantation into immunodeficient mice revealed that K19(+) cells reproduced, differentiated into K19(-) cells, and generated large tumors at a high frequency in vivo. K19(+) cells were found to be involved in EMT and the activation of TGFb/Smad signaling, and these properties were suppressed by K19 knockdown or treatment with a TGFbR1 inhibitor. The TGFbR1 inhibitor also showed high therapeutic effect against K19(+) tumor in the mouse xenograft model. Immunohistochemistry of HCC specimens showed that compared with K19(-) patients, K19(+) patients had significantly poorer recurrence-free survival and higher tumor TGFbR1 expression.. K19 is a new CSC marker associated with EMT and TGFb/Smad signaling, and it would thus be a good therapeutic target for TGFbR1 inhibition.

Topics: Animals; Antimetabolites, Antineoplastic; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Fluorouracil; Humans; Keratin-19; Liver Neoplasms; Male; Mice, Inbred NOD; Mice, SCID; Multivariate Analysis; Neoplastic Stem Cells; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

TGF-β plays a key role in the progression of various tumors. The main objective of our study was to investigate whether TGF-β is able to regulate N-nitrosodiethylamine (DEN)-induced hepatocellular carcinoma (HCC) progression in a mouse model by inducing Treg cell polarization.. HCC progression, TGF-β and Foxp3 expression levels, serum TGF-β, IL10 and GP73 levels as well as percentage of Treg cells were analyzed in healthy, HCC and HCC+SM-16 mouse groups. The effect of TGF-β on Treg cell polarization in vitro was measured by flow cytometric analysis. The expression of TGF-β and IL10 was identified by IHC in HCC patients and the correlation between TGF-β and IL10 was also assessed.. TGF-β expression is up-regulated in a DEN-induced HCC mouse model. TGF-β can promote the differentiation of Foxp3(+)CD4(+) T cells (Treg cells) in vitro. However, blocking the TGF-β pathway with a specific TGF-β receptor inhibitor, SM-16, reduced HCC progression and the percentage of Treg cells in liver tissue. The correlation between TGF-β and Treg cells was also confirmed in HCC patients and the expression of both TGF-β and IL-10 was shown to be associated with HCC progression.. TGF-β is necessary for HCC progression, acting by inducing Treg cell polarization.

Topics: Animals; Azabicyclo Compounds; Carcinoma, Hepatocellular; Cell Differentiation; Cell Polarity; Cell Proliferation; Diethylnitrosamine; Disease Models, Animal; Disease Progression; Enzyme-Linked Immunosorbent Assay; Forkhead Transcription Factors; Interleukin-10; Liver Neoplasms; Male; Membrane Proteins; Mice; Phosphoproteins; Receptors, Transforming Growth Factor beta; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

Hepatocellular carcinoma is one of the most aggressive cancers with poor prognosis worldwide. Tumor progression remains a significant cause of high mortality in patients with hepatocellular carcinoma. However, the molecular mechanism underlying tumor progression of hepatocellular carcinoma has not been completely unraveled currently. The aim of this study was to gain insight into the molecular mechanisms of tumor progression of hepatocellular carcinoma.. We performed microarray analysis on 24 tissue specimens obtained at the time of surgical resection or liver transplantation from 24 patients with hepatocellular carcinoma downloaded from the Gene Expression Omnibus database.. Our analysis indicated that several differentially expressed genes might play crucial roles in the progression of hepatocellular carcinoma, such as GADD45G, SPTBN1, CDC27, TPD52 and INSIG1. GADD45G and SPTBN1 not only contribute to tumor progression in hepatocellular carcinoma, but also correlate with poor prognosis in esophageal squamous cell carcinoma and pancreatic cancer respectively. Futhermore, we performed pathway enrichment analysis and found enriched pathways, including "Proteasome", "Alanine, aspartate and glutamate metabolism", "TGF-beta signaling pathway", "Wnt signaling pathway", and so on.. Our findings confirmed the presence of multiple molecular alterations during tumor progression and indicated the differentially expressed genes might be involved in tumor progression though multiple pathways. Genes GADD45G and SPTBN1 might correlate with poor prognosis in hepatocellular carcinoma as has already been shown for other malignancies of the gastrointestinal tract.

Topics: Carcinoma, Hepatocellular; Cluster Analysis; Databases, Genetic; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Protein Array Analysis; Signal Transduction; Transforming Growth Factor beta

EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high-density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH-1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1-EVI1 or MDS1, was overexpressed in JHH-1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des-γ-carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15(INK) (4B) by transforming growth factor (TGF)-β and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-β-treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-β-mediated growth inhibition of HCC cells.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromosomes, Human, Pair 3; DNA Copy Number Variations; DNA-Binding Proteins; Female; Gene Amplification; Gene Expression; Humans; Liver Neoplasms; Male; MDS1 and EVI1 Complex Locus Protein; Middle Aged; Proto-Oncogenes; Transcription Factors; Transforming Growth Factor beta

Ski and SnoN proteins function as transcriptional co-repressors in the TGF-β pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-β signalling, malignant transformation, and alterations in cell proliferation.. We carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-β, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-β/Smad-driven gene transcription.. Ski and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-β-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-β-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-β-target genes.. Normal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells.. This work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.

Topics: Actins; Animals; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cytoskeleton; DNA-Binding Proteins; Hepatocytes; Intracellular Signaling Peptides and Proteins; Polymerization; Protein Stability; Proto-Oncogene Proteins; Rats; Smad Proteins; Transforming Growth Factor beta

The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process.. The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series.. We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.. Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

Topics: Actins; Adult; Aged; Antigens, Neoplasm; Biomarkers; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Cohort Studies; Epithelial Cell Adhesion Molecule; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Keratin-19; Liver Neoplasms; Male; Microscopy, Fluorescence; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Odds Ratio; Prognosis; Recombinant Proteins; Stem Cells; Transforming Growth Factor beta

To investigate biological mechanisms underlying pyruvate kinase M2 isoform (PKM2) regulation of cell migration and invasion in hepatocellular carcinoma cells.. HepG2 and Huh-7 hepatocellular carcinoma cell lines were stably transfected and cultured in DMEM (HyClone, Logan, UT, United States). To investigate the effects of PKM2 on cellular proliferation, hepatocellular carcinoma cells were subjected to the Cell Counting Kit-8 (Dojindo, Kamimashiki-gun, Kumamoto, Japan). And investigate the effects of PKM2 on cell signal pathway related with migration and invasion, Western immunoblotting were used to find out the differential proteins. All the antibody used was purchaseed from Cell Signal Technology. In order to explore cell motility used Transwell invasion and wound healing assays. The transwell plate with 0.5 mg/mL collagen type I (BD Bioscience, San Jose, CA)-coated filters. The wound-healing assay was performed in 6-well plates. Total RNA was extracted using TRIzol reagent (Invitrogen, CA, United States) and then reverse transcription was conducted. Quantitative reverse transcription-polymerase chain reaction (PCR) analysis was performed with the ABI 7500 real-time PCR system (Applied Biosystems). We further use digital gene expression tag profiling and identification of differentially expressed genes.. The cells seeded in four 96-well plates were measured OD450 by conducted Cell Counting Kit-8. From this conduction we observed that both HepG2 and Huh-7 hepatocellular carcinoma cells with silenced PKM2 turn on a proliferate inhibition; however, cell migration and invasion were enhanced compared with the control upon stimulation with epidermal growth factor (EGF). Our results indicate that the knockdown of PKM2 decreased the expression of E-cadherin and enhanced the activity of the EGF/EGFR signaling pathway, furthermore up-regulate the subsequent signal molecular the PLCγ1 and extracellular signal-regulated kinase 1/2 expression in the hepatocellular carcinoma cell lines HepG2 and Huh-7, which regulates cell motility. These variations we observed were due to the activation of the transforming growth factor beta (TGFβ) signaling pathway after PKM2 knockdown. We also found that the expression of TGFBRI was increased and the phosphorylation of Smad2 was enhanced. Taken together, our findings demonstrate that PKM2 can regulate cell motility through the EGF/EGFR and TGFβ/TGFR signaling pathways in hepatocellular carcinoma cells.. PKM2 play different roles in modulating the proliferation and metastasis of hepatocellular carcinoma cells, and this finding could help to guide the future targeted therapies.

Topics: Carcinoma, Hepatocellular; Carrier Proteins; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Proteins; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Time Factors; Transfection; Transforming Growth Factor beta

Mitochondrial dysfunction has been found to be associated with various pathological conditions, particularly cancer. However, the mechanisms underlying tumor malignancy induced by mitochondrial dysfunction are not fully understood. In the present study, the effects of mitochondrial dysfunction on epithelial-mesenchymal transition (EMT), were investigated using mitochondrial-depleted ρ(0) cells derived from the Hep3B hepatocarcinoma cell line. The Hep3B/ρ(0) cells displayed the EMT phenotype with more aggressive migration and higher invasiveness compared to their parental cells. The Hep3B/ρ(0) cells also showed typical expression pattern of EMT markers such as vimentin and E-cadherin. These phenotypes in Hep3B/ρ(0) cells were mediated by increased transforming growth factor-β (TGF-β) through the canonical Smad-dependent signaling pathway. Additionally, TGF-β signaling was activated via induction of c-Jun/AP-1 expression and activity. Therefore, mitochondrial dysfunction induces EMT through TGF-β/Smad/Snail signaling via c-Jun/AP-1 activation. These results indicate that mitochondrial dysfunction plays an important role in the EMT process and could be a novel therapeutic target for malignant cancer therapy.

Topics: Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Mitochondria; Signal Transduction; Smad Proteins; Snail Family Transcription Factors; Transcription Factor AP-1; Transcription Factors; Transforming Growth Factor beta

Accumulating evidence has revealed that fucoidan exhibits anti-tumor activities by arresting cell cycle and inducing apoptosis in many types of cancer cells including hepatocellular carcinoma (HCC). Exploring its effect on microRNA expression, we found that fucoidan markedly upregulated miR-29b of human HCC cells. The induction of miR-29b was accompanied with suppression of its downstream target DNMT3B in a dose-dependent manner. The reduction of luciferase activity of DNMT3B 3'-UTR reporter by fucoidan was as markedly as that by miR-29b mimic, indicating that fucoidan induced miR-29b to suppress DNMT3B. Accordingly, the mRNA and protein levels of MTSS1 (metastasis suppressor 1), a target silenced by DNMT3B, were increased after fucoidan treatment. Furthermore, fucoidan also down-regulated TGF-β receptor and Smad signaling of HCC cells. All these effects leaded to the inhibition of EMT (increased E-cadherin and decreased N-cadherin) and prevention of extracellular matrix degradation (increased TIMP-1 and decreased MMP2, 9), by which the invasion activity of HCC cells was diminished. Our results demonstrate the profound effect of fucoidan not only on the regulation of miR-29b-DNMT3B-MTSS1 axis but also on the inhibition of TGF-β signaling in HCC cells, suggesting the potential of using fucoidan as integrative therapeutics against invasion and metastasis of HCC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3B; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Microfilament Proteins; MicroRNAs; Neoplasm Proteins; Polysaccharides; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Hepatocellular carcinoma (HCC) is the second-leading cause of cancer-related death worldwide, and the factors influencing HCC progression are poorly understood. Here we reveal that HCC progression via induction of epithelial-mesenchymal transition (EMT) is closely associated with the expression of CD36/fatty acid translocase and elevated free fatty acid (FFA) levels. Although obesity is manifested as elevated FFA levels, the degree of EMT was not associated with the body mass index of the patients, highlighting the specific roles of CD36 and FFA uptake. Treatment of human liver cancer cell lines with FFAs exacerbated the EMT phenotype, whereas chemical inhibition of CD36 mitigated these effects. Furthermore, the Wnt and TGF-β signaling pathways were activated upon FFA treatment, potentially acting as upstream activators of the EMT program. These results provide the first direct evidence associating CD36 and elevated FFAs with HCC progression.

Topics: Biomarkers; Carcinoma, Hepatocellular; CD36 Antigens; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Fatty Acids, Nonesterified; Gene Expression; Humans; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Transforming growth factor-β (TGF-β) signaling plays a key role in progression and metastasis of HCC. This study was undertaken to gain the proof of concept of a small-molecule inhibitor of TGF-β type I receptor kinase, EW-7197 as a potent anti-cancer therapy for HCC. We identified tissue inhibitors of metalloproteinases-1 (TIMP-1) as one of the secreted proteins of hepatic stellate cells (HSCs) and a key mediator of TGF-β-mediated crosstalk between HSCs and HCC cells. TGF-β signaling led to increased expression of TIMP-1, which activates focal adhesion kinase (FAK) signaling via its interaction with CD63. Inhibition of TGF-β signaling using EW-7197 significantly attenuated the progression and intrahepatic metastasis of HCC in an SK-HEP1-Luc orthotopic-xenograft mouse model. In addition, EW-7197 inhibited TGF-β-stimulated TIMP-1 secretion by HSCs as well as the TIMP-1-induced proliferation, motility, and survival of HCC cells. Further, EW-7197 interrupted TGF-β-mediated epithelial-to-mesenchymal transition and Akt signaling, leading to significant reductions in the motility and anchorage-independent growth of HCC cells. In conclusion, we found that TIMP-1 mediates TGF-β-regulated crosstalk between HSCs and HCC cells via FAK signaling. In addition, EW-7197 demonstrates potent in vivo anti-cancer therapeutic activity and may be a potential new anti-cancer drug of choice to treat patients with liver cancer.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Communication; Cell Line, Transformed; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Disease Progression; Epithelial-Mesenchymal Transition; Female; Focal Adhesion Protein-Tyrosine Kinases; Hepatic Stellate Cells; Heterografts; Humans; Liver Neoplasms; Mice; Proto-Oncogene Proteins c-akt; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Triazoles

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-β (TGF-β) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-β. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-β/Smad signaling.. NRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-β signaling were analyzed by Western blotting and confocal immunofluorescence microscopy.. We show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-β/Smad signaling while NRP2 expression has no impact on TGF-β signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-β signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-β signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens.. These data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-β-dependent HCC progression.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Movement; Humans; Liver Neoplasms; Neuropilin-2; Phenotype; Signal Transduction; Sulfhydryl Reagents; Tissue Array Analysis; Transforming Growth Factor beta; Tumor Cells, Cultured

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a poor patient survival. Expression of TGF-β1 is up-regulated in HCC and is thought to play a crucial role in the occurrence and development of HCC. However, the mechanism of TGF-β1-mediated facilitation of malignant growth and invasion remains unclear, although some previous studies highlighted a potential involvement of the connective tissue growth factor (CTGF). Here we demonstrate that the in vitro migration of the HCC cell line SMMC-7721 is increased in the presence of recombinant TGF-β1, and that this effect is reversed by the specific inhibitor SB431542. Furthermore, TGF-β1 treatment up-regulated the expression of its own mRNA as well as the expression of CTGF mRNA. The TGF-β1-stimulated migration of SMMC-7721 cells was diminished by siRNA silencing of CTGF. These in vitro observations were validated in a murine xenograft model. In particular, silencing of CTFG diminished the TGF-β1-induced tumorigenesis in experimental animals. In conclusion, TGF-β1 plays a critical role in HCC migration and invasion, and this effect is dependent on CTGF.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Connective Tissue Growth Factor; Humans; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Transforming Growth Factor beta; Up-Regulation

RFA (radiofrequency ablation) is an established therapy for HCC (hepatocellular carcinoma). The multikinase inhibitor sorafenib prolongs survival in advanced HCC. We examined the effects of RFA alone and in combination with sorafenib on a bystanding tumour in a two-tumour rat model of HCC. A total of 80 rats were implanted with two liver tumours and randomized to four treatment groups: vehicle and sham operation (control), sorafenib and sham operation (Sora/Sham), vehicle and RFA (Vh/RFA), and sorafenib and RFA (Sora/RFA) (n=10/group per time point). RFA or sham-operation was performed on the left lobe tumour on day 15. Animals were killed at day 18 and day 30. Non-RFA-targeted right lobe tumours were analysed for angiogenesis, growth factors [HGF (hepatocyte growth factor), EGF (epidermal growth factor) and VEGF (vascular endothelial growth factor)] and infiltrating immune cells (CD3 and CD68). At day 30, the non-RFA-targeted tumours were significantly smaller in all three treatment groups compared with control (Sora/Sham P≤0.0001, Vh/RFA P=0.005 and Sora/RFA P≤0.0001). The smallest tumours were observed in animals treated with a combination of sorafenib and RFA, whereas the size reduction seen in the RFA-only group indicated an RFA-mediated distant suppression of tumour growth. Growth factor measurement revealed transiently decreased EGF levels after RFA (P=0.008), whereas sorafenib treatment decreased HGF levels (P=0.001). MVD (microvessel density) was reduced by sorafenib (P=0.002) despite increased VEGF levels (P≤0.0001). The immune parameters revealed augmented T-cells and IL-10 (interleukin 10) levels in all three treatment groups; sorafenib additionally increased macrophage numbers (P≤0.0001). RFA and sorafenib alone resulted in significant volume reduction of the non-RFA-targeted tumour; this effect was enhanced when both modalities were combined.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Catheter Ablation; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Hepatocyte Growth Factor; Interleukin-10; Liver Neoplasms, Experimental; Macrophages; Niacinamide; Phenylurea Compounds; Rats; Sorafenib; T-Lymphocytes; Transforming Growth Factor beta; Vascular Endothelial Growth Factors

Previous studies showed Compound Astragalus and Salvia miltiorrhiza extract (CASE), extract from Astragalus membranaceus and Salvia miltiorhiza, significantly suppresses hepatocellular carcinoma (HCC) in rats induced by diethylinitrosamine (DEN), and in vitro experiments further demonstrated that CASE's anti-HepG2 cell invasion is associated with transforming growth factor-β (TGF-β). We hypothesized that CASE's suppression of HCC is modulated by TGF-β/Smad signaling, and we conducted this in vivo study to test this hypothesis.. Rats were divided into the normal control, the DEN group, and three CASE (60, 120, and 240 mg/kg) treatment groups. The expression of phosphorylation(p) Smad both at C-terminal and linker region, plasminogen activator inhibitor 1, and Smad4 and Smad7 of liver tissues were measured and compared across the five groups.. The positive staining of pSmad2L and pSmad3L increased both in hepatoma nodule areas and adjacent relatively normal liver tissues in rats treated with DEN, while the positive staining of pSmad2C and pSmad3C increased only in relatively normal liver tissues adjacent to hepatoma tissues. The elevated expression of pSmad2C, pSmad2L, pSmad3L, Smad4, and plasminogen activator inhibitor 1 proteins were suppressed by CASE in a dose-dependent manner. CASE treatment also significantly reduced the intranuclear amounts of pSmad2L and pSmad3L, and upregulated the elevation of pSmad3C positive cells and protein expression in a dose-dependent manner.. The results suggest that CASE significantly suppresses HCC progression by mediating TGF-β/Smad signaling, especially by modulating Smad3 phosphorylation both at the C-terminal and linker region.

Topics: Animals; Astragalus Plant; Carcinoma, Hepatocellular; Diethylnitrosamine; Dose-Response Relationship, Drug; Gene Expression; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Male; Neoplasm Invasiveness; Phosphorylation; Plant Extracts; Plasminogen Activator Inhibitor 1; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

The two isoforms of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), 1 with a long cytoplasmic domain (CEACAM1-L) and 1 with a short (CEACAM1-S), are involved in different signaling pathways. β2-spectrin (β2SP) is an adaptor protein that plays critical roles in the proper control of Smad access to activate receptors involved in regulation of TGF-β signaling. In this study, we examined the association between CEACAM1 isoform balance and hepatocellular carcinoma (HCC) malignant potential and investigated the possibility of a molecular interaction between CEACAM1 and β2SP.. Immunohistochemical analysis was carried out with CEACAM1-L or CEACAM1-S antibodies on 154 HCC tissues to correlate with the factors of malignancy. Invasion assay was performed for the effect of CEACAM1 expression on HCC cell lines. Moreover, immunohistochemical analysis and immunoprecipitation analysis were performed to investigate the association between CEACAM1 isoform balance and β2SP.. In immunohistochemical analysis, CEACAM1-L expression dominance was a risk factor for HCC recurrence (p = 0.04) and was significantly associated with a shorter survival compared with CEACAM1-S expression dominance. Invasion assay indicated that CEACAM1-4L-transfected HLF and PLC/PRF/5 cells showed significantly increased invasion (p < 0.0001) and CEACAM1-4S-transfected HLF cells showed significantly decreased invasion. Immunohistochemical analysis of β2SP suggested that the HCCs with CEACAM1-L-dominant expression were more strongly stained with β2SP than the HCCs with CEACAM1-S-dominant expression (p = 0.013), and coprecipitation assays indicated that CEACAM1-L could bind to β2SP.. CEACAM1-L may enhance the HCC invasiveness through an interaction with β2SP and subsequent effects on TGF-β signaling.

Topics: Aged; Antigens, CD; Apoptosis; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Line, Tumor; Cell Proliferation; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Protein Isoforms; Retrospective Studies; Signal Transduction; Smad3 Protein; Spectrin; Survival Rate; Transfection; Transforming Growth Factor beta

Interpatient heterogeneity of hepatocellular carcinoma has been in-depth addressed. Intrapatient heterogeneity is less known. Four clones were freshly isolated from an Edmondson grade I HCV-associated hepatocellular carcinoma. Biochemical approaches, functional assays and cytogenetics were used. Albumin inducibility was uncoupled from canonical cytokeratin profiles, suggesting pathological combinations of hepatospecific and biliary markers. Poor differentiation and TGFβ's proproliferative effect on all clones were observed. TGFβ, Interferon α and doxorubicin sensitivity levels were found highly heterogeneous. Progenitor and stem cells markers OV6 and EpCAM were mutually exclusively expressed. All clones were CD44+, while none expressed CD90, CD133, or CD117. Three clones displayed a liver progenitor OV6+ phenotype, and were susceptible to hepatocytic differentiation, among which one fibroblastoid clone displayed intrahepatic parenchymal engraftment capability. A fourth clone, the less motile, displayed a cancer stem cell EpCAM+ phenotype, was essentially β-catenin negative, and was as expected devoid of hepatocytic differentiation capability, yet the most sensitive to doxorubicin treatment. Cytogenetics evidenced in all clones a t(12;22)(p11;q11) translocation found in several myelodysplastic syndromes. All clones, that probably derive from EpCAM+ tumor cells, display aberrant E-cadherin cytosolic localization. Because of their diverse pathophysiolocal features, these freshly isolated, low population doubling-defined, HCC clones may provide novel opportunities to tackle HCC heterogeneity in a single patient background for therapy improvement purposes, especially regarding recently developed targeted strategies.

Topics: Aged; Animals; Antigens, Neoplasm; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinogenicity Tests; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Differentiation; Cell Proliferation; Clone Cells; Epithelial Cell Adhesion Molecule; Female; Hematologic Neoplasms; Humans; Hyaluronan Receptors; Karyotyping; Liver Neoplasms; Mice, Nude; Stem Cells; Transforming Growth Factor beta; Translocation, Genetic; Tumor Cells, Cultured

The effects of TGF-β on dendritic cells (DCs) in the tumor microenvironment are not well-understood. In this study, we investigated the effect of TGF-β on the induction of programmed death ligand-1 (PD-L1) expression in DCs and the underlying mechanism, and we further investigated the influence of the DCs with PD-L1 expression altered by TGF-β on T-cell immunity. We determined that TGF-β increased the expression of PD-L1 and signal transducers and activators of transcription 3 (STAT3) in DCs in both a time- and dose-dependent manner, and the expression of PD-L1 was decreased significantly after STAT3 blockade. In addition, TGF-β-treated DCs induced the apoptosis of T cells and increased the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Furthermore, the cytotoxicity of T cells against mice hepatocellular carcinoma cells (Hepa) was obviously suppressed. These results suggest that PD-L1 may play an important role in TGF-β-induced immune dysfunction, which finally results in a failure in the anti-tumor responses, and the TGF-β-STAT3-PD-L1 signaling pathway may contribute to novel therapeutic targets for the tumor based on DCs.

Topics: Animals; B7-H1 Antigen; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Clonal Anergy; Coculture Techniques; Dendritic Cells; Liver Neoplasms; Male; Mice, Inbred BALB C; STAT3 Transcription Factor; T-Lymphocytes; Transforming Growth Factor beta

The nonsense mutations of the hepatitis B virus (HBV) surface (S) gene have been reported to have oncogenic potential. We have previously identified several transforming nonsense mutations of the HBV S gene from hepatocarcinoma (HCC) patients. Among them, the sW182* mutant (the stop codon for tryptophan 182) showed the most potent oncogenicity in a mouse xenograft model using stably transfected mouse fibroblast cells. This study is aimed at understanding the molecular mechanisms leading to the oncogenic activity of the sW182* mutant. A gene expression microarray in combination with gene set enrichment analysis (GSEA) revealed differentially expressed gene sets in the sW182* cells, including those related to cell-cycle regulation, deoxyribonucleic acid repair, and genome instability. Of the differentially expressed genes, the transforming growth factor-β-induced (TGFBI) gene was further validated to be dysregulated in the sW182* cells. This dysregulation was accompanied by hypermethylation of the TGFBI promoter. The level of cyclin D1, a negatively regulated TGFBI target, was highly elevated in the sW182* mutant cells, which is consistent with the potent oncogenicity. Furthermore, frequent abnormal mitosis and multinucleation were observed in the mutant cells. Exogenous expression of TGFBI alleviated the oncogenic activity of the sW182* cells. In human HBV-related HCC cancerous tissue, expression of TGFBI was downregulated in 25 of the 55 (45%) patients examined, suggesting that TGFBI dysregulation could occur in HBV-related HCC development in some cases. These results suggest that dysregulation of TGFBI is involved in the oncogenic activity of the sW182* mutant of the hepatitis B virus S gene.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line; Codon, Nonsense; Cyclin D1; DNA Methylation; DNA Repair; Down-Regulation; Extracellular Matrix Proteins; Gene Expression; Genomic Instability; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Liver Neoplasms; Mice; Mice, Nude; NIH 3T3 Cells; Promoter Regions, Genetic; Transforming Growth Factor beta

Mucin1 (MUC1) is a transmembrane glycoprotein that plays a key role as an oncogene in the tumorigenesis of many human adenocarcinomas. However, the role of MUC1 in human hepatocellular carcinoma (HCC) progression remains unclear. In the present study, we silenced MUC1 to investigate its effect on the human HCC cell line SMMC-7721 and found that knockdown of MUC1 significantly inhibited cell proliferation, enhanced cell-cell aggregation and induced apoptosis. No significant differences were found in in vitro migration or invasion. We also observed that knockdown of MUC1 decreased the translocation of β‑catenin to the nucleus, reduced the activity of T cell factor and blocked the expression of cyclin D1 and c-Myc. In addition, MUC1 knockdown enhanced the expression of E-cadherin, a molecular chaperone of β‑catenin that plays an important role in cell-cell aggregation. In vivo assays demonstrated that there was no tumor growth in mice injected with MUC1-silenced cells. Global gene expression analysis showed that a series of genes encoding molecules in the Wnt/β‑catenin, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), insulin, transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF) signaling pathways were all influenced by the knockdown of MUC1, and these may contribute to the phenotypic alterations observed. Collectively, our results indicate that MUC1 plays a key role in HCC tumorigenesis.

Topics: Animals; Apoptosis; beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Gene Knockdown Techniques; Humans; Liver Neoplasms; Mice; Mitogen-Activated Protein Kinases; Mucin-1; NF-kappa B; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL-11 mRNA, autocrine induction of IL-11, and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for antimetastatic therapies.

Topics: Animals; Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Homeodomain Proteins; Humans; Interleukin-11; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Prognosis; Repressor Proteins; RNA, Long Noncoding; RNA, Messenger; STAT3 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Up-Regulation; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

The transition from liver fibrosis to hepatocellular carcinoma (HCC) has been suggested to be a continuous and developmental pathological process. MicroRNAs (miRNAs) are recently discovered molecules that regulate the expression of genes involved in liver disease. Many reports demonstrate that miR-483-5p and miR-483-3p, which originate from miR-483, are up-regulated in HCC, and their oncogenic targets have been identified. However, recent studies have suggested that miR-483-5p/3p is partially down-regulated in HCC samples and is down-regulated in rat liver fibrosis. Therefore, the aberrant expression and function of miR-483 in liver fibrosis remains elusive. In this study, we demonstrate that overexpression of miR-483 in vivo inhibits mouse liver fibrosis induced by CCl4 . We demonstrate that miR-483-5p/3p acts together to target two pro-fibrosis factors, platelet-derived growth factor-β and tissue inhibitor of metalloproteinase 2, which suppress the activation of hepatic stellate cells (HSC) LX-2. Our work identifies the pathway that regulates liver fibrosis by inhibiting the activation of HSCs.

Topics: Animals; Carbon Tetrachloride; Carcinoma, Hepatocellular; Cells, Cultured; Coculture Techniques; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Liver Neoplasms; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Platelet-Derived Growth Factor; Rats; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of cancer stem cells (CSCs)-mediated post-surgical recurrence. For this reason, targeting the CSCs or the cancer cells with CSCs-like properties has become a new approach for the treatment of HCC. GLA exhibits anti-tumor effects in that it attenuates the proliferation, migration, invasion, and angiogenesis of human cancer cells. However, the functions of GLA in the regulation of CSCs-like properties in HCC cells, and the molecular mechanisms underlying in remain obscure. Here we found that GLA attenuated the CSCs-like properties by the microRNA-148a (miR-148a)-mediated inhibition of transforming growth factor beta (TGF-β)/SMAD2 signal pathway in HCC cell lines (HepG2, Huh-7, and MHCC97H). Indeed, GLA inhibited the activations/expressions of both TGFβ-induced and the endogenous SMAD2. Further, GLA improved the expression of miR-148a in a dose/time-dependent manner. MiR-148a, which targeted the SMAD2-3'UTR, decreased the expression and function of SMAD2. Knockdown of miR-148a abolished the GLA-induced inhibition of TGF-β/SMAD2 signal pathway and the CSCs-like properties in HCC cells. Our study found a novel mechanism that GLA inhibits the CSCs-like properties of HCC cells by miR-148a-mediated inhibition of TGF-β/SMAD2 signal pathway, which may help to identify potential targets for the therapies of HCC.

Topics: Antineoplastic Agents; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Liver Neoplasms; MicroRNAs; Neoplastic Stem Cells; Phenols; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Sorafenib is a multi-kinase inhibitor approved for hepatocellular carcinoma, but rarely causes tumor regression in patients with chronic liver diseases. To investigate whether growth factor-mediated signaling is involved in sorafenib resistance, HepG2 and PLC/PRF/5 hepatoma cells were exposed to epidermal growth factor (EGF), hepatocyte growth factor (HGF) or transforming growth factor-β (TGF-β) prior to treatment with sorafenib. Furthermore, to identify an effective combination treatment with sorafenib, growth factor-sensitized cells were treated with sorafenib alone or in combination with celecoxib, lovastatin or valproic acid (VPA). Trypan blue staining and Annexin V assays showed that the cytotoxic effect of sorafenib was inhibited by 15-54% in cells sensitized to TGF-β (P<0.05). Western blotting analysis showed that TGF-β significantly activated extracellular signal-regulated kinase (ERK)-mediated AKT signaling, and sorafenib failed to suppress both ERK and AKT in TGF-β-sensitized cells. The decreased anti-tumor effect of sorafenib was rescued by chemical inhibition of ERK and AKT. When TGF-β-sensitized cells were treated with sorafenib plus VPA, the levels of phosphorylated ERK and AKT were considerably suppressed and the numbers of dead cells were increased by 3.7-5.7-fold compared with those exposed to sorafenib alone (P<0.05). Moreover, low dose sorafenib-induced cell migration was effectively suppressed by combination treatment with sorafenib and VPA. Collectively, TGF-β/ERK/AKT signaling might play a critical role in sorafenib resistance in hepatoma cells, and combination treatment with VPA may be effective against this drug resistance.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Liver Neoplasms; MAP Kinase Signaling System; Niacinamide; Phenylurea Compounds; Proto-Oncogene Proteins c-akt; Signal Transduction; Sorafenib; Transforming Growth Factor beta; Treatment Failure; Treatment Outcome; Valproic Acid

TGF-β signaling promotes metastasis by controlling the expression of downstream target genes. In this issue of Cancer Cell, Yuan and colleagues discover a novel TGF-β-induced lncRNA, lncRNA-ATB, which stimulates EMT through sequestering miR-200s and facilitates colonization by stabilizing IL-11 mRNA, thus promoting both early and late steps of cancer metastasis.

Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Male; RNA, Long Noncoding; Transforming Growth Factor beta

Distinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-β) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-β establishes and modifies the key features of these cell subpopulations is not fully understood.. In the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-β is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-β is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-β, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases.. Our study reveals a self-perpetuating crosstalk between TGF-β signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-β to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.

Topics: AC133 Antigen; Animals; Antigens, CD; Carcinoma, Hepatocellular; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome, Human; Glycoproteins; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Neoplastic Stem Cells; NIH 3T3 Cells; Peptides; Sequence Analysis, DNA; Transforming Growth Factor beta

Transcriptional intermediary factor 1 gamma (TIF1γ) may play either a potential tumor-suppressor or -promoter role in cancer. Here we report on a critical role of TIF1γ in the progression of hepatocellular carcinoma (HCC). Reduced expression of TIF1γ was detected in HCC, especially in advanced HCC tissues, compared to adjacent noncancerous tissues. HCC patients with low TIF1γ expression had shorter overall survival times and higher recurrence rates than those with high TIF1γ expression. Reduced TIF1γ expression was an independent and significant risk factor for recurrence and survival after curative resection. In HCC cells, TIF1γ played a dual role: It promoted tumor growth in early-stage HCC, but not in advanced-stage HCC, whereas it inhibited invasion and metastasis in both early- and advanced-stage HCC. Mechanistically, we confirmed that TIF1γ inhibited transforming growth factor-β/ Drosophila mothers against decapentaplegic protein (TGF-β/Smad) signaling through monoubiquitination of Smad4 and suppressed the formation of Smad2/3/4 complex in HCC cells. TGF-β-inducing cytostasis and metastasis were both inhibited by TIF1γ in HCC. We further proved that TIF1γ suppressed cyotstasis-related TGF-β/Smad downstream c-myc down-regulation, as well as p21/cip1 and p15/ink4b up-regulation in early-stage HCC. Meanwhile, TGF-β inducible epithelial-mesenchymal transition and TGF-β/Smad downstream metastatic cascades, including phosphatase and tensin homolog deleted on chromosome ten down-regulation, chemokine (CXC motif) receptor 4 and matrix metalloproteinase 1 induction, and epidermal growth factor receptor- and protein kinase B-signaling transactivation, were inhibited by TIF1γ. In addition, we found that the down-regulation of TIF1γ in HCC was caused by hypermethylation of CpG islands in the TIF1γ promoter, and demonstrated that the combination of TIF1γ and phosphorylated Smad2 was a more powerful predictor of poor prognosis.. TIF1γ regulates tumor growth and metastasis through inhibition of TGF-β/Smad signaling and may serve as a novel prognostic biomarker in HCC.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; CpG Islands; DNA Methylation; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Humans; Liver; Liver Neoplasms, Experimental; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Smad2 Protein; Transcription Factors; Transforming Growth Factor beta

S-allylmercaptocysteine (SAMC), one of the water-soluble organosulfur garlic derivatives, can induce the apoptosis of many types of cancer cells through the MAPK signaling pathway. The TGF-β signaling pathway also plays a pivotal role in the process of oncogenesis, and has a certain crosstalk with the MAPK pathway. In the present study, hepatocellular carcinoma cell line HepG2 with an intact TGF-β signal and colon cancer cell line SW620 with an imperfect TGF-β signal were selected to ascertain whether SAMC induces the apoptosis of cancer cells by TGF-β signaling. In both cell lines treated with MAPK inhibitors and SAMC, an increased apoptosis rate was observed by electron microscopy, TUNEL and flow cytometric assays. Immunohistochemistry and western blot assays showed that SAMC induced the apoptosis of cancer cells by activating TGF-β1, TβRII, p-smad2/3, smad4 and smad7 signals, and promoting Bim expression while decreasing Bcl-2 expression and finally activating the mitochondrial apoptosis pathway proteins caspase-3 and caspase-9 in the HepG2 cell line. In contrast, in the SW620 cell line, the apoptosis induced by SAMC only affected TGF-β1 and smad7 signals, and promoted the expression of Bax and Bad and finally activated the mitochondrial apoptosis pathway protein caspase-9. When we compare the apoptosis rate in both cell lines, a significantly lower apoptosis rate was noted in the SW620 cell line than the rate noted in the HepG2 cell line. In summary, SAMC induces the apoptosis of cancer cells by activating the TGF-β signaling pathway, after MAPK signaling is inhibited.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cysteine; Drug Synergism; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Protein Kinase Inhibitors; Signal Transduction; Transforming Growth Factor beta

STAT3 is highly activated in a wide variety of cancers and functions to promote tumor survival. We previously reported that blocking STAT3 activation inhibited human hepatocellular carcinoma (HCC) growth in vitro, but whether this treatment also triggered antitumor immune responses in vivo remained unknown. In this study, we found that blocking the STAT3 pathway in HCC cells dramatically inhibited murine HCC growth in vivo and prolonged survival of tumor-bearing mice. Importantly, the presence of STAT3-blocked HCC augmented NK cell cytotoxicity against HCC and increased expression of molecules associated with NK cell activation and cytotoxicity. In T cell-deficient nude mice, a unique NK cell-mediated antitumor function against STAT3-blocked HCC was suggested. NK cells were shown to be necessary and sufficient in NK or T cell depletion experiments, or by adoptively transferring NK cells. Furthermore, regulatory T cells and immunosuppressive IL-10 and TGF-β cytokines were reduced in mice bearing STAT3-blocked HCC cells, suggesting that these factors may be involved in HCC-induced NK cell suppression. These findings indicate that blocking STAT3 in HCC cells can initiate innate immunity in vivo.

Topics: Adoptive Transfer; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Immune Tolerance; Immunity, Innate; Interleukin-10; Killer Cells, Natural; Liver Neoplasms; Lymphocyte Activation; Lymphocyte Depletion; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Oligodeoxyribonucleotides; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

PIWIL2 is widely expressed in various tumours and implicated in playing a role in tumourigenesis. For a more thorough study of PIWIL2 functions in tumour cells, we aimed to establish PIWIL2-specific transcript knock-down/knockout HepG2 cell lines using transcription activator-like effector nuclease (TALEN) technology. Furthermore, we proposed to use the cell models to explore PIWIL2 functions in TGF-β signalling in HepG2 cells. HepG2s are human hepatocellular carcinoma cells.. We established PIWIL2 knock-down or knock-out cell lines in HepG2 using TALEN technology. Next, we sought to use the cell line models to investigate effects of full length PIWIL2-specific transcripts in cell proliferation induced by TGF-β.. First, we established PIWIL2-specific transcript mono-allele and bi-allele knockout HepG2 cell lines. By using the cell line models, we found that specific transcript knockdown of full length PIWIL2 can suppress cell proliferation, while ectopic expression of PIWIL2 enhanced proliferation of HepG2 by suppressing the TGF-β pathway. Furthermore, we demonstrated that PIWIL2 can interact with HSP90 to prevent formation of HSP90-TβR complexes, which promote degradation of TβRs.. Taken together, our study revealed critical negative regulation of TGF-β signalling by PIWIL2 in HepG2 tumour cells, and provided an effective strategy to study specific gene transcript functions in cells.

Topics: Argonaute Proteins; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Hep G2 Cells; HSP90 Heat-Shock Proteins; Humans; Liver Neoplasms; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Sequence Alignment; Signal Transduction; Transforming Growth Factor beta

Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site, invade surrounding tissue, and establish distant metastases. A hallmark of EMT is the loss of E-cadherin expression, and one major signal for the induction of EMT is TGFβ, which is dysregulated in up to 40% of hepatocellular carcinoma (HCC). We have constructed an EMT network of 70 nodes and 135 edges by integrating the signaling pathways involved in developmental EMT and known dysregulations in invasive HCC. We then used discrete dynamic modeling to understand the dynamics of the EMT network driven by TGFβ. Our network model recapitulates known dysregulations during the induction of EMT and predicts the activation of the Wnt and Sonic hedgehog (SHH) signaling pathways during this process. We show, across multiple murine (P2E and P2M) and human HCC cell lines (Huh7, PLC/PRF/5, HLE, and HLF), that the TGFβ signaling axis is a conserved driver of mesenchymal phenotype HCC and confirm that Wnt and SHH signaling are induced in these cell lines. Furthermore, we identify by network analysis eight regulatory feedback motifs that stabilize the EMT process and show that these motifs involve cross-talk among multiple major pathways. Our model will be useful in identifying potential therapeutic targets for the suppression of EMT, invasion, and metastasis in HCC.

Topics: Animals; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Hedgehog Proteins; Humans; Liver Neoplasms; Mice; Transcriptional Activation; Transforming Growth Factor beta; Wnt Signaling Pathway

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of metastasis and recurrence, which are primarily attributed to the angiogenesis. So, the anti-angiogenesis treatment has become the new approach for HCC therapy. In addition to treating leukemia, arsenic trioxide (As2O3) also suppresses other solid tumors, including HCC. However, the roles of As2O3 in the angiogenesis potential of HCC cells remain unclear. In our present study, As2O3 attenuated the angiogenic ability by the microRNA-491 (miR-491)-mediated inhibition of TGF-β/SMAD3/NF-κB signal pathway in MHCC97H and MHCC97L cells. Briefly, in these cells, As2O3 improved the expression of miR-491 via DNA-demethylation; miR-491, which targeted the SMAD3-3'-UTR, decreased the expression/function of SMAD3, leading to the inactivation of NF-κB/IL-6/STAT-3 signaling; knockdown of miR-491 abolished the As2O3-induced inhibitions of the TGF-β/SMAD3/NF-κB pathway, the VEGF secretion, and the angiogenesis. By understanding a novel mechanism whereby As2O3 inhibits the angiogenic potential in HCC cells, our study would help in the design of future strategies of developing As2O3 as a potential chemopreventive agent when used alone or in combination with other current anticancer drugs.

Topics: 3' Untranslated Regions; Angiogenesis Inhibitors; Arsenic Trioxide; Arsenicals; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Methylation; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Liver Neoplasms; MicroRNAs; Neovascularization, Pathologic; Neovascularization, Physiologic; NF-kappa B; Oxides; RNA Interference; Signal Transduction; Smad3 Protein; Time Factors; Transfection; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Galangin can suppress hepatocellular carcinoma (HCC) cell proliferation. In this study, we demonstrated that galangin induced autophagy by activating the transforming growth factor (TGF)-β receptor/Smad pathway and increased TGF-β receptor I (RI), TGF-βRII, Smad1, Smad2, Smad3 and Smad4 levels but decreased Smad6 and Smad7 levels. Autophagy induced by galangin appears to depend on the TGF-β receptor/Smad signalling pathway because the down-regulation of Smad4 by siRNA or inhibition of TGF-β receptor activation by LY2109761 blocked galangin-induced autophagy. The down-regulation of Beclin1, autophagy-related gene (ATG) 16L, ATG12 and ATG3 restored HepG2 cell proliferation and prevented galangin-induced apoptosis. Our findings indicate a novel mechanism for galangin-induced autophagy via activation of the TGF-β receptor/Smad pathway. The induction of autophagy thus reflects the anti-proliferation effect of galangin on HCC cells.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Carcinoma, Hepatocellular; Cell Proliferation; Dose-Response Relationship, Drug; Flavonoids; Hep G2 Cells; Humans; Liver Neoplasms; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Smad Proteins; Transfection; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with hepatocellular carcinoma (HCC) development and progression. DNAJC6 (DNA/HSP40 homolog subfamily C member 6) encodes auxilin, which is responsible for juvenile Parkinsonism with phenotypic variability. However, the role of DNAJC6 in HCC development and progression is limited. Here, we report that DNAJC6 is up-regulated in HCC tissues and up-regulation of DNAJC6 expression predicts poor outcome in patients with HCC. Furthermore, overexpression of DNAJC6 enhances the ability for acquisition of mesenchymal traits, enhanced cell proliferation and invasion. DNAJC6 positively regulated expression of EMT-related transcription factor, also activating transforming growth factor β (TGF-β) pathway to contribute to EMT. Our findings demonstrated an important function of DNAJC6 in the progression of HCC by induction of EMT, and they implicate DNAJC6 as a marker of poor outcome in HCC.

Topics: Aged; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HSP40 Heat-Shock Proteins; Humans; Liver Neoplasms; Mice; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Transforming Growth Factor beta; Treatment Outcome; Up-Regulation

Transforming growth factor-β (TGF-β) has been shown to play a central role in the promotion of cell motility, but its functional mechanism has remained unclear. With the aim of investigating the diagnostic and treatment modalities for patients with hepatocellular carcinoma (HCC), the signaling pathway that may contribute to TGF-β-mediated cell invasion in hepatoma cells was evaluated.. Three hepatoma cell lines, HepG2, PLC/PRF/5, and HLF, were treated with TGF-β, and the involvement of the non-canonical TGF-β pathway was analyzed by cell migration assays. HepG2 cells were treated with a p21-activated kinase-2 (PAK2)-targeting small interfering RNA and analyzed for their cell motility. The relationships between the PAK2 status and the clinicopathological characteristics of 62 HCC patients were also analyzed.. The cell migration assays showed that Akt is a critical regulator of TGF-β-mediated cell migration. Western blotting analyses showed that TGF-β stimulated Akt and PAK2 in all three hepatoma cell lines, and phosphorylated PAK2 was blocked by Akt inhibitor. Suppression of PAK2 expression by small interfering RNA resulted in increased focal adhesions with significantly repressed cell migration in the presence of TGF-β. Clinicopathological analyses showed that the phosphorylation level of PAK2 was closely associated with tumor progression, metastasis, and early recurrence of HCC.. PAK2 may be a critical mediator of TGF-β-mediated hepatoma cell migration, and may represent a potential target for the treatment of HCC.

Topics: Aged; Carcinoma, Hepatocellular; Cell Movement; Female; Humans; Liver Neoplasms; Male; p21-Activated Kinases; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Tumor recurrence and metastases are the major obstacles to improving the prognosis of patients with hepatocellular carcinoma (HCC). To identify novel risk factors associated with HCC recurrence and metastases, we have established a panel of recurrence-associated microRNAs (miRNAs) by comparing miRNA expression in recurrent and nonrecurrent human HCC tissue samples using microarrays (recurrence is defined as recurrent disease occurring within a 2-year time point of the original treatment). Among the panel, expression of the miR-216a/217 cluster was consistently and significantly up-regulated in HCC tissue samples and cell lines associated with early tumor recurrence, poor disease-free survival, and an epithelial-mesenchymal transition (EMT) phenotype. Stable overexpression of miR-216a/217-induced EMT increased the stem-like cell population, migration, and metastatic ability of epithelial HCC cells. Phosphatase and tensin homolog (PTEN) and mothers against decapentaplegic homolog 7 (SMAD7) were subsequently identified as two functional targets of miR-216a/217, and both PTEN and SMAD7 were down-regulated in HCC. Ectopic expression of PTEN or SMAD7 partially rescued miR-216a/217-mediated EMT, cell migration, and stem-like properties of HCC cells. Previously, SMAD7 was shown to be a transforming growth factor beta (TGF-β) type 1 receptor antagonist. Here, we further demonstrated that overexpression of miR-216a/217 acted as a positive feedback regulator for the TGF-β pathway and the canonical pathway involved in the activation of phosphoinositide 3-kinase/protein kinase K (PI3K/Akt) signaling in HCC cells. Additionally, activation of the TGF-β- and PI3K/Akt-signaling pathways in HCC cells resulted in an acquired resistance to sorafenib, whereas blocking activation of the TGF-β pathway overcame miR-216a/217-induced sorafenib resistance and prevented tumor metastases in HCC.. Overexpression of miR-216a/217 activates the PI3K/Akt and TGF-β pathways by targeting PTEN and SMAD7, contributing to hepatocarcinogenesis and tumor recurrence in HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; In Vitro Techniques; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Smad7 Protein; Sorafenib; Transforming Growth Factor beta; Up-Regulation

The tumor-draining lymph node (TDLN) is the critical and initial site of the immune decision made between activation and tolerance to tumor antigens. Tumor-reactive lymphadenopathy in TDLN has been observed for decades, but the profiles of immune regulation in these nodes remain unclear.. Both regulatory T cells (Tregs) and effector T cells were examined using 6 × 10(5) Hepa1-6 hepatocellular carcinoma cells implanted in footpads of syngeneic C57BL/6J mice, which formed TDLN. FOXP3(+) Tregs and CD8(+) T cells in TDLN were detected by immunohistochemical staining. The frequency of CD4(+)FOXP3(+) T cells and FOXP3 mRNA expression were determined by flow cytometry and real-time quantitative polymerase chain reaction. The interferon γ secretion ability of CD8(+) T cells in TDLN was measured by enzyme-linked immunospot technique.. There was significant expansion of Tregs and CD8(+) T cells in the tumor-draining popliteal lymph node compared with nondraining popliteal lymph node and spleen in the same mouse. Tregs were diffusely distributed in the CD8(+) T cell compartment. The CD8(+) T cells primed in TDLN showed a strong ability of interferon γ secretion via in vitro stimulation.. These findings support the notion that Tregs suppress CD8(+) T cells by secreting cytokines such as transforming growth factor β and interleukin 10, but do not make CD8(+) T cells lose function in the TDLN. Deletion of Tregs at the secondary lymphoid organs could be crucial for the establishment of a tumor-specific immunotherapy.

Topics: Animals; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Disease Models, Animal; Female; Forkhead Transcription Factors; Immune Tolerance; Interferon-gamma; Interleukin-10; Liver Neoplasms; Lymph Nodes; Mice; Mice, Inbred C57BL; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Parathyroid hormone-related protein (PTHrP) is a polyhormone secretory protein that plays fundamental roles in the development and function of various tissues. Transforming growth factor (TGF)-β is an important tumor suppressor that induces cell cycle arrest and apoptosis. Increased PTHrP expression has been implicated in TGF-β-induced growth inhibition in human hepatocellular carcinoma cells. However, whether PTHrP is involved in TGF-β-induced apoptosis remains unknown. Using Hep3B and HuH-7, two human hepatocellular carcinoma cell lines, the current study examined the hypothesis that TGF-β-induced apoptosis is mediated by the induction of PTHrP expression. We found that (1) TGF-β induces PTHrP mRNA expression, protein expression and secretion in a time-dependent fashion; (2) knockdown of PTHrP gene expression or neutralization of secreted PTHrP isoforms blocks TGF-β-induced apoptosis; and (3) TGF-β-induced PTHrP expression is Smad3-dependent. Thus, we have identified PTHrP as a novel mediator for TGF-β-induced apoptosis in Hep3B cells. Our findings provide further insights into the mechanisms through which TGF-β conveys tumor suppression activity.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Expression; Humans; Liver Neoplasms; Parathyroid Hormone-Related Protein; RNA, Messenger; Transforming Growth Factor beta

Smad7 is a principal inhibitor of the TGFβ-Smad signalling pathway. We have investigated the functional significance of Smad7 in hepatocellular carcinoma (HCC). Smad7 knockout (KO) and wild-type (WT) mice were injected with diethylnitrosamine (DEN) to induce HCC. The effects of Smad7 on cellular features were examined in HCC cells, using a Smad7 over-expression or deletion approach. Signalling pathway components modulated by Smad7 in HCC were evaluated using luciferase reporter assay and co-immunoprecipitation. Smad7 was down-regulated in human HCCs compared with the adjacent normal tissues (p < 0.001). Smad7 KO mice were more susceptible to DEN-induced HCC than WT mice (78% versus 22%, p < 0.05). HCCs from KO mice displayed a greater proliferation activity (p < 0.05) and a reduced apoptotic index compared with WT littermates (p < 0.05). Deletion of Smad7 promoted cell proliferation in primary cultured HCC cells. In addition, over-expression of Smad7 in HCC cell lines markedly suppressed cell growth (p < 0.0001) and colony formation (p < 0.01). Cell cycle analysis revealed an increase in the G1 phase and a reduction in the S-phase populations, accompanied by up-regulation of p27(Kip1) and down-regulation of cyclin D1. Smad7 increased cell apoptosis (p < 0.01) by mediating an intrinsic [caspase-9, caspase-3 and poly(ADP-ribose) polymerase] apoptotic pathway. Moreover, Smad7 inhibited NF-κB signalling by interacting with TAB2, an upstream activator of NF-κB, and inhibited TGFβ signalling by suppressing phosphorylation of Smad3. In conclusion, loss of Smad7 enhances susceptibility to HCC. Smad7 suppresses HCC cell growth by inhibiting proliferation and G1 -S phase transition and inducing apoptosis through attenuation of NF-κB and TGFβ signalling. Smad7 acts as a potential tumour suppressor in liver.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Proliferation; Diethylnitrosamine; G1 Phase; Genes, Reporter; Genetic Predisposition to Disease; Hep G2 Cells; Hepatocytes; Humans; Immunoprecipitation; Liver Neoplasms, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Phenotype; Primary Cell Culture; S Phase; Signal Transduction; Smad7 Protein; Time Factors; Transfection; Transforming Growth Factor beta

The role of transforming growth factor-beta (TGF-β) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-β signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-β's role in regulating HCC malignancy. Here, TGF-β pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-β receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-β receptor II (TβRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-β and growth-inhibited by exogenous TGF-β. However, stable knockdown of TβRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-β signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-β and the survival activity induced by autocrine TGF-β revealing a delicate selection of the two opposing activities of TGF-β during HCC evolution.

Topics: Animals; Apoptosis; Carcinogenesis; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Gene Knockdown Techniques; Humans; Liver Neoplasms; Mice; Neoplasm Metastasis; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Melanoma differentiation associated gene-7 (MDA-7)/interleukin‑24 (IL-24) has been considered as a tumor-suppressor gene, which suppresses the growth and induces the apoptosis of cancer cells. In the present study, we investigated the effect and mechanisms of MDA-7/IL-24 regarding the inhibition of metastasis of HepG2 and BEL-7402 human hepatocellular carcinoma (HCC) cells in vitro. We established MDA-7/IL-24-overexpressing HepG2 and BEL-7402 cell lines and found that MDA-7/IL-24 overexpression inhibited tumor cell adhesion and invasion, and induced G2/M arrest in tumor cells. To explore its mechanism of action, western blotting and real-time-PCR assay were used to investigate the expression of E-cadherin, CD44, ICAM-1, matrix metalloproteinase (MMP)-2 and -9, CyclinB, Twist, survivin, p-ERK and p-Akt. ELISA assay was used to measure the secretion of TGF-β, and a reporter gene assay was used to detected the transcriptional activity of NF-κB and AP-1 in HepG2 and BEL-7402 cells. The results showed that MDA-7/IL-24 overexpression decreased the expression of CD44, ICAM-1, MMP-2/-9, CyclinB, Twist, survivin, TGF-β and p-Akt, transcriptional activity of NF-κB, and increased the expression of E-cadherin and p-ERK and transcriptional activity of AP-1 in HepG2 and BEL-7402 cells. Our results revealed that MDA-7/IL-24 mediated the inhibition of adhesion and invasion in HepG2 and BEL-7402 cells by suppressing metastasis-related gene expression. Thus, MDA-7/IL-24 may be used as a novel cancer-suppressor gene for the therapy of human HCC.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Down-Regulation; G2 Phase; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Hep G2 Cells; Humans; Interleukins; Liver Neoplasms; Neoplasm Invasiveness; NF-kappa B; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.

Topics: alpha-Fetoproteins; Area Under Curve; Biomarkers; Carcinoma, Hepatocellular; Computational Biology; Culture Media, Conditioned; Culture Media, Serum-Free; Female; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Viral; Hepatitis B virus; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Matrix Metalloproteinase 1; Middle Aged; Organ Culture Techniques; Osteopontin; Pregnancy-Specific beta 1-Glycoproteins; Proteome; Proteomics; ROC Curve; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is increasing in frequency in the U.S. The major reason for the low postoperative survival rate of HCC is widespread intrahepatic metastasis or invasion, and activation of TGFβ signaling is associated with the invasive phenotype. This study aims at determining the novel function of miR-127 in modulating HCC migration. Overexpression of miR-127 inhibits HCC cell migration, invasion and tumor growth in nude mice. MiR-127 directly represses matrix metalloproteinase 13 (MMP13) 3'UTR activity and protein expression, and diminishes MMP13/TGFβ-induced HCC migration. In turn, TGFβ decreases miR-127 expression by enhancing c-Jun-mediated inhibition of miR-127 promoter activity. In contrast, p53 transactivates miR-127 promoter and induces miR-127 expression, which is antagonized by c-Jun. The inhibition of miR-127 by c-Jun is through TGFβ-mediated ERK and JNK pathways. The lower miR-127 expression shows a negative correlation with the higher MMP13 expression in a subset of human HCC specimens. This is the first report elucidating a feedback regulation between miR-127 and the TGFβ/c-Jun cascade in HCC migration via MMP13 that involves a crosstalk between the oncogene c-Jun and tumor suppressor p53.

Topics: 3' Untranslated Regions; Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Down-Regulation; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Liver Neoplasms; MAP Kinase Signaling System; Matrix Metalloproteinase 13; Mice; Mice, Nude; MicroRNAs; Models, Biological; Molecular Sequence Data; Neoplasm Invasiveness; Proto-Oncogene Proteins c-jun; Signal Transduction; Transforming Growth Factor beta

We investigated blocking the TGF-β signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761) and a neutralizing humanized antibody (D10) against TGF-βRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-β receptor cycling in cancer cells.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Hepatocellular; Connective Tissue Growth Factor; Female; Hep G2 Cells; Homeodomain Proteins; Humans; Liver Neoplasms; Male; Middle Aged; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Quinolines; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Transcription Factors; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Liver fibrosis is a severe, life-threatening clinical condition resulting from nonresolving hepatitis of different origins. IL-17A is critical in inflammation, but its relation to liver fibrosis remains elusive. We find increased IL-17A expression in fibrotic livers from HBV-infected patients undergoing partial hepatectomy because of cirrhosis-related early-stage hepatocellular carcinoma in comparison with control nonfibrotic livers from uninfected patients with hepatic hemangioma. In fibrotic livers, IL-17A immunoreactivity localizes to the inflammatory infiltrate. In experimental carbon tetrachloride-induced liver fibrosis of IL-17RA-deficient mice, we observe reduced neutrophil influx, proinflammatory cytokines, hepatocellular necrosis, inflammation, and fibrosis as compared with control C57BL/6 mice. IL-17A is produced by neutrophils and T lymphocytes expressing the Th17 lineage-specific transcription factor Retinoic acid receptor-related orphan receptor γt. Furthermore, hepatic stellate cells (HSCs) isolated from naive C57BL/6 mice respond to IL-17A with increased IL-6, α-smooth muscle actin, collagen, and TGF-β mRNA expression, suggesting an IL-17A-driven fibrotic process. Pharmacologic ERK1/2 or p38 inhibition significantly attenuated IL-17A-induced HSC activation and collagen expression. In conclusion, IL-17A(+) Retinoic acid receptor-related orphan receptor γt(+) neutrophils and T cells are recruited into the injured liver driving a chronic, fibrotic hepatitis. IL-17A-dependent HSC activation may be critical for liver fibrosis. Thus, blockade of IL-17A could potentially benefit patients with chronic hepatitis and liver fibrosis.

Topics: Actins; Adult; Animals; Carbon Tetrachloride Poisoning; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Collagen; Cytokines; Female; Gene Expression Regulation; Hemangioma; Hepatectomy; Hepatic Stellate Cells; Hepatitis B, Chronic; Hepatitis, Animal; Humans; Interleukin-17; Liver Cirrhosis; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; Middle Aged; Neutrophils; Nuclear Receptor Subfamily 1, Group F, Member 3; Protein Kinase Inhibitors; Receptors, Interleukin-17; Recombinant Proteins; Th17 Cells; Transforming Growth Factor beta

Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133(+) TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG-dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2(+/-) mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.

Topics: AC133 Antigen; Adaptor Proteins, Signal Transducing; Animals; Antigens, CD; Antineoplastic Agents; Base Sequence; Carcinoma, Hepatocellular; Cell Separation; Drug Resistance, Neoplasm; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycoproteins; Homeodomain Proteins; Humans; Inhibitory Concentration 50; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Nanog Homeobox Protein; Neoplastic Stem Cells; Niacinamide; Oncogenes; Peptides; Phenylurea Compounds; Phosphoproteins; RNA-Binding Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; Smad Proteins; Sorafenib; Spheroids, Cellular; Toll-Like Receptor 4; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays; YAP-Signaling Proteins

Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; DNA Primers; Humans; Liver Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-α stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Class Ia Phosphatidylinositol 3-Kinase; Gene Expression Regulation, Neoplastic; Hepatocytes; HSP20 Heat-Shock Proteins; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; MicroRNAs; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Transforming Growth Factor beta

Our previous studies demonstrated that autocrine motility factor/phosphoglucose isomerase (AMF/PGI) possesses tumorigenic activities through the modulation of intracellular signaling. We then investigated the effects of ursolic acid (UA), oleanolic acid (OA), tangeretin, and nobiletin against AMF/PGI-mediated oncogenesis in cultured stable Huh7 and Hep3B cells expressing wild-type or mutated AMF/PGI and in a mouse model in this study. The working concentrations of the tested compounds were lower than their IC10 , which was determined by Brdu incorporation and colony formation assay. Only UA efficiently suppressed the AMF/PGI-induced Huh7 cell migration and MMP-3 secretion. Additionally, UA inhibited the AMF/PGI-mediated protection against TGF-β-induced apoptosis in Hep3B cells, whereas OA, tangeretin, and nobiletin had no effect. In Huh7 cells and tumor tissues, UA disrupted the Src/RhoA/PI 3-kinase signaling and complex formation induced by AMF/PGI. In the Hep3B system, UA dramatically suppressed AMF/PGI-induced anti-apoptotic signaling transmission, including Akt, p85, Bad, and Stat3 phosphorylation. AMF/PGI enhances tumor growth, angiogenesis, and pulmonary metastasis in mice, which is correlated with its enzymatic activity, and critically, UA intraperitoneal injection reduces the tumorigenesis in vivo, enhances apoptosis in tumor tissues and also prolongs mouse survival. Combination of sub-optimal dose of UA and cisplatin, a synergistic tumor cell-killing effects was found. Thus, UA modulates intracellular signaling and might serve as a functional natural compound for preventing or alleviating hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Cell Proliferation; Cisplatin; Disease Models, Animal; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Flavones; Fluorescent Antibody Technique; Glucose-6-Phosphate Isomerase; Humans; Immunoprecipitation; Liver Neoplasms; Luciferases; Lung Neoplasms; Male; Matrix Metalloproteinase 3; Mice; Mice, Nude; Neovascularization, Pathologic; Oleanolic Acid; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta; Triterpenes; Tumor Cells, Cultured; Ursolic Acid

Sorafenib has been shown to improve survival rate of hepatocellular carcinoma (HCC) patients significantly. Decline of tumor infiltrated regulatory T cells (TITs) may account for the activity of sorafenib partially. In this study, the underlying mechanism of sorafenib reducing TITs was investigated.. Tumor infiltrated mononuclear cells (TIMs), which were isolated form 19 HCC patients with or without sorafenib therapy, were analyzed by flow cytometry. TGF-β signal pathways were analyzed by immunoblotting. In vitro test, naïve T cells were induced to regulatory T cells (Tregs) with or without sorafenib. After 3 days of culture, percentage of Tregs from CD4+ cells and TGF-β signal pathways were analyzed. Meanwhile, TIMs from HCC patients without sorafenib treatment were cultured in the presence of sorafenib, and then the percentage of Foxp3 expressing cells from TIMs was analyzed.. TITs were increased in HCC patients compared with controls. However, after sorafenib therapy, TITs were decreased significantly and TGF-β signal pathways were down-regulated. Additionally, in the presence of sorafenib, induction of Tregs was inhibited and TGF-β signal pathways in resulting cells were down-regulated. However, sorafenib treatment did not affect the percentage of Foxp3 expressing cells from TIMs in vitro.. Sorafenib reducing TITs in HCC patients are associated with down-regulation of TGF-β signal. This finding may help us for better understanding the activity of sorafenib in HCC patients.

Topics: Adult; Aged; Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Down-Regulation; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Leukocytes, Mononuclear; Liver Neoplasms; Male; Middle Aged; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Signal Transduction; Sorafenib; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity.. Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression.. We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity.. Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Transformed; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genetic Therapy; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver Neoplasms; Mice; Transforming Growth Factor beta; Tumor Microenvironment

The immunosuppressive network within the tumor microenvironment is one of the major obstacles to the success of cancer immunotherapy. γδ T cells are attractive effectors for cancer immunotherapy. Nevertheless, the promising anti-tumor effect in vitro is partially if not totally mitigated in vivo. Thus, understanding the immune status of tumor-infiltrating γδ T cells is essential for orchestrating effective immunotherapy strategies. In this study, we have investigated the immunophenotype and function of γδ T cells in hepatocellular carcinoma (HCC) patients.. The phenotype of γδ T cells in peripheral blood, and peritumoral and tumoral tissues of HCC patients (n=61) was characterized by flow cytometry. Functional analysis of the HCC-infiltrating γδ T cells was conducted directly after γδ T cell isolation.. The infiltration of γδ T cells in tumoral tissues was significantly reduced compared to paired peritumoral tissues. Impairment in degranulation of the granule pathway and downregulation of IFN-γ secretion were also demonstrated in HCC-infiltrating γδ T cells, which was in agreement with the results of gene microarray analysis, and further strengthened by the compromised specific cytotoxicity and IFN-γ secretion in vitro. Moreover, isolated HCC-infiltrating CD4(+)CD25(+) regulatory T cells (Treg cells) directly suppressed the cytotoxic function and IFN-γ secretion of γδ T cells in a TGFβ- and IL-10-dependent manner.. The effector function of γδ T cells was substantially impaired in HCC, which is partially mediated by Treg cells. We propose a new mechanism by which immune privilege develops within the tumor milieu.

Topics: Carcinoma, Hepatocellular; Case-Control Studies; Cell Line, Tumor; Cell Movement; Female; Flow Cytometry; Humans; Immunophenotyping; Immunotherapy; In Vitro Techniques; Interferon-gamma; Interleukin-10; Liver Neoplasms; Male; Middle Aged; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is activated in different cytokine signaling pathways. Deletion of Tak1 from hepatocytes results in spontaneous development of hepatocellular carcinoma (HCC), liver inflammation, and fibrosis. TGF-β activates TAK1 and Smad signaling, which regulate cell death, proliferation, and carcinogenesis. However, it is not clear whether TGF-β signaling in hepatocytes, via TGF-β receptor-2 (Tgfbr2), promotes HCC and liver fibrosis.. We generated mice with hepatocyte-specific deletion of Tak1 (Tak1ΔHep), as well as Tak1/Tgfbr2DHep and Tak1/Smad4ΔHep mice. Tak1flox/flox, Tgfbr2ΔHep, and Smad4ΔHep mice were used as controls, respectively. We assessed development of liver injury, inflammation, fibrosis, and HCC. Primary hepatocytes isolated from these mice were used to assess TGF-β-mediated signaling.. Levels of TGF-β, TGF-βR2, and phospho-Smad2/3 were increased in HCCs from Tak1ΔHep mice, which developed liver fibrosis and inflammation by 1 month and HCC by 9 months. However, Tak1/Tgfbr2ΔHep mice did not have this phenotype, and their hepatocytes did not undergo spontaneous cell death or compensatory proliferation. Hepatocytes from Tak1ΔHep mice incubated with TGF-β did not activate p38, c-Jun N-terminal kinase, or nuclear factor-κB; conversely, TGF-β-mediated cell death and phosphorylation of Smad2/3 were increased, compared with control hepatocytes. Blocking the Smad pathway inhibited TGF-β-mediated death of Tak1-/- hepatocytes. Accordingly, disruption of Smad4 reduced the spontaneous liver injury, inflammation, fibrosis, and HCC that develops in Tak1ΔHep mice. Levels of the anti-apoptotic protein Bcl-xL, β-catenin, connective tissue growth factor, and vascular endothelial growth factor were increased in HCC from Tak1ΔHep mice, but not in HCCs from Tak1/Tgfbr2ΔHep mice. Injection of N-nitrosodiethylamine induced HCC formation in wild-type mice, but less in Tgfbr2ΔHep mice.. TGF-β promotes development of HCC in Tak1ΔHep mice by inducing hepatocyte apoptosis and compensatory proliferation during early phases of tumorigenesis, and inducing expression of anti-apoptotic, pro-oncogenic, and angiogenic factors during tumor progression.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; DNA Mutational Analysis; DNA, Neoplasm; Gene Deletion; Hepatocytes; Liver Cirrhosis; Liver Neoplasms; Liver Neoplasms, Experimental; MAP Kinase Kinase Kinases; Mice; Mice, Transgenic; Phenotype; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic "BH3-domain only" PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-β (TGF-β) through the intrinsic pathway and to tumor necrosis factor-α (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-β or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autocrine Communication; Benzenesulfonates; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Knockdown Techniques; Humans; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Proto-Oncogene Proteins; Pyridines; Signal Transduction; Sorafenib; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Small fish models have been used for decades in carcinogenicity testing. Demonstration of common morphological changes associated with specific mechanisms is a clear avenue by which data can be compared across divergent phyletic levels. Dimethylnitrosamine, used in rats to model human alcoholic cirrhosis and hepatic neoplasia, is also a potent hepatotoxin and carcinogen in fish. We recently reported some striking differences in the mutagenicity of DMN in lambda cII transgenic medaka fish vs. Big Blue(®) rats, but the pre-neoplastic and neoplastic commonalities between the two models are largely unknown. Here, we focus on these commonalities, with special emphasis on the TGF-β pathway and its corresponding role in DMN-induced hepatic neoplasia. Similar to mammals, hepatocellular necrosis, regeneration, and dysplasia; hepatic stellate cell and "spindle cell" proliferation; hepatocellular and biliary carcinomas; and TGF-β1 expression by dysplastic hepatocytes all occurred in DMN-exposed medaka. Positive TGF-β1 staining increased with increasing DMN exposure in bile preductular epithelial cells, intermediate cells, immature hepatocytes and fewer mature hepatocytes. Muscle specific actin identified hepatic stellate cells in DMN-exposed fish. Additional mechanistic comparisons between animal models at different phyletic levels will continue to facilitate the interspecies extrapolations that are so critical to toxicological risk assessments.

Topics: Animals; Animals, Genetically Modified; Biliary Tract Neoplasms; Biomarkers; Carcinogenicity Tests; Carcinogens; Carcinoma, Hepatocellular; Cell Proliferation; Chemical and Drug Induced Liver Injury; Dimethylnitrosamine; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fish Diseases; Liver; Liver Neoplasms; Male; Mutagenicity Tests; Oryzias; Rats; Signal Transduction; Species Specificity; Transforming Growth Factor beta

Recently, it has been reported that a small population of cancer stem cells (CSCs) play a role in resistance to chemotherapy and radiation therapy. We reported that CD13(+) liver CSCs survive in hypoxic lesions after chemotherapy, presumably through increased expression of CD13/Aminopeptidase N, which is a scavenger enzyme in the reactive oxygen species (ROS) metabolic pathway. On the other hand, the concept of epithelial-mesenchymal transition (EMT) was indicated by a recent study showing an increased plasticity linked to the cellular "stemness" of CSCs.. To study the relationship between CSCs and EMT, we examined biological characteristics of liver cancer cell lines with EMT by exposing transforming growth factor-β (TGF-β).. We showed that a TGF-β-induced EMT-like phenomenon is associated with increased CD13 expression in liver cancer cells. This phenomenon prevents further increases in the ROS level as well as the induction of apoptosis, promoting the survival of CD13(+) CSCs, whereas inhibition of CD13 stimulates apoptosis. Immunohistochemical analysis also indicated that after chemotherapy, CD13 was coexpressed with N-cadherin in surviving cancer cells within fibrous capsules. We have demonstrated that CD13 expression plays a role in supporting the survival of CSCs and that there is an EMT-associated reduction in ROS elevation.. This novel and consistent linkage between functional CSC markers and the EMT phenomenon suggests a bona fide candidate for targeted therapy for EMT-mediated invasion and metastasis of liver cancer.

Topics: Apoptosis; Cadherins; Carcinoma, Hepatocellular; CD13 Antigens; Cell Line, Tumor; Chi-Square Distribution; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Liver Neoplasms; Neoplastic Stem Cells; Polycomb Repressive Complex 1; Reactive Oxygen Species; Receptor, Notch1; RNA, Messenger; Statistics, Nonparametric; Transforming Growth Factor beta; Tumor Stem Cell Assay

Hepatocellular carcinoma (HCC) is the most common liver cancer. Therapeutic results are usually unsatisfactory because liver tumors recur often. Immunologic factors may be related to the recurrence of HCC; however, this possibility is mentioned only rarely.. Thirty HCC patients undergoing hepatectomies were divided into 3 groups according to the diameters of their HCCs: group A (n = 8), diameter ≤3 cm; group B (n = 8), diameter >3 cm and ≤5 cm; and group C (n = 14), diameter >5 cm. T-lymphocytes from peripheral blood, nontumor liver tissue, and the HCC were analyzed.. The percentage of CD25+ in the CD4+ T cells did not differ between the peripheral blood and the nontumor liver tissue among the 3 groups. CD25+ cells were increased in the tumor tissue in group C patients (range, 6-41%; median, 22.9%; P = .003), compared to group A patients. The percentage of CD25+ in the CD4+ T cells in tumor tissue was positively correlated with tumor sizes (r = 0.556). These CD4+ CD25+ lymphocytes produced transforming growth factor-β and interferon-γ but not interleukin-10, and were anergic to plate-coated monoclonal antibodies (anti-CD3/anti-CD28). The characteristics of these antibodies were comparable to those of regulatory T cells. When the infiltration lymphocytes including CD4+ CD25+ T cells were added to the mixed lymphocyte reaction activated by autologous tumor lysate-pulsed dendritic cells, the proliferation of lymphocytes was inhibited.. The increase of CD4+ CD25+ T cells in the tumor microenvironment correlates with tumor sizes. These CD4+ CD25+ regulatory T cells appeared to suppress the immune response activated by dendritic cells.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; Cell Proliferation; Dendritic Cells; Female; Forkhead Transcription Factors; Hepatectomy; Humans; Immunity, Cellular; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Liver Neoplasms; Male; Middle Aged; Prognosis; Retrospective Studies; Transforming Growth Factor beta; Tumor Microenvironment

The purposes of the present study were to investigate the modifying effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a genotoxic carcinogen produced during cooking of protein-rich foods, and elucidate underlying mechanisms in a two-stage hepatocarcinogenesis mice model. Six-week-old B6C3F1 mice were subjected to two-thirds partial hepatectomy at the beginning of the study, followed by an intraperitoneal injection of diethylnitrosamine on day 1. Starting 1 week later, they were fed diets containing IQ at doses of 30, 100, or 300 ppm for 39 weeks. A dose-dependent trend for increase in eosinophilic altered foci as well as eosinophilic hepatocellular adenomas was observed, along with significant elevation in the incidence of hepatocellular carcinomas in the 100- and 300-ppm IQ groups as compared with initiation control group. Furthermore, IQ elevated the protein expression levels of Wnt1, transforming growth factor-β (TGF-β), TGF-β receptors 1 and 2 (TβR1 and TβR2), and phosphorylated c-Jun (p-c-Jun), while suppressing those of E-cadherin and p21(WAF1/Cip1). Moreover, translocation of β-catenin to the nuclei as well as upregulated nuclear expression of c-Myc and cyclin D1, which are downstream targets of β-catenin and p-c-Jun, were detected at 100 and 300 ppm. These findings suggest that IQ exerts dose-dependent promoting effects on mice hepatocarcinogenesis by activating TGF-β and Wnt/β-catenin signaling pathways and inhibiting cell adhesion.

Topics: Animals; beta Catenin; Blotting, Western; Body Weight; Carcinoma, Hepatocellular; Cell Adhesion; Cell Transformation, Neoplastic; Diethylnitrosamine; Dose-Response Relationship, Drug; Drinking; Eating; Hepatectomy; Immunohistochemistry; Liver; Liver Neoplasms, Experimental; Male; Mice; Organ Size; Quinolines; Time Factors; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway

Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC.. We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion.. HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β). Neutralization of both growth factors shows different roles for VEGF and TGFβ in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle.. These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Polarity; Disease Progression; Glycoproteins; Hepacivirus; Hepatitis C; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Tight Junctions; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Virus Replication

The aim of our study was to improve the detection of HCC by measuring alpha-fetoprotein (AFP) in addition to other molecular markers by estimating the plasma concentration of transforming growth factor beta (TGF-β) and epidermal growth factor receptor (EGFR). In particular, the role of hepatitis C and B viruses (HCV and HBV) infection was evaluated with relation to TGF-β and EGFR plasma concentration.. Eighty-five patients with liver disease, 54 with hepatocellular carcinoma (HCC), 16 with liver metastasis (LM), 15 with liver cirrhosis (LC) and 30 healthy volunteers were evaluated. AFP, TGF-β and EGFR were detected with enzyme-linked immunoassay (ELISA) in plasma of all study participants.. The mean values of TGF-β and EGFR in all patients were much higher than in control group, p<0.0001. In HCC patients the levels of TGF-β and EGFR were much higher than in LM and LC patients. Moreover, TGF-β and EGFR were significantly higher in the presence of both viruses or only in the presence of HCV, p=0.002. No decrease or increase of AFP was noted in these patients.. Our data suggest the reliability of TGF-β and EGFR in detecting HCC, in particular when the carcinogenesis is affected by virus infection.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Hepatocellular; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Female; Hepacivirus; Hepatitis C; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Prognosis; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the major primary liver cancers in adults. The phenotypic overlap between HCC and CC has been shown to comprise a continuous liver cancer spectrum. As a proof of this concept, a recent study demonstrated a genomic subtype of HCC that expressed CC-like gene expression traits, such as CC-like HCC, which revealed the common genomic trait of stem-cell-like properties and aggressive clinical outcomes. Scirrhous HCC (S-HCC), a rare variant of HCC, is characterized by abundant fibrous stroma and has been known to express several liver stem/progenitor cell markers. This suggests that S-HCC may harbor common intermediate traits between HCC and CC, including stem-cell traits, which are similar to those of CC-like HCC. However, the molecular and pathological characteristics of S-HCC have not been fully evaluated. By performing gene-expression profiling and immunohistochemical evaluation, we compared the morphological and molecular features of S-HCC with those of CC and HCC. S-HCC expresses both CC-like and stem-cell-like genomic traits. In addition, we observed the expression of core epithelial-mesenchymal transition (EMT)-related genes, which may contribute to the aggressive behavior of S-HCC. Overexpression of transforming growth factor beta (TGF-β) signaling was also found, implying its regulatory role in the pathobiology of S-HCC.. We suggest that the fibrous stromal component in HCC may contribute to the acquisition of CC-like gene-expression traits in HCC. The expression of stem-cell-like traits and TGF-β/EMT molecules may play a pivotal role in the aggressive phenotyping of S-HCC. (HEPATOLOGY 2012;55:1776-1786).

Topics: Adult; Aged; Aged, 80 and over; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Cholangiocarcinoma; Epithelial-Mesenchymal Transition; Female; Fibrosis; Gene Expression Profiling; Humans; Liver Neoplasms; Male; Middle Aged; Signal Transduction; Transforming Growth Factor beta

The study was aimed to investigate the relationship between plasma transforming growth factor beta 1 (TGF-β(1)) expression and the characteristics of hepatocellular carcinoma (HCC).. Five hundred and seventy-one patients with HCC were subjected. Plasma TGF-β(1) levels were measured by enzyme-linked immunosorbent assay at diagnosis and compared in accordance with clinical and radiological characteristics.. Plasma TGF-β(1) levels were significantly higher in the diffuse infiltrative type (n = 159) than in the nodular type of HCC (n = 412; 3.94 ± 0.34 vs. 3.79 ± 0.29 log(10) pg/ml; p < 0.001). They were much higher in patients with portal vein thrombosis or extrahepatic metastasis than in those without (3.88 ± 0.34 vs. 3.81 ± 0.29 log(10) pg/ml, p = 0.008; 3.94 ± 0.35 vs. 3.82 ± 0.30 log(10) pg/ml, p = 0.013, respectively). Also, plasma TGF-β(1) levels showed a positive correlation with the size of HCC (r = 0.014, p < 0.001). Additionally, plasma TGF-β(1) levels were inversely related to the survival periods (p < 0.001).. TGF-β(1) was overexpressed in invasive types of HCC and it may be involved in the rapid progression of HCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Cell Proliferation; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide. In Egypt, the disease is usually detected in an advanced stage at which no treatment may be effective including surgery. Early detection of the disease is thus an important goal allowing the patient to be treated before the enlargement of the tumor or its metastasis to distant organs. Tumor markers are serological agents which serum level may be useful in predicting the presence of the tumor at early stages. Alpha fetoprotein (AFP) which is the golden marker for HCC is of low sensitivity, therefore, additional markers such as alpha-L-fucosidase (AFU), transforming growth factors alpha and beta (TGF-α and TGF-β) and interleukin-8 (IL-8) are suggested to be simultaneously evaluated in order to enhance the detection of HCC. A total of 96 patients with different liver diseases such as HCC, hepatitis C virus (HCV), hepatitis B virus (HBV) and cirrhotic patients are included in this study. Sixteen healthy volunteers are used as a control group. In patients with HCC each of AFP, AFU, TGF-α and TGF-β recorded significantly higher levels than the other patient groups and controls. HCC patients recorded significantly lower level of IL-8 compared to the other patient groups but significantly higher than the control. For AFP, AFU, TGF-α, TGF-β and IL-8, at the optimal cut-off values (obtained from the receiver operating characteristic (ROC) curves), the calculated sensitivities are 46%, 72.97%, 67.56%, 54.05% and 83.8%, respectively. The simultaneous evaluation using all of the suggested markers resulted in increasing the sensitivity up to 100%. It thus recommended that, if patients with cirrhosis, as high risk patients, are subjected to regular examination using these markers in addition to AFP, HCC may be detected by 100% sensitivity in an early stage and as a consequence an effective treatment can be achieved.

Topics: Adult; Aged; alpha-Fetoproteins; alpha-L-Fucosidase; Biomarkers, Tumor; Carcinoma, Hepatocellular; Case-Control Studies; Early Detection of Cancer; Egypt; Female; Fibrosis; Hepatitis, Viral, Human; Humans; Interleukin-8; Liver Neoplasms; Male; Middle Aged; ROC Curve; Transforming Growth Factor alpha; Transforming Growth Factor beta

FNDC3B was recently identified in an oncogenomic screen for amplified oncogenes in hepatocellular carcinoma. It is located at 3q26 and is amplified in over 20% of cancers, usually as part of a broad amplified region encompassing the entire 3q arm. Consistent with an oncogenic role in multiple cancer types, we show here that overexpression of FNDC3B is capable of malignantly transforming mammary and kidney epithelial cells in addition to hepatocytes. To explore how FNDC3B transforms cells, we determined the cellular localization of its gene product and the cancer pathways that it activates. We found that the FNDC3B oncoprotein localizes to the Golgi network, and that its correct localization is essential for its transforming function. We found that overexpression of FNDC3B induces the epithelial-to-mesenchymal transition (EMT) and activates several cancer pathways, including PI3-kinase/Akt, Rb1 and TGFβ signaling. For TGFβ signaling, we analyzed the point in the pathway at which FNDC3B operates and obtained evidence that it induces expression of all three TGFβ ligands and also promotes TGFBR1 cell-surface localization. We found that RNAi-mediated knockdown of FNDC3B in cancer cells with 3q amplification suppressed their clonogenicity and tumorigenicity, but that the same RNAi knockdown had no effect on single-copy 3q cancer cells. These results indicate that FNDC3B is an important oncogenic driver gene of the 3q amplicon, adding to the growing list of oncogenic drivers within this commonly amplified region.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 3; Epithelial-Mesenchymal Transition; Fibronectins; Gene Dosage; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genetic Vectors; Golgi Apparatus; Humans; Immunoprecipitation; Oncogenes; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Retroviridae; RNA Interference; Signal Transduction; Transcriptional Activation; Transfection; Transforming Growth Factor beta

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC.

Topics: Base Sequence; Carcinoma, Hepatocellular; DNA Primers; Humans; Hyaluronan Receptors; Liver Neoplasms; Mesoderm; Phenotype; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Molecular signatures causing hepatocellular carcinoma (HCC) from chronic infection of hepatitis B virus (HBV) or hepatitis C virus (HCV) are not clearly known. Using microarray datasets composed of HCV-positive HCC or HBV-positive HCC, pathways that could discriminate tumor tissue from adjacent non-tumor liver tissue were selected by implementing nearest shrunken centroid algorithm. Cancer-related signaling pathways and lipid metabolism-related pathways were predominantly enriched in HCV-positive HCC, whereas functionally diverse pathways including immune-related pathways, cell cycle pathways, and RNA metabolism pathways were mainly enriched in HBV-positive HCC. In addition to differentially involved pathways, signaling pathways such as TGF-β, MAPK, and p53 pathways were commonly significant in both HCCs, suggesting the presence of common hepatocarcinogenesis process. The pathway clustering also verified segregation of pathways into the functional subgroups in both HCCs. This study indicates the functional distinction and similarity on the pathways implicated in the development of HCV- and/or HBV-positive HCC.

Topics: Carcinoma, Hepatocellular; Cell Cycle; DNA; Gene Expression Regulation, Neoplastic; Hepacivirus; Hepatitis B; Hepatitis B virus; Hepatitis C; Humans; Liver; Liver Neoplasms; Microarray Analysis; Mitogen-Activated Protein Kinases; Multigene Family; Sequence Analysis, DNA; Signal Transduction; Transforming Growth Factor beta

Intrahepatic malignant tumours include hepatocellular carcinomas (HCC), cholangiocarcinomas (CC) and combined hepatocholangiocarcinomas (cHCC-CC), a group of rare and poorly characterized tumours that exhibit both biliary and hepatocytic differentiation. The aim of the study was to characterize the molecular pathways specifically associated with cHCC-CC pathogenesis. We performed a genome-wide transcriptional analysis of 20 histologically defined cHCC-CC and compared them with a series of typical HCC and of CC. Data were analysed by gene set enrichment and integrative genomics and results were further validated in situ by tissue microarray using an independent series of 152 tumours. We report that cHCC-CC exhibit stem/progenitor features, a down-regulation of the hepatocyte differentiation program and a commitment to the biliary lineage. TGFβ and Wnt/β-catenin were identified as the two major signalling pathways activated in cHCC-CC. A β-catenin signature distinct from that observed in well-differentiated HCC with mutant β-catenin was found in cHCC-CC. This signature was associated with microenvironment remodelling and TGFβ activation. Furthermore, integrative genomics revealed that cHCC-CC share characteristics of poorly differentiated HCC with stem cell traits and poor prognosis. The common traits displayed by CC, cHCC-CC and some HCC suggest that these tumours could originate from stem/progenitor cell(s) and raised the hypothesis of a potential continuum between intrahepatic CC, cHCC-CC and poorly differentiated HCC.

Topics: beta Catenin; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Cell Differentiation; Cholangiocarcinoma; Extracellular Matrix; Gene Expression Profiling; Humans; Liver Neoplasms; Neoplastic Stem Cells; Prognosis; Signal Transduction; Tissue Array Analysis; Transforming Growth Factor beta; Wnt Signaling Pathway

Loss of signal transducer and activator of transcription 5 (STAT5) from liver tissue results in steatosis and enhanced cell proliferation. This study demonstrates that liver-specific Stat5-null mice develop severe hepatic steatosis as well as hepatocellular carcinomas at 17 months of age, even in the absence of chemical insults. To understand STAT5's role as a tumor suppressor, we identified and investigated new STAT5 target genes. Expression of Nox4, the gene encoding the reactive oxygen species (ROS)-generating enzyme NOX4, was induced by growth hormone through STAT5. In addition, the genes encoding the proapoptotic proteins PUMA and BIM were induced by growth hormone through STAT5, which bound to GAS motifs in the promoter regions of all three genes. We further show that STAT5-induced activation of Puma and Bim was dependent on NOX4. Treatment of mice with transforming growth factor-β, an inducer of apoptosis, resulted in cleaved caspase-3 in control but not in liver-specific Stat5-null mice. This study demonstrates for the first time that cytokines through STAT5 regulate the expression of the ROS-generating enzyme NOX4 and key proapoptotic proteins.. STAT5 harnesses several distinct signaling pathways in the liver and thereby functions as a tumor suppressor. Besides suppressing the activation of STAT3, STAT5 induces the expression of proapoptotic genes and the production of ROS.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carbon Tetrachloride; Carcinoma, Hepatocellular; Caspase 3; Cells, Cultured; Fatty Liver; Fibroblasts; Gene Expression Regulation, Neoplastic; Growth Hormone; Hepatocytes; Liver; Liver Neoplasms; Membrane Proteins; Mice; NADPH Oxidase 4; NADPH Oxidases; Promoter Regions, Genetic; Proto-Oncogene Proteins; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; STAT5 Transcription Factor; Transforming Growth Factor beta; Tumor Suppressor Proteins

Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt signaling. Underlying biologic mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer.. The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs), and CTNNB1-mutation data (91 HCCs). Effects of sorafenib on Wnt signaling were evaluated in four liver cancer cell lines with active Wnt signaling and a tumor xenograft model.. Evidence for Wnt activation was observed for 315 (49.1%) cases, and was further classified as CTNNB1 class (138 cases [21.5%]) or Wnt-TGFβ class (177 cases [27.6%]). CTNNB1 class was characterized by upregulation of liver-specific Wnt-targets, nuclear β-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, whereas Wnt-TGFβ class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear β-catenin. Sorafenib decreased Wnt signaling and β-catenin protein in HepG2 (CTNNB1 class), SNU387 (Wnt-TGFβ class), SNU398 (CTNNB1-mutation), and Huh7 (lithium-chloride-pathway activation) cell lines. In addition, sorafenib attenuated expression of liver-related Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1 class-specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals.. Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGFβ), accounting for half of all HCC patients. Sorafenib modulates β-catenin/Wnt signaling in experimental models that harbor the CTNNB1 class signature.

Topics: Animals; Antineoplastic Agents; beta Catenin; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cluster Analysis; Female; Gene Expression Profiling; Genomics; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Reproducibility of Results; Sorafenib; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway

We have reported the synthesis of a novel salen ligand and its mononuclear Pd-salen complexes derived from 2-{[2-hydroxy-3-{[(E)-(2-hydroxyphenyl)methylidene]amino}propyl)imino]methyl}phenol. The newly synthesized and isolated Pd(II) complexes have been identified and fully characterized by various physico-chemical studies viz., elemental analyses, IR, UV-Vis, (1)H, (13)C NMR spectroscopy, electron spray ionization mass spectrometry (ESI-MS) and TGA/DTA studies. The molecular structure of the salen ligand has been ascertained by single-crystal XRD and it is coordinated to Pd(II) ion through two nitrogen and two oxygen atoms. The UV-Vis data clearly suggest a square-planar environment around both the Pd(II) ions. The DNA binding studies of the synthesized compounds has been investigated by electron spectroscopy and fluorescence measurements. The results suggest that Pd(II) complexes bind to DNA strongly as compared to the free ligand. The free salen ligand and its Pd(II) complexes have also been tested against human hepatoma cancer cell line (Huh7) and results manifested exceptional anti-proliferative effects of the Pd(II) complexes. The anti-proliferative activity of Pd(II) complexes has been modulated by specific regulatory genes.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Coordination Complexes; Crystallography, X-Ray; DNA; Ethylenediamines; Humans; Hydrogen Bonding; Liver Neoplasms; Molecular Conformation; Palladium; Transforming Growth Factor beta; Tumor Suppressor Proteins

In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodeling and wound healing. Tumors apply persistent mechanical and pathological stress to tissues and causes continual infiltration of MSCs. Here, we demonstrate that MSCs promote human hepatocellular carcinoma (HCC) metastasis under the influence of inflammation. The metastasis promoting effect could be imitated with the supernatant of MSCs pretreated with IFNγ and TNFα. Interestingly, treatment of HCC cells with the supernatant leads to epithelial-mesenchymal transition (EMT), an effect related to the production of TGFβ by cytokines stimulated MSCs. Importantly, the levels of MSCs expressing SSEA4 in clinical HCC samples significantly correlated with poor prognosis of HCC, and EMT of HCC was strongly associated with a shorter cancer-free interval (CFI) and a worse overall survival (OS). Therefore, our results suggest that MSCs in tumor inflammatory microenvironment could promote tumor metastasis through TGFβ-induced EMT.

Topics: Aged; Carcinoma, Hepatocellular; Cytokines; Epithelial-Mesenchymal Transition; Female; Humans; Inflammation; Liver Neoplasms; Male; Mesenchymal Stem Cells; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Phenotype; Proportional Hazards Models; Transforming Growth Factor beta; Treatment Outcome; Wound Healing

Portal vein tumor thrombus (PVTT) is strongly correlated to a poor prognosis for patients with hepatocellular carcinoma (HCC). In this study, we uncovered a causative link between hepatitis B virus (HBV) infection and development of PVTT. Mechanistically, elevated TGF-β activity, associated with the persistent presence of HBV in the liver tissue, suppresses the expression of microRNA-34a, leading to enhanced production of chemokine CCL22, which recruits regulatory T (Treg) cells to facilitate immune escape. These findings strongly suggest that HBV infection and activity of the TGF-β-miR-34a-CCL22 axis serve as potent etiological factors to predispose HCC patients for the development of PVTT, possibly through the creation of an immune-subversive microenvironment to favor colonization of disseminated HCC cells in the portal venous system.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Chemokine CCL22; Female; Hepatitis B; Hepatitis B virus; Humans; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; Portal Vein; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Neutrophil infiltration has been linked to clinical outcome of various cancer types. However, its role in hepatocellular carcinoma (HCC) is unclear. In this study, we investigated prognostic values for intratumoral and peritumoral neutrophils in HCC patients undergoing curative resection.. The expression of CD66b, CD8, TGF-beta, and CD34 was assessed by immunohistochemistry in tissue microarrays containing paired intratumoral and peritumoral tissues from 197 patients receiving curative resection for HCC. Prognostic values for these and other clinicopathologic factors were evaluated.. Intratumoral CD66b(+) neutrophils significantly correlated with CD8(+) T cells (r=0.240, p=0.004), TGF-beta expression (p=0.012), BCLC stage (p=0.016), and early recurrence (p=0.041). Increased intratumoral neutrophils were significantly associated with decreased RFS/OS (p=0.001 and p<0.001, respectively) in univariate analysis and were identified as an independent prognostic factor (HR=1.845, 95% CI=1.169-2.911, p=0.008 for RFS; HR=2.578, 95% CI=1.618-4.106, p<0.001 for OS) in multivariate analysis. Intratumoral neutrophil-to-CD8(+) T cell ratio (iNTR) better predicted the outcome in terms of minimum p values. Intratumoral neutrophils were also demonstrated to be statistically predictive for RFS/OS in the normal AFP subgroup, small HCC subgroup, and validation cohort. However, peritumoral neutrophils were not associated with the outcome of HCC.. The presence of intratumoral neutrophils was a poor prognostic factor for HCC after resection. Intratumoral neutrophil-to-CD8(+) T cell ratio was a better predictor of outcome.

Topics: Antigens, CD; Antigens, CD34; Carcinoma, Hepatocellular; CD8 Antigens; Cell Adhesion Molecules; Cohort Studies; Female; GPI-Linked Proteins; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neutrophil Infiltration; Neutrophils; Prognosis; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) induces apoptosis in hepatocytes, a process that is inhibited by the epidermal growth factor receptor (EGFR) pathway. The aim of this work was to ablate EGFR in hepatocellular carcinoma (HCC) cells to understand its role in impairing TGF-β-induced cell death.. Response to TGF-β in terms of apoptosis was analyzed in different HCC cell lines and the effect of canceling EGFR expression was evaluated.. TGF-β induces apoptosis in some HCC cells (such as Hep3B, PLC/PRF/5, Huh7, or SNU449), but it also mediates survival signals, coincident with the up-regulation of EGFR ligands. Inhibition of the EGFR, either by targeted knock-down with specific siRNA or by pharmacological inhibition, significantly enhances apoptotic response. TGF-β treatment in EGFR targeted knock-down cells correlates with higher levels of the NADPH oxidase NOX4 and changes in the expression profile of BCL-2 and IAP families. However, other HCC cells, such as HepG2, which show over activation of the Ras/ERKs pathway, SK-Hep1, with an endothelial phenotype, or SNU398, where the TGF-β-Smad signaling is altered, show apoptosis resistance that is not restored through EGFR blockade.. The inhibition of EGFR in HCC may enhance TGF-β-induced pro-apoptotic signaling. However, this effect may only concern those tumors with an epithelial phenotype which do not bear alterations in TGF-β signaling nor exhibit an over-activation of the survival pathways downstream of the EGFR.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, bcl-2; Humans; Liver Neoplasms; RNA, Small Interfering; Signal Transduction; Transcriptome; Transforming Growth Factor beta

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.

Topics: Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclic GMP; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nickel; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; S-Nitroso-N-Acetylpenicillamine; Thapsigargin; Transfection; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) is an important regulator of cell growth, and loss of TGF-β signaling is a hallmark of carcinogenesis. The Smad3/4 adaptor protein β2-spectrin (β2SP) is emerging as a potent regulator of tumorigenesis through its ability to modulate the tumor suppressor function of TGF-β. However, to date the role of the TGF-β signaling pathway at specific stages of the development of hepatocellular carcinoma (HCC), particularly in relation to the activation of other oncogenic pathways, remains poorly delineated. Here we identify a mechanism by which β2SP, a crucial Smad3 adaptor, modulates cyclin dependent kinase 4 (CDK4), cell cycle progression, and suppression of HCC. Increased expression of β2SP inhibits phosphorylation of the retinoblastoma gene product (Rb) and markedly reduces CDK4 expression to a far greater extent than other CDKs and cyclins. Furthermore, suppression of CDK4 by β2SP efficiently restores Rb hypophosphorylation and cell cycle arrest in G(1) . We further demonstrate that β2SP interacts with CDK4 and Smad3 in a competitive and TGF-β-dependent manner. In addition, haploinsufficiency of cdk4 in β2sp(+/-) mice results in a dramatic decline in HCC formation compared to that observed in β2sp(+/-) mice.. β2SP deficiency leads to CDK4 activation and contributes to dysregulation of the cell cycle, cellular proliferation, oncogene overexpression, and the formation of HCCs. Our data highlight CDK4 as an attractive target for the pharmacologic inhibition of HCC and demonstrate the importance of β2sp(+/-) mice as a model of preclinical efficacy in the treatment of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin-Dependent Kinase 4; Haploinsufficiency; Liver Neoplasms; Mice; Smad3 Protein; Spectrin; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β (TGF-β) is implicated in hepatocarcinogenesis and hepatocellular carcinoma (HCC) metastasis. HAb18G/CD147, which belongs to the CD147 family, is an HCC-associated antigen that has a crucial role in tumor invasion and metastasis. The goal of this study was to investigate the role of HAb18G/CD147 during EMT in hepatocarcinogenesis. Human normal hepatic cell lines QZG and L02, primary mouse hepatocytes and nude mouse models were used to determine the role of HAb18G/CD147 in EMT, and the involvement of the TGF-β-driven pathway. A dual-luciferase reporter assay and ChIP were used to investigate the transcriptional regulation of the CD147 gene. Samples from patients with liver disease were assessed to determine the relationship between HAb18G/CD147 and typical markers for EMT. Our results show that upregulation of HAb18G/CD147 is induced by TGF-β coupled with downregulation of E-cadherin and upregulation of N-cadherin and vimentin. The expression of HAb18G/CD147 is controlled by the cell survival PI3K/Akt/GSK3β signaling pathway, and is directly regulated by the transcription factor Slug. Transfection of CD147 also induces an elevated expression of TGF-β. CD147-transfected hepatocytes have mesenchymal phenotypes that accelerate tumor formation and tumor metastasis in vivo. Immunohistochemistry analysis shows a negative correlation between HAb18G/CD147 and E-cadherin expression (r(s)=-0.3622, P=0.0105), and a positive correlation between HAb18G/CD147 and Slug expression (r(s)=0.3064, P=0.0323) in human HCC tissues. Our study uncovers a novel role of HAb18G/CD147 in mediating EMT in the process of HCC progression and showed that CD147 is a Slug target gene in the signaling cascade TGF-β→PI3K/Akt→GSK3β→Snail→Slug→CD147. Our results suggest that CD147 may be a potential target for the treatment and prevention of HCC.

Topics: Animals; Basigin; Cadherins; Carcinoma, Hepatocellular; Cell Line; Cell Transformation, Neoplastic; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mice; Mice, Nude; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

HCC (hepatocellular carcinoma) is among the most common and lethal cancers worldwide with a poor prognosis mainly due to a high recurrence rate and chemotherapy resistance. ATO (arsenic trioxide) is a multi-target drug that has been effectively used as an anticancer drug in acute promyelocytic leukaemia. However, a Phase II trial involving patients with HCC indicates that the use of arsenic as a single agent is not effective against HCC. TGIF (TG-interacting factor) is a transcriptional co-repressor that interferes with TGF-β (transforming growth factor-β) signalling which plays a growth-inhibitory role in HCC. In the present study, we demonstrated that ATO induced hepatocellular apoptosis via TGF-β/Smad signalling and led to downstream induction of p21(WAF1/CIP1) (p21). However, ATO could also induce TGIF expression via a post-transcriptional regulation mechanism to antagonize this effect. Using a biotin-labelled RNA probe pull-down assay and in vivo RNA immunoprecipitation analysis, we identified that HuR (human antigen R) bound to the TGIF mRNA 3'-UTR (3'-untranslated region) and prevented it from degradation. ATO treatment increased the interaction between HuR and TGIF mRNA, and reduction of HuR expression inhibited ATO-induced TGIF expression. Moreover, the EGFR (epidermal growth factor receptor)/PI3K (phosphoinositide 3-kinase)/Akt pathway was shown to mediate the post-transcriptional regulation of TGIF in response to ATO. Finally, we also demonstrated that the down-regulation of TGIF could sensitize ATO-induced HepG2 cell apoptosis. Collectively, we propose that the EGFR/PI3K/Akt pathway may regulate the post-transcriptional regulation of TGIF expression to antagonize ATO-induced apoptosis in HCC. Blockage of the PI3K/Akt pathway or TGIF expression combined with ATO treatment may be a promising strategy for HCC therapy.

Topics: 3' Untranslated Regions; Antigens, Surface; Apoptosis; Arsenic Trioxide; Arsenicals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; ELAV Proteins; ELAV-Like Protein 1; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Liver Neoplasms; Models, Biological; Oxides; Phosphatidylinositol 3-Kinases; Repressor Proteins; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

Cirrhotic septa harbor vessels and inflammatory, fibrogenic, and ductular epithelial cells, collectively referred to as the ductular reaction (DR). Lack of the DR in the stromal compartment around hepatocellular carcinoma (HCC) has been documented; however, the relationship of epithelial keratin 19 (K19) structures to progression of intralesional carcinogenesis has not been explored. K19 immunoreactivity in the stromal compartment around 176 nodules in cirrhotic explants was examined. Quantitative differences (P < 0.0001) were manifested in three distinct histologically identifiable patterns: "complex" around cirrhotic nodules (CN), "attenuated" around dysplastic nodules (DN), and "absent" around HCC. Markers of necrosis or apoptosis could not explain the perinodular K19 epithelial loss; however, multicolor immunolabeling for K19, vimentin, E-Cadherin, SNAIL, and fibroblast-specific protein 1 (FSP-1) demonstrated discrepancies in immunophenotype and cytomorphologic features. Variability of cellular features was accompanied by an overall decrease in epithelial markers and significantly increased fractions of SNAIL- and FSP-1-positive cells in the DR around DN when compared with CN (P < 0.0001). Immunolabeling of transforming growth factor-β signaling components (TGFβR1, SMAD3, and pSMAD2/3) demonstrated increased percentages of pSMAD2/3 around DN when compared with CN (P < 0.0001). These findings collectively suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. Paracrine signaling is proposed as a link that emphasizes the importance of the epithelial-stromal compartment in malignant progression of HCC in cirrhosis.

Topics: Apoptosis; Biopsy; Carcinoma, Hepatocellular; Epithelium; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Keratin-19; Liver Cirrhosis; Liver Neoplasms; Models, Biological; Necrosis; Predictive Value of Tests; Signal Transduction; Transforming Growth Factor beta

Prognostic prediction is a vital component in clinical management of hepatocellular carcinoma (HCC) patients. Rapidly evolving genomic assays serve as flexible sources to discover molecular signatures that sensitively and specifically predict clinical outcome of the patients. Studies have identified molecular signatures of HCC tumors that depict biological aggressiveness yielded through activation of specific genes and molecular pathways such as MYC, AKT, TGF-beta, and IGF2 as well as inactivation of TP53 pathway. Despite the promise for tumor-derived signatures' role in therapeutic target discovery, their value as prognostic marker seems to be limited especially in early-stage HCC, which has been increasing as a result of successful implementation HCC surveillance for patients with liver cirrhosis. In contrast, non-tumor, diseased liver tissues turn out to be a rich source of molecular information to capture propensity to hepatocarcinogenesis and metastasis through dyregulation of growth signaling and inflammatory/oxidative stress/immune response. In addition, the liver-derived signatures hold prognostic relevance irrespective of HCC stage, suggesting their role as prognostic markers. Furthermore, they may also be utilized for development of HCC chemoprevention therapy.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Gene Expression Regulation; Genes, myc; Genes, p53; Humans; Insulin-Like Growth Factor II; Liver Neoplasms; Predictive Value of Tests; Prognosis; Proto-Oncogene Proteins c-akt; Sensitivity and Specificity; Signal Transduction; Transforming Growth Factor beta

Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.. Gene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation.. TGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Homeodomain Proteins; Humans; Liver Neoplasms; Mice; Nanog Homeobox Protein; Neoplastic Stem Cells; Promoter Regions, Genetic; Signal Transduction; Smad Proteins; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Radix Glycyrrhizae polysaccharide (GP) possesses multiple pharmacological activities. However, the effect of GP on CD4+CD25+ regulatory T (Treg) cells has not been elucidated. This study aimed to investigate the effects of GP on Treg cells and Th1/Th2 cytokines in H22 hepatocarcinoma tumor-bearing mice. The results demonstrated that GP inhibits tumor progression. In the lymph nodes of the tumor microenvironment and spleen, the proportion of Treg cells was significantly higher in the tumor-bearing mice. GP administration down-regulated the population of Treg cells (P < 0.01) and decreased lymph node Foxp3 and IL-10 mRNA expression (P < 0.01). In addition, GP treatment decreased IL-10 and TGF-β level (P < 0.01) and increased IL-2 and IL-12p70 level in serum (P < 0.01). In conclusion, GP reduced the proportion of Treg cells and Foxp3 lowered expression in Treg cells, and up-regulated Th1/Th2 cytokine ratio in serum in the tumor bearing mice, which might partially cause the inhibition of tumor growth.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Proliferation; Cytokines; Down-Regulation; Drugs, Chinese Herbal; Female; Forkhead Transcription Factors; Glycyrrhiza; Interleukin-10; Interleukin-12; Liver Neoplasms; Mice; Mice, Inbred BALB C; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Polysaccharides; RNA, Messenger; T-Lymphocytes, Regulatory; Th1 Cells; Th1-Th2 Balance; Th2 Cells; Transforming Growth Factor beta

Tumor-stroma interactions in hepatocellular carcinoma (HCC) are of key importance to tumor progression. In this study, we show that HCC invasive cells produce high levels of connective tissue growth factor (CTGF) and generate tumors with a high stromal component in a xenograft model. A transforming growth factor beta (TGF-beta) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF-beta-dependent down-regulation of CTGF diminished tumor growth, intravasation, and metastatic dissemination of HCC cells by inhibiting cancer-associated fibroblast proliferation. By contrast, noninvasive HCC cells were found to produce low levels of CTGF. Upon TGF-beta1 stimulation, noninvasive HCC cells form tumors with a high stromal content and CTGF expression, which is inhibited by treatment with LY2109761. In addition, the acquired intravasation and metastatic spread of noninvasive HCC cells after TGF-beta1 stimulation was blocked by LY2109761. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to a significant reduction of HCC growth and dissemination. Interestingly, patients with high CTGF expression had poor prognosis, suggesting that treatment aimed at reducing TGF-beta-dependent CTGF expression may offer clinical benefits.. Taken together, our preclinical results indicate that LY2109761 targets the cross-talk between HCC and the stroma and provide a rationale for future clinical trials.

Topics: Carcinoma, Hepatocellular; Connective Tissue Growth Factor; Disease Progression; Down-Regulation; Humans; Liver Neoplasms; Pyrazoles; Pyrroles; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta cooperates with oncogenic Ras to activate nuclear beta-catenin during the epithelial to mesenchymal transition of hepatocytes, a process relevant in the progression of hepatocellular carcinoma (HCC). In this study we investigated the role of beta-catenin in the differentiation of murine, oncogene-targeted hepatocytes and in 133 human HCC patients scheduled for orthotopic liver transplantation. Transforming growth factor-beta caused dissociation of plasma membrane E-cadherin/beta-catenin complexes and accumulation of nuclear beta-catenin in Ras-transformed, but otherwise normal hepatocytes in p19(ARF)-/- mice. Both processes were inhibited by Smad7-mediated disruption of transforming growth factor-beta signaling. Overexpression of constitutively active beta-catenin resulted in high levels of CK19 and M2-PK, whereas ablation of beta-catenin by axin overexpression caused strong expression of CK8 and CK18. Therefore, nuclear beta-catenin resulted in dedifferentiation of neoplastic hepatocytes to immature progenitor cells, whereas loss of nuclear beta-catenin led to a differentiated HCC phenotype. Poorly differentiated human HCC showed cytoplasmic redistribution or even loss of E-cadherin, suggesting epithelial to mesenchymal transition. Analysis of 133 HCC patient samples revealed that 58.6% of human HCC exhibited strong nuclear beta-catenin accumulation, which correlated with clinical features such as vascular invasion and recurrence of disease after orthotopic liver transplantation. These data suggest that activation of beta-catenin signaling causes dedifferentiation to malignant, immature hepatocyte progenitors and facilitates recurrence of human HCC after orthotopic liver transplantation.

Topics: Animals; beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Differentiation; Cell Membrane; Cell Nucleus; Epithelium; Female; Hepatocytes; Humans; Liver; Liver Neoplasms; Liver Transplantation; Male; Mesoderm; Mice; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Phenotype; Protein Transport; Signal Transduction; Smad7 Protein; Stem Cells; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) results from the cumulative effects of deregulated tumor suppressor genes and oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and their downstream signaling pathway components. The overexpression of transforming growth factor alpha (TGF-alpha) and the inhibition of TGF-beta signaling are especially common in human liver cancer. Thus, we assessed whether TGF-alpha overexpression and TGF-beta signaling inactivation cooperate in hepatocarcinogenesis using an in vivo mouse model, MT1/TGFa;AlbCre/Tgfbr2(flx/flx) mice ("TGFa;Tgfbr2(hepko)"), which overexpresses TGF-alpha and lacks a TGF-beta receptor in the liver. TGF-beta signaling inactivation did not alter the frequency or number of cancers in mice with overexpression of TGF-alpha. However, the tumors in the TGFa;Tgfbr2(hepko) mice displayed increased proliferation and increased cdk2, cyclin E and cyclin A expression as well as decreased Cdkn1a/p21 expression compared to normal liver and compared to the cancers arising in the TGF-alpha overexpressing mice with intact TGF-beta receptors. Increased phosphorylated ERK1/2 expression was also present in the tumors from the TGFa;Tgfbr2(hepko) mice and correlated with downregulated Raf kinase inhibitor protein expression, which is a common molecular event in human HCC. Thus, TGF-beta signaling inactivation appears to cooperate with TGF-alpha in vivo to promote the formation of liver cancer that recapitulates molecular features of human HCC.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Integrases; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta

To identify microRNAs (miRNAs) that may have a causal role in hepatocarcinogenesis, we used an animal model in which C57BL/6 mice fed choline-deficient and amino acid defined (CDAA) diet develop preneoplastic lesions at 65 weeks and hepatocellular carcinomas after 84 weeks. miRNA expression profiling showed significant upregulation of miR-181b and miR-181d in the livers of mice as early as 32 weeks that persisted at preneoplastic stage. The expression of tissue inhibitor of metalloprotease 3 (TIMP3), a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic transforming growth factor (TGF)beta and its downstream mediators Smad 2, 3 and 4 and increase in phospho-Smad2 in the liver nuclear extract correlated with elevated miR-181b/d in mice fed CDAA diet. The levels of the precursor and mature miR-181b were augmented on exposure of hepatic cells to TGFbeta and were significantly reduced by small interference RNA-mediated depletion of Smad4, showing the involvement of TGFbeta signaling pathway in miR-181b expression. Ectopic expression and depletion of miR-181b showed that miR-181b enhanced matrix metallopeptidases (MMP)2 and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of hepatocellular carcinoma (HCC) cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice. miR-181b also enhanced resistance of HCC cells to the anticancer drug doxorubicin. On the basis of these results, we conclude that upregulation of miR-181b at early stages of feeding CDAA diet promotes hepatocarcinogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Movement; Colony-Forming Units Assay; Diet; Disease Models, Animal; Humans; Liver; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; MicroRNAs; Neoplasm Staging; Tissue Inhibitor of Metalloproteinase-3; Tissue Inhibitor of Metalloproteinase-4; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Up-Regulation

Hepcidin is the master regulatory hormone of systemic iron metabolism. Hepcidin deficiency causes common iron overload syndromes whereas its overexpression is responsible for microcytic anemias. Hepcidin transcription is activated by the bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways, whereas comparatively little is known about how hepcidin expression is inhibited. By using high-throughput siRNA screening we identified SMAD7 as a potent hepcidin suppressor. SMAD7 is an inhibitory SMAD protein that mediates a negative feedback loop to both transforming growth factor-beta and BMP signaling and that recently was shown to be coregulated with hepcidin via SMAD4 in response to altered iron availability in vivo. We show that SMAD7 is coregulated with hepcidin by BMPs in primary murine hepatocytes and that SMAD7 overexpression completely abolishes hepcidin activation by BMPs and transforming growth factor-beta. We identify a distinct SMAD regulatory motif (GTCAAGAC) within the hepcidin promoter involved in SMAD7-dependent hepcidin suppression, demonstrating that SMAD7 does not simply antagonize the previously reported hemojuvelin/BMP-responsive elements. This work identifies a potent inhibitory factor for hepcidin expression and uncovers a negative feedback pathway for hepcidin regulation, providing insight into a mechanism how hepcidin expression may be limited to avoid iron deficiency.

Topics: Animals; Antimicrobial Cationic Peptides; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Down-Regulation; Feedback, Physiological; Hepatocytes; Hepcidins; Humans; Interleukin-6; Iron; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. CD133, a transmembrane glycoprotein, is an important cell surface marker for both stem cells and cancer stem cells in various tissues including liver. CD133 expression has been recently linked to poor prognosis in HCC patients. CD133+ liver cancer cells are characterized by resistance to chemotherapy, self-renewal, multilineage potential, increased colony formation, and in vivo cancer initiation at limited dilution. Recent studies demonstrate that CD133 expression is regulated by DNA methylation. In this study, we explored the role of transforming growth factor beta (TGFbeta), a multifunctional cytokine that plays a critical role in chronic liver injury, in the regulation of CD133 expression. TGFbeta1 is capable of up-regulating CD133 expression specifically within the Huh7 HCC cell line in a time- and dose-dependent manner. Most important, TGFbeta1-induced CD133+ Huh7 cells demonstrate increased tumor initiation in vivo. Forced expression of inhibitory Smads, including Smad6 and Smad7, attenuated TGFbeta1-induced CD133 expression. Within CD133- Huh7 cells, TGFbeta1 stimulation inhibited the expression of DNA methyltransferases (DNMT) 1 and DNMT3beta, which are critical in the maintenance of regional DNA methylation, and global DNMT activity in CD133- Huh7 cells was inhibited by TGFbeta1. DNMT3beta inhibition by TGFbeta1 was partially rescued with overexpression of inhibitory Smads. Lastly, TGFbeta1 treatment led to significant demethylation in CD133 promoter-1 in CD133- Huh7 cells.. TGFbeta1 is able to regulate CD133 expression through inhibition of DNMT1 and DNMT3beta expression and subsequent demethylation of promoter-1. TGFbeta1-induced CD133+ Huh7 cells are tumorigenic. The mechanism by which TGFbeta induces CD133 expression is partially dependent on the Smads pathway.

Topics: AC133 Antigen; Antigens, CD; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3B; Epigenesis, Genetic; Glycoproteins; Humans; Liver Neoplasms; Neoplastic Stem Cells; Peptides; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

Hepatic stem cells (HSCs) are involved in repair of liver injury. Stem cells may have inhibitory effects on tumor cell growth and apoptosis. However, it is unknown whether HSCs regulate the biological functions of hepatocarcinoma cells, especially tumor cell growth and apoptosis. The present study was designed to determine the effects of hepatocytic precursor (stem-like) WB-F344 cells on the growth and apoptosis of hepatoma CBRH-7919 cells. Using a Transwell chamber culture system, we co-cultured WB-F344 cells and CBRH-7919 cells in serum-free conditioned medium at 3 different ratios: 1:1 (2 x 10(5): 2 x 10(5) cells/well), 1:5 (4 x 10(4): 2 x 10(5) cells/well), and 5:1 (2 x 10(5): 4 x 10(4) cells/well). We determined the effects of stem cells on tumor cells using in vivo xenograft assay in nude mice and determining gene expression by RT-PCR and Western blot analyses. With the increment proportion of the WB-F344 cells in the co-culture system, tumor formation was inhibited in nude mice. Moreover, down-regulation of bone morphogenetic protein 4 (BMP4), Bcl-2, and c-Myc and upregulation of PTEN also occurred along with the inhibitory effects. Western blotting showed that the TGF-beta/Smad pathway played a prominent role in tumor inhibition, which may have been mediated by the cytokines released from the stem cells. In conclusion, hepatocytic precursor (stem-like) WB-F344 cells inhibit the tumorigenicity of hepatoma CBRH-7919 cells, and the effect is mediated by TGF-beta/Smad signaling pathway.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Coculture Techniques; Flow Cytometry; Hepatocytes; Liver Neoplasms, Experimental; Male; Mice; Mice, Nude; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad4 Protein; Stem Cells; Survival Rate; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) occurs mainly in the liver associated with chronic hepatitis and hepatic cirrhosis as a result of prolonged viral infection. Transforming growth factor-beta (TGF-beta) induces the fibrosis in hepatic cirrhosis, although it is also an inhibitor of hepatocyte proliferation. To understand the role of TGF-beta signaling in HCC progression, we analyzed gene expression in HCC cells in relation to TGF-beta signaling using a two-way clustering algorithm. By the analysis, five HCC cell lines were classified into two groups according to their metastatic capacity. TGF-beta receptor II (TGFBR2) was downregulated in metastatic cells, which did not show a response to TGF-beta. Immunohistochemistry demonstrated clear membrane distribution of TGFBR2 in noncancerous hepatocytes, whereas reduced TGFBR2 expression was observed in 34 of 136 HCCs. In clinical cases, reduced TGFBR2 expression correlated with larger tumor size (P<0.001), poor differentiation (P<0.001), portal vein invasion (P=0.002), intrahepatic metastasis (IM) (P<0.001), and shorter recurrence-free survival (P=0.022). In conclusion, reduced TGFBR2 expression was associated with aggressive features of HCC such as IM, and may represent an immunohistochemical biomarker to detect aggressive HCC.

Topics: Aged; Carcinoma, Hepatocellular; Down-Regulation; Female; Hepatitis, Chronic; Hepatocytes; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is considered as a disease of dysfunction of the stem cells. Studies on stem cells have demonstrated that Oct4 plays a pivotal role in embryo regulation. In order to understand the role of Oct4 in HCC and the relationship among Oct4 and wnt/β-catenin and TGF-β signal pathways, we have detected the expression of Oct4, Nanog, Sox2, STAT3 as well as the genes in wnt/β-catenin, and TGF-β families in HCC cell lines and in tumor specimens from HCC patients. The authors found that Oct4 was expressed in all of the four HCC cell lines and the tumor specimens from HCC patients. Some other genes were also expressed in them with different level including Nanog, Sox2, STAT3 and TCF3, wnt10b, β-catenin, ELF, Smad3 and Smad4. The ability of the clone formation and migration of the HepG2 decreased after Oct4 was knockdowned. Silencing of Oct4 and TCF3 in HCC cell line HepG2 revealed that there were complicated relationships among Oct4, wnt/β-catenin family and TGF-β family genes. Knockdowning Oct4 reduced the expression of TGF-β family genes ELF, Smad3, Smad4 and wnt/β-catenin family genes, wnt10b, and β-catenin but increased TCF3. In reverse, knockdowning TCF3 led to the increased expression of Oct4 and TGF-β family genes. In conclusion, the expression of Oct4 in HCC may play an important role as in stem cell. Because Oct4 improves not only the function of wnt/β-catenin, but also the TGF-β signal pathways, the significance of its expression in HCC might be more complicated than we evinced before.

Topics: Base Sequence; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Primers; Gene Knockdown Techniques; Humans; Liver Neoplasms; Octamer Transcription Factor-3; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16(Ink4a)) are frequently inactivated in these cancers. We tested whether transforming growth factor-beta (TGF-beta) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF-beta signaling that leads to small mothers against decapentaplegic (Smad)-targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF-beta1 (1-5 ng/mL) treatment. Upon treatment, c-myc was down-regulated, p21(Cip1) and p15(Ink4b) were up-regulated, and cells were arrested at G(1). The expression of p16(Ink4a) was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21(Cip1) and p15(Ink4b) recapitulated TGF-beta1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N-acetyl-L-cysteine, or silencing of the NOX4 gene, rescued p21(Cip1) and p15(Ink4b) accumulation as well as the growth arrest in response to TGF-beta. Human HCC tumors raised in immunodeficient mice also displayed TGF-beta1-induced senescence. More importantly, peritumoral injection of TGF-beta1 (2 ng) at 4-day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF-beta receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth.. TGF-beta induces p53-independent and p16(Ink4a)-independent, but Nox4-dependent, p21(Cip1)-dependent, p15(Ink4b)-dependent, and ROS-dependent senescence arrest in well-differentiated HCC cells. Moreover, TGF-beta-induced senescence in vivo is associated with a strong antitumor response against HCC.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p21; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Mice; Mice, Nude; NADPH Oxidase 4; NADPH Oxidases; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.

Topics: Cancer Vaccines; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Chemokine CXCL10; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Hep G2 Cells; Humans; Interferon-gamma; Interleukin-10; Interleukin-12; RNA; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, through a mechanism mediated by reactive oxygen species (ROS) production. Numerous tumoral cells develop mechanisms to escape from the TGF-beta-induced tumor suppressor effects. In this work we show that in FaO rat hepatoma cells inhibition of the epidermal growth factor receptor (EGFR) with the tyrphostin AG1478 enhances TGF-beta-induced cell death, coincident with an elevated increase in ROS production and GSH depletion. These events correlate with down-regulation of genes involved in the maintenance of redox homeostasis, such as gamma-GCS and MnSOD, and elevated mitochondrial ROS. Nonetheless, not all the ROS proceed from the mitochondria. Emerging evidences indicate that ROS production by TGF-beta is also mediated by the NADPH oxidase (NOX) system. TGF-beta-treated FaO cells induce nox1 expression. However, the treatment with TGF-beta and AG1478 greatly enhanced the expression of another family member: nox4. NOX1 and NOX4 targeted knock-down by siRNA experiments suggest that they play opposite roles, because NOX1 knockdown increases caspase-3 activity and cell death, whilst NOX4 knock-down attenuates the apoptotic process. This attenuation correlates with maintenance of GSH and antioxidant enzymes levels. In summary, EGFR inhibition enhances apoptosis induced by TGF-beta in FaO rat hepatoma cells through an increased oxidative stress coincident with a change in the expression pattern of NOX enzymes.

Topics: Animals; Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Isoenzymes; Liver Neoplasms; Mitochondria; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 4; NADPH Oxidases; Oxidative Stress; Protein Kinase Inhibitors; Quinazolines; Rats; Reactive Oxygen Species; RNA, Small Interfering; Transforming Growth Factor beta; Tyrphostins

betaig-h3 is a TGF-induced extracellular matrix (ECM) protein. Our previous evidence suggests that beta ig-h3 may promote adhesion and invasion potential of human hepatoma cells, but the mechanism underlying this process is still unknown. The present study identifies a pivotal role for molecules of the beta ig-h3 signal transduction pathway. We demonstrated that beta ig-h3 co-immunoprecipitated with alpha 3beta 1 integrin in human 7721 hepatoma cells. The addition of alpha 3beta 1 integrin antibodies inhibited the elevated adhesion and migration in beta ig-h3-over-expressing 7721 cells, but not in beta ig-h3 siRNA transfected 7721 cells. The expression and activity of integrin downstream molecules FAK and paxillin show a positive correlation with beta ig-h3 expression in different human hepatoma cells. Levels of focal adhesions and stress fibers were decreased in beta ig-h3 siRNA transfected 7721 cells. We suggest that by interaction with alpha 3beta 1 integrin, beta ig-h3 activates FAK-paxillin signaling linkage, leads to cytoskeleton reorganization, and thus enhances the metastatic potentials of human hepatoma cells.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cytoskeleton; Extracellular Matrix Proteins; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Integrin alpha3beta1; Liver Neoplasms; Neoplasm Invasiveness; Paxillin; Transfection; Transforming Growth Factor beta

Immunosuppressive therapy after liver transplantation for hepatocellular carcinoma (HCC) is one of the major contributory factors for HCC recurrence and metastasis. Sirolimus, a potent immunosuppressant, has been reported to be an effective inhibitor in a variety of tumors. The present study is designed to explore whether sirolimus could block the growth and metastatic progression of HCC.. MHCC97H cells were used as targets to explore the effect of sirolimus on cell cycle progression, apoptosis, proliferation, and its antiangiogenic mechanism. LCI-D20, a highly metastatic model of human HCC in nude mice, was also used as the model tumor to explore the effect of sirolimus on tumor growth and metastatic progression.. In vitro, sirolimus induced cell cycle arrest at the G1 checkpoint and blocked proliferation of MHCC97H cells but did not induce apoptosis. In vivo, sirolimus prevented tumor growth and metastatic progression in LCI-D20. Intratumoral microvessel density and circulating levels of VEGF in tumor-bearing mice were also significantly reduced in sirolimus treatment group. Quantitative RT-PCR showed that sirolimus down-regulated the mRNA expression of VEGF and HIF-1a, but not of bFGF, and TGF-b in MHCC97H cells. Furthermore, western blot analysis confirmed that sirolimus also decreased expression of HIF-1a at protein level, in parallel with the down-regulation of the levels of VEGF protein excretion in a time-dependent manner as compared to untreated control cells following anoxia.. The immunosuppressive macrolide sirolimus prevents the growth and metastatic progression of HCC, and suppresses VEGF synthesis and secretion by downregulating HIF-1a expression. Sirolimus may be useful for clinical application in patients who received a liver transplant for HCC.

Topics: Actins; Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Line, Tumor; DNA Primers; Fibroblast Growth Factor 2; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; RNA, Messenger; Sirolimus; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with few effective therapeutic options for advanced disease. At least 40% of HCCs are clonal, potentially arising from STAT3+, NANOG+ and OCT3/4+ liver progenitor/stem cell transformation, along with inactivation of transforming growth factor-beta (TGF-beta) signaling. Here we report significantly greater signal transducer and activator of transcription 3 (STAT3) and tyrosine phosphorylated STAT3 in human HCC tissues (P<0.0030 and P<0.0455, respectively) than in human normal liver. Further, in HCC cells with loss of response to TGF-beta, NSC 74859, a STAT3-specific inhibitor, markedly suppresses growth. In contrast, CD133(+) status did not affect the response to STAT3 inhibition: both CD133(+) Huh-7 cells and CD133(-) Huh-7 cells are equally sensitive to NSC 74859 treatment and STAT3 inhibition, with an IC(50) of 100 muM. Thus, the TGF-beta/beta2 spectrin (beta2SP) pathway may reflect a more functional 'stem/progenitor' state than CD133. Furthermore, NSC 74859 treatment of Huh-7 xenografts in nude mice significantly retarded tumor growth, with an effective dose of only 5 mg/kg. Moreover, NSC 74859 inhibited tyrosine phosphorylation of STAT3 in HCC cells in vivo. We conclude that inhibiting interleukin 6 (IL6)/STAT3 in HCCs with inactivation of the TGF-beta/beta2SP pathway is an effective approach in management of HCCs. Thus, IL6/STAT3, a major signaling pathway in HCC stem cell renewal and proliferation, can provide a novel approach to the treatment of specific HCCs.

Topics: AC133 Antigen; Aminosalicylic Acid; Aminosalicylic Acids; Animals; Antigens, CD; Apoptosis; Benzenesulfonates; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Colony-Forming Units Assay; Female; Glycoproteins; Humans; Immunoenzyme Techniques; Liver Neoplasms; Mice; Mice, Nude; Peptides; Phosphorylation; STAT3 Transcription Factor; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor beta (TGF-beta) signaling involves both tumor suppression and oncogenesis. TGF-beta activates TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3-mediated signaling between tumor suppression and oncogenesis in HBx-expressing hepatocytes indicated that TbetaRI-dependent pSmad3C transmitted a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L- and pSmad3C-mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx-activated JNK/pSmad3L/c-Myc oncogenic pathway was enhanced, while the TbetaRI/pSmad3C/p21(WAF1) tumor-suppressive pathway was impaired as human and mouse HBx-associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor-suppressive pSmad3C developed HCC within 12 years.. HBx shifts hepatocytic TGF-beta signaling from the tumor-suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV-infected liver specimens should prove clinically useful for predicting risk of HCC.

Topics: Adult; Animals; Carcinoma, Hepatocellular; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; DNA, Viral; Female; Hepatitis B, Chronic; Hepatocytes; Humans; JNK Mitogen-Activated Protein Kinases; Liver Cirrhosis; Liver Neoplasms; Male; Mice; Mice, Transgenic; Middle Aged; Phosphorylation; Signal Transduction; Smad3 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Viral Regulatory and Accessory Proteins; Young Adult

Liver fibrosis can be induced by environmental chemicals or toxicants, and finally stimulates fibrogenic cytokines expression, such as transforming growth factor-beta (TGF-beta) and its downstream mediator connective tissue growth factor (CTGF). 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a metabolite of arachidonic acid, can act as a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, and function as either anti-inflammatory or inflammatory agents in different cell types. In this study, CTGF was detected in three human hepatoma cell lines, Hep3B, HepG2, and Huh-7, and it was up-regulated by TGF-beta. 15d-PGJ(2) significantly inhibited TGF-beta-induced CTGF protein and mRNA expressions, and promoter activity in hepatoma cells. 15d-PGJ(2) suppressed TGF-beta-induced Smad2 phosphorylation, however enhancing the phosphorylation of ERK, c-Jun N-terminal kinase (JNK), and p38 in TGF-beta-treated Hep3B cells. Other PPAR ligands like the PPARgamma agonist, troglitazone; the PPARalpha agonist, Wy-14643, and bezafibrate were also able to inhibit TGF-beta-induced CTGF. The results suggest that 15d-PGJ(2) inhibits TGF-beta-induced CTGF expression by inhibiting the phosphorylation of Smad2, which is independent of PPAR, and 15d-PGJ(2) might also act through a PPAR-dependent mechanism in human hepatoma cells. 15d-PGJ(2) might have a beneficent effect on prevention of liver fibrosis induced by environmental toxicants.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Connective Tissue Growth Factor; Fibrosis; Gene Expression; Hepatic Stellate Cells; Hepatocytes; Humans; Immunologic Factors; Liver Cirrhosis; Phosphorylation; Prostaglandin D2; RNA, Messenger; Smad2 Protein; Transforming Growth Factor beta; Up-Regulation

In breast carcinomas, prolonged signalling through the TGF-beta receptor promotes latent tumour progression, metastasis and the epithelial-to-mesenchymal transition of tumour cells. Previously, it has been found that the 154 amino acid RING finger protein, RNF11, was overexpressed in high-grade breast tumours and was capable of modulating TGF-beta signalling.. Utilizing cellular and biochemical assays, key interactions and molecular roles for the RNF11 protein in the TGF-beta pathway were explored.. It is shown that RNF11 is required for TGF-beta signalling and is capable of enhancing the Smad-TGF-beta signalling pathway directly. Further, that endogenous RNF11 and Smad4 proteins associate and co-localize in a TGF-beta-enhanced manner. This study indicates that RNF11 induces an increase in Smad4 protein levels. In functional assays, it is observed that RNF11 enhances Smad4-dependant TGF-beta signalling and that RNF11 alone can recapitulate Smad4-dependant apoptosis in cellular assays.. RNF11 acts directly on Smad4 to enhance Smad4 function, and plays a role in prolonged TGF-beta signalling and possibly in latent tumour progression.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Carcinoma, Hepatocellular; Carrier Proteins; Cells, Cultured; DNA-Binding Proteins; Fluorescent Antibody Technique; Humans; Immunoblotting; Immunoprecipitation; Kidney; Liver Neoplasms; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Ubiquitin

Carbonic anhydrase IX (CAIX) is a membrane-associated carbonic anhydrase (CA) that is overexpressed in a variety of tumor types and associated with increased metastasis, giving a poor prognosis. Transcriptional regulation of transmembrane protein CAIX is complex. We describe further characterization of the 1.2kb hCA9 promoter, and the effect of TGF-beta on the transcriptional activity and expression of hCAIX in Hep3B cells. Transcriptional activity of different promoter regions of hCA9 promoter showed the presence of negative regulatory region between -300bp and -500bp of hCAIX promoter. The -116/+38 region was enough for basal transcriptional activity in Hep3B cells. TGF-beta upregulates all promoter regions of hCA9 with the highest beig for -466/+38 that has a negatively regulated region. The transcriptional activation of hCA9 promoter by TGF-beta is consistent with hCAIX mRNA levels revealed by RT-PCR and hCAIX protein expression levels by flow cytometry in Hep3B cells.

Topics: Antigens, Neoplasm; Carbonic Anhydrase IX; Carbonic Anhydrases; Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1; Liver Neoplasms; Promoter Regions, Genetic; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Up-Regulation

Plasminogen activator inhibitor type I (PAI-1) is a marker of the fibrinolytic system and serves as a possible predictor for hepatic metabolic syndromes. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, is a drug used for treatment of hyperlipidemia. Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of crucial genes involved in various metabolic pathways. In this study, we show that fenofibrate increased SHP gene expression in cultured liver cells and in the normal and diabetic mouse liver by activating the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway in a PPARalpha-independent manner. Administration of transforming growth factor beta (TGF-beta) or a methionine-deficient and choline-deficient (MCD) diet to induce the progressive fibrosing steatohepatitis model in C57BL/6 mice was significantly reversed by fenofibrate via AMPK-mediated induction of SHP gene expression with a dramatic decrease in PAI-1 messenger RNA (mRNA) and protein expression along with other fibrotic marker genes. No reversal was observed in SHP null mice treated with fenofibrate. Treatment with another PPARalpha agonist, WY14643, showed contrasting effects on these marker gene expressions in wild-type and SHP null mice, demonstrating the specificity of fenofibrate in activating AMPK signaling. Fenofibrate exhibited a differential inhibitory pattern on PAI-1 gene expression depending on the transcription factors inhibited by SHP.. By demonstrating that a PPARalpha-independent fenofibrate-AMPK-SHP regulatory cascade can play a key role in PAI-1 gene down-regulation and reversal of fibrosis, our study suggests that various AMPK activators regulating SHP might provide a novel pharmacologic option in ameliorating hepatic metabolic syndromes.

Topics: AMP-Activated Protein Kinases; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytokines; Fenofibrate; Male; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; PPAR alpha; Pyrimidines; Rats; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

The transforming growth factor receptor III (TGFbetaRIII) is the most abundant and essential TGF-beta binding protein that functions as a co-receptor with other receptors in TGF-beta signaling. In earlier studies, expression of TGFbetaRIII was reported to be decreased in a variety of human cancers. Functional assessment of TGFbetaRIII was performed in many previously studied cancers but not in hepatocellular carcinoma. Therefore, in this study, we investigated the expression and genetic alterations of TGFbetaRIII in hepatocellular carcinoma (HCC) by quantitative real-time PCR (qRT-PCR) and single-strand conformation polymorphism (SSCP) analysis. The qRT-PCR showed down-regulation of TGFbetaRIII in the tumor samples. To investigate whether genetic alterations mediated decreased expression of TGFbetaRIII, we performed mutation analysis of 67 human HCC tissues by SSCP and direct sequencing. We found five previously reported and one novel single nucleotide polymorphisms in exons 2, 3, 5, 13 and 14, but no mutations were detected. These polymorphisms were not associated with amino acid changes except for a base change found in exon 2 (TCC-->TTC, S15F). The loss of heterozygosity (LOH) analysis performed on 10 tumors and corresponding normal pairs, showed a low rate of LOH (2/10). The results of this study suggest that TGFbetaRIII is transcriptionally down-regulated in hepatocellular carcinoma. In addition, genetic alterations did not appear to be associated with the reduced expression level of TGFbetaRIII. To clarify the role of TGFbetaRIII in hepatocellular tumor development and progression, functional analysis is needed in future studies.

Topics: Adolescent; Adult; Aged; Carcinoma, Hepatocellular; Child; Child, Preschool; Down-Regulation; Female; Humans; Liver Neoplasms; Loss of Heterozygosity; Male; Middle Aged; Polymorphism, Single Nucleotide; Proteoglycans; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, and prior attempts to develop genomic-based classification for HCC have yielded highly divergent results, indicating difficulty in identifying unified molecular anatomy. We performed a meta-analysis of gene expression profiles in data sets from eight independent patient cohorts across the world. In addition, aiming to establish the real world applicability of a classification system, we profiled 118 formalin-fixed, paraffin-embedded tissues from an additional patient cohort. A total of 603 patients were analyzed, representing the major etiologies of HCC (hepatitis B and C) collected from Western and Eastern countries. We observed three robust HCC subclasses (termed S1, S2, and S3), each correlated with clinical parameters such as tumor size, extent of cellular differentiation, and serum alpha-fetoprotein levels. An analysis of the components of the signatures indicated that S1 reflected aberrant activation of the WNT signaling pathway, S2 was characterized by proliferation as well as MYC and AKT activation, and S3 was associated with hepatocyte differentiation. Functional studies indicated that the WNT pathway activation signature characteristic of S1 tumors was not simply the result of beta-catenin mutation but rather was the result of transforming growth factor-beta activation, thus representing a new mechanism of WNT pathway activation in HCC. These experiments establish the first consensus classification framework for HCC based on gene expression profiles and highlight the power of integrating multiple data sets to define a robust molecular taxonomy of the disease.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cohort Studies; Gene Expression Profiling; Humans; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-beta in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-beta-induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-beta-induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-beta, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-beta and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-beta-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitochondria; NADPH Oxidase 4; NADPH Oxidases; Oxidative Stress; Protein Kinase Inhibitors; Transforming Growth Factor beta; Up-Regulation

GLI2 (GLI-Kruppel family member 2), a zinc finger transcription factor that mediates Hedgehog signaling, is implicated in the progression of an ever-growing number of human malignancies, including prostate and pancreatic cancer, as well as basal cell carcinoma of the skin. Its expression is up-regulated by transforming growth factor-beta (TGF-beta) in a variety of cell types, both normal and transformed. We report herein that TGF-beta-driven GLI2 expression is transcriptional and does not result from stabilization of GLI2 transcripts. We describe the characterization of the 5'-flanking sequence of human GLI2 mRNA, the identification of a transcription start site, the cloning of approximately 1,600 bp of the regulatory promoter region and the identification and functional analysis of a TGF-beta-responsive region mapped to a 91-bp sequence between nucleotides -119 and -29 of the promoter. This region harbors SMAD and lymphoid enhancer factor/T cell factor binding sites that allow functional cooperation between SMAD3 and beta-catenin, recruited to the promoter in response to TGF-beta to drive GLI2 gene transcription.

Topics: Base Sequence; beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cells, Cultured; Chromatin Immunoprecipitation; Cloning, Molecular; Humans; Keratinocytes; Kruppel-Like Transcription Factors; Liver Neoplasms; Luciferases; Molecular Sequence Data; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Regulatory Sequences, Nucleic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad3 Protein; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Zinc Finger Protein Gli2

The epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E-cadherin and nonmembranous beta-catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down-regulation of E-cadherin, nonmembranous expression of beta-catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis C-associated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh-7). Overexpression of Snail or/and Twist in Huh-7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppress E-cadherin transcription.. Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays.

Topics: Adult; Aged; Animals; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Epithelial Cells; Female; gamma Catenin; Humans; Liver Neoplasms; Male; Mesoderm; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Nuclear Proteins; Retrospective Studies; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous; Twist-Related Protein 1; Vimentin

Topics: beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cell Differentiation; Epithelial Cells; Humans; Liver Neoplasms; Mesoderm; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates cell growth, differentiation, and apoptosis of various types of cells. Autophagy is emerging as a critical response of normal and cancer cells to environmental changes, but the relationship between TGF-beta signaling and autophagy has been poorly understood. Here, we showed that TGF-beta activates autophagy in human hepatocellular carcinoma cell lines. TGF-beta induced accumulation of autophagosomes and conversion of microtubule-associated protein 1 light chain 3 and enhanced the degradation rate of long-lived proteins. TGF-beta increased the mRNA expression levels of BECLIN1, ATG5, ATG7, and death-associated protein kinase (DAPK). Knockdown of Smad2/3, Smad4, or DAPK, or inhibition of c-Jun NH(2)-terminal kinase, attenuated TGF-beta-induced autophagy, indicating the involvement of both Smad and non-Smad pathway(s). TGF-beta activated autophagy earlier than execution of apoptosis (6-12 versus 48 h), and reduction of autophagy genes by small interfering RNA attenuated TGF-beta-mediated growth inhibition and induction of proapoptotic genes Bim and Bmf, suggesting the contribution of autophagy pathway to the growth-inhibitory effect of TGF-beta. Additionally, TGF-beta also induced autophagy in some mammary carcinoma cells, including MDA-MB-231 cells. These findings show that TGF-beta signaling pathway activates autophagy in certain human cancer cells and that induction of autophagy is a novel aspect of biological functions of TGF-beta.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Bcl-2-Like Protein 11; Beclin-1; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Membrane Proteins; Microtubule-Associated Proteins; Proto-Oncogene Proteins; Receptors, Transforming Growth Factor beta; Smad Proteins; Transfection; Transforming Growth Factor beta; Ubiquitin-Activating Enzymes

Topics: Aminosalicylic Acids; Benzenesulfonates; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Quinolines; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The clinical heterogeneity of HCC, and the lack of good diagnostic markers and treatment strategies, has rendered the disease a major challenge. Patients with HCC have a highly variable clinical course, indicating that HCC comprises several biologically distinctive subgroups reflecting a molecular heterogeneity of the tumors. Transforming growth factor beta (TGF-beta) is known to exhibit tumor stage dependent suppressive (that is, growth inhibition) and oncogenic (that is, invasiveness) properties. Here, we asked if a TGF-beta specific gene expression signature could refine the classification and prognostic predictions for HCC patients. Applying a comparative functional genomics approach we demonstrated that a temporal TGF-beta gene expression signature established in mouse primary hepatocytes successfully discriminated distinct subgroups of HCC. The TGF-beta positive cluster included two novel homogeneous groups of HCC associated with early and late TGF-beta signatures. Kaplan-Meier plots and log-rank statistics indicated that the patients with a late TGF-beta signature showed significantly (P < 0.005) shortened mean survival time (16.2 +/- 5.3 months) compared to the patients with an early (60.7 +/- 16.1 months) TGF-beta signature. Also, tumors expressing late TGF-beta-responsive genes displayed invasive phenotype and increased tumor recurrence. We also showed that the late TGF-beta signature accurately predicted liver metastasis and discriminated HCC cell lines by degree of invasiveness. Finally, we established that the TGF-beta gene expression signature possessed a predictive value for tumors other than HCC.. These data demonstrate the clinical significance of the genes embedded in TGF-beta expression signature for the molecular classification of HCC.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatocytes; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Knockout; Neoplasm Recurrence, Local; Phenotype; Predictive Value of Tests; Prognosis; Sensitivity and Specificity; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) plays a dual and complex role in human cancer. In this report, we observe a specific set of MicroRNAs (miRNAs) changed in response to TGF-beta in human hepatocellular carcinoma (HCC) cells by miRNA microarray screening. A cluster of miRNA, miR-23a approximately 27a approximately 24, is induced in an early stage by TGF-beta in Huh-7 cells. Knockdown of Smad4, Smad2 or Smad3 expression by RNA interference can attenuate the response of miR-23a approximately 27a approximately 24 to TGF-beta addition, indicating that this induction is dependent on Smad pathway. We also explore that miR-23a approximately 27a approximately 24 can function as an antiapoptotic and proliferation-promoting factor in liver cancer cells. In addition, expression of this miRNA cluster is found to be remarkably upregulated in HCC tissues versus normal liver tissues. These findings suggest a novel, alternative mechanism through which TGF-beta could induce specific miRNA expression to escape from tumor-suppressive response in HCC cells.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Humans; Lentivirus; Liver Neoplasms; MicroRNAs; Oligonucleotide Array Sequence Analysis; Smad Proteins; Transfection; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. We recently identified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as an important mediator of TGF-beta-induced apoptosis in hepatoma cells. In this study, we have further explored the mechanism by which TGF-beta up-regulates TRAIL expression. The 5'-flanking region of the TRAIL gene was isolated and characterized. Deletion mutants of the 5'-untranslated region of the TRAIL gene revealed a region comprising nucleotides -1950 to -1100 responsible for TRAIL induction following treatment with TGF-beta. Within this region, we have identified an activator protein-1 (AP-1) site indispensable for TGF-beta-mediated induction of TRAIL. Activation of this AP-1 site is mediated by a JunD.FosB heterodimer. Expression of DNSmad4, DNJunD, or DNFosB significantly impairs TGF-beta-mediated activation of the TRAIL promoter. Furthermore, with tRNA interference targeting Smad4, junD, FosB, we could abolish TRAIL expression and, subsequently, TGF-beta-induced TRAIL-mediated apoptosis in hepatoma cells. Our results reveal a new AP-1 site within the TRAIL promoter functionally involved in TGF-beta-induced TRAIL expression and apoptosis in hepatomas and thus provide evidence for the underlying mechanism by which TGF-beta might regulate cell death in liver cancer.

Topics: Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA, Neoplasm; Gene Silencing; Humans; Liver Neoplasms; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Smad Proteins; Smad4 Protein; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor AP-1; Transforming Growth Factor beta

We have previously demonstrated that 40%-70% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) within 15 months, revealing the importance of the transforming growth factor-beta (TGF-beta) signaling pathway in suppressing tumorigenesis in the liver. The current study was carried out to investigate mechanisms by which embryonic liver fodrin (ELF), a crucial Smad3/4 adaptor, suppresses liver tumor formation. Histological analysis of hyperplastic liver tissues from elf(+/-) mice revealed abundant newly formed vascular structures, suggesting aberrant angiogenesis with loss of ELF function. In addition, elf(+/-) mice displayed an expansion of endothelial progenitor cells. Ectopic ELF expression in fetal bovine heart endothelial (FBHE) cells resulted in cell cycle arrest and apoptosis. Further analysis of developing yolk sacs of elf(-/-) mice revealed a failure of normal vasculature and significantly decreased endothelial cell differentiation with embryonic lethality. Immunohistochemical analysis of hepatocellular cancer (HCC) from the elf(+/-) mice revealed an abnormal angiogenic profile, suggesting the role of ELF as an angiogenic regulator in suppressing HCC. Lastly, acute small interfering RNA (siRNA) inhibition of ELF raised retinoblastoma protein (pRb) levels nearly fourfold in HepG2 cells (a hepatocellular carcinoma cell line) as well as in cow pulmonary artery endothelial (CPAE) cells, respectively.. Taken together these results, ELF, a TGF-beta adaptor and signaling molecule, functions as a critical adaptor protein in TGF-beta modulation of angiogenesis as well as cell cycle progression. Loss of ELF in the liver leads the cancer formation by deregulated hepatocyte proliferation and stimulation of angiogenesis in early cancers. Our studies propose that ELF is potentially a powerful target for mimetics enhancing the TGF-beta pathway tumor suppression of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cattle; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Endothelium, Vascular; Hepatocytes; Humans; Liver Neoplasms; Mice; Mice, Knockout; Microfilament Proteins; Neovascularization, Pathologic; Prognosis; Retinoblastoma Protein; Signal Transduction; Transforming Growth Factor beta

The members of the platelet-derived growth factor (PDGF) and the transforming growth factor-beta (TGFbeta) pathways are important in the induction of liver fibrosis and cirrhosis; however, their role in the subsequent progression to hepatocellular carcinoma (HCC) remains elusive. Our study provides new insights into mechanisms of dysregulation of PDGFs, TGFbeta and signal transducer and activator of transcription (STAT) pathways in the pathogenesis of methyl-deficient rodent liver carcinogenesis, a remarkably relevant model to the development of HCC in humans. We demonstrated a progressive increase in the Pdgfs and TGFbeta expression in preneoplastic tissue and liver tumors indicating their promotional role in carcinogenesis, particularly in progression of liver fibrosis and cirrhosis. However, activation of the STAT3 occurred only in fully developed HCC and was associated with downregulation of the Socs1 gene. The inhibition of the Socs1 expression in HCC was associated with an increase in histone H3 lysine 9, H3 lysine 27, and H4 lysine 20 trimethylation at the Socs1 promoter, but not with promoter methylation. The results of our study suggest the following model of events in hepatocarcinogenesis: during early stages, overexpression of the Socs1 effectively inhibits TGFbeta- and PDGF-induced STAT3 activation, whereas, during the advanced stages of hepatocarcinogenesis, the Socs1 downregulation resulted in loss of its ability to attenuate the signal from the upregulated TGFbeta and PDGFs leading to oncogenic STAT3 activation and malignant cell transformation. This model illustrates that the Socs1 acts as classic tumor suppressor by preventing activation of the STAT3 and downregulation of Socs1 and consequent activation of STAT3 may be a crucial events leading to formation of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Diet; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation; Humans; Liver; Liver Neoplasms; Male; Methionine; Methylation; Platelet-Derived Growth Factor; Promoter Regions, Genetic; Random Allocation; Rats; Rats, Inbred F344; Receptors, Platelet-Derived Growth Factor; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Transforming Growth Factor beta

The transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes through an oxidative stress process. Here, we have analyzed the role of different NADPH oxidase isoforms in the intracellular signalling induced by TGF-beta in hepatocytes, to later explore whether this mechanism is altered in liver tumor cells.. Primary cultures of rat and human hepatocytes, HepG2 and Hep3B cells were used in in vitro studies to analyze the TGF-beta response.. TGF-beta-induced apoptosis in rat hepatocytes does not require Rac-dependent NADPH oxidases. TGF-beta upregulates the Rac-independent Nox4, which correlates with its pro-apoptotic activity. Regulation of Nox4 occurs at the transcriptional level and is counteracted by intracellular survival signals. siRNA targeted knock-down of Nox4 attenuates NADPH oxidase activity, caspase activation and cell death in rat hepatocytes. NOX4 upregulation by TGF-beta is also observed in human hepatocytes, coincident with apoptosis. In human hepatocellular carcinoma (HCC) cell lines, NOX4 upregulation by TGF-beta is only observed in cells that are sensitive to its cytotoxic effect, such as Hep3B cells. siRNA targeted knock-down of NOX4 in these cells impairs TGF-beta-induced apoptosis.. Upregulation of NOX4 by TGF-beta is required for its pro-apoptotic activity in hepatocytes. Impairment of this TGF-beta-induced response might confer apoptosis resistance in HCC cells.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Fetus; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Hepatocytes; Humans; Liver Neoplasms; NADPH Oxidase 4; NADPH Oxidases; Rats; Rats, Wistar; RNA, Small Interfering; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Hepatitis C virus (HCV) becomes chronic in about 85 % of infected individuals, whereas only 15 % of infected people clear spontaneously the virus. The progression of hepatitis C to chronic status is associated to a profound down-regulation of CD4 and CD8 multispecific immune response. This immune defect may participate to the immune tolerance of VHC and consequently to its persistence. Recent findings indicate that T regulatory cells as Tr1 play an inhibitory role on T helper responses notably in the context of auto-immune or inflammatory disorders. The existence of immunosuppressive mechanisms supported by Tr1 lymphocytes and their IL-10 production represent an attractive hypothesis. We have previously evaluated the existence of regulatory T cells (Tr1) via high production of IL-10, in liver biopsies of three well-defined cohorts of HCV-1b infected patients. To this purpose, we compared liver biopsies of chronically infected patients including patients without liver lesions, with cirrhosis and with hepatocellular carcinoma (HCC). Using quantitative real time PCR, the results obtained demonstrate, an increased expression of interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta)_, in liver biopsies with more severe fibrosis. This observation was correlated with an increased expression during the pathogenesis progression, of the three specific markers of the Tr1 cells sub-population, recently described and confirming the Tr1 phenotype. Evidence of regulatory T cells installation in the liver of chronically infected patient and increased frequency in cirrhosis and HCC suggest a main role of these cells in the aggravation of the liver pathology. This study should bring insight of T regulatory cell implications in VHC persistence and in the pathology progression.

Topics: Adult; Aged; Antigens, CD; Biopsy; Carcinoma, Hepatocellular; Cytokines; Disease Progression; DNA Primers; Hepacivirus; Hepatitis C; Hepatitis C, Chronic; Humans; Immune Tolerance; Interleukin-10; Liver; Liver Cirrhosis; Liver Neoplasms; Middle Aged; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Cytokines play a key role in regulation of immunity and inflammation. The aim of the study was to detect serum levels of TGF-beta, IL-10, and sTNFalphaRII in patients with type C chronic liver disease (CLD) and to correlate these with biochemical and histopathological parameters used to assess the severity of the disease. Blood samples were aseptically collected from 90 CLD patients. Cytokine levels were also followed up in 39 chronic hepatitis cases. Levels of the cytokines in 90 CLD patients were significantly higher than in controls. In the follow up patients, 12 were non-responders and the serum levels of cytokines were still elevated after therapy whereas in 27 responders cytokine levels were significantly reduced after therapy and correlated well with biochemical and histopathological parameters. It is inferred that cytokine levels reflect the level of inflammation in chronic hepatitis C virus (HCV) infection and can be used as indirect markers to assess the severity of liver disease.

Topics: Adult; Antiviral Agents; Biomarkers; Carcinoma, Hepatocellular; Disease Progression; Female; Hepatitis C, Chronic; Humans; Interleukin-10; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Receptors, Tumor Necrosis Factor, Type II; Severity of Illness Index; Transforming Growth Factor beta; Treatment Outcome

Topics: Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Hepatitis C, Chronic; Humans; Liver Neoplasms; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) treatment is challenging because the mechanisms underlying tumor progression are still largely unknown. Transforming growth factor (TGF)-beta1 is considered a crucial molecule in HCC tumorigenesis because increased levels of patients' serum and urine are associated with disease progression. The aim of the present study was to investigate the inhibition of TGF-beta signaling and its impact on HCC progression. Human HCC cell lines were treated with a TGF-beta receptor kinase inhibitor (LY2109761) whose selectivity was determined in a kinase assay. Exogenous TGF-beta1 phosphorylates the TGF-beta receptor, consequently activating Smad-2, whereas the drug selectively blocks this effect and dephosphorylates autocrine p-Smad-2 at concentrations ranging from 0.001 to 0.1 microM. A cytotoxic effect documented by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), trypan blue, and propidium iodide staining assays was observed at 10microM, whereas the drug inhibits (P < 0.001) the migration of HCC cells on fibronectin, laminin-5, and vitronectin and invasion through Matrigel (P < 0.001) at concentrations up to 0.1 microM. LY2109761 up-regulates (P < 0.001) E-cadherin mRNA and protein levels. This increase was localized at the cellular membrane where E-cadherin mediates anchorage that is cell-cell dependent. Consistently, a functional monoclonal antibody that inhibits E-cadherin-dependent cell-cell contact restores the migratory and invasive activity. Finally, nonmetastatic HCC tissues from 7 patients were cultured with TGF-beta1 in the presence or absence of LY2109761. E-cadherin expression was reduced by TGF-beta1 and was significantly (P < 0.0001) increased by LY2109761 treatment, measured by quantitative real-time PCR on microdissected tissues and by immunohistochemistry on serial sections. In 72 patients, E-cadherin tissue expression was more weakly expressed in metastatic than in nonmetastatic HCC (P < 0.0001).. LY2109761 blocks migration and invasion of HCC cells by up-regulating E-cadherin, suggesting that there could be a mechanistic use for this molecule in clinical trials.

Topics: Aged; Antineoplastic Agents; Benzenesulfonates; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Niacinamide; Phenylurea Compounds; Pyridines; Sorafenib; Transforming Growth Factor beta

We previously demonstrated that CD4(+)CD25(+) T regulatory cells (Tregs), important for the maintenance of immune tolerance and prevention of autoimmune disease, from patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) exhibit reduced Foxp3 expression and Treg suppressor function compared with healthy donors. Since TGF-beta signaling has been previously reported to be critical for both Foxp3 expression and Treg function, we examined whether this signaling pathway was dysregulated in patients with HAM/TSP. Levels of TGF-beta receptor II (TGF-betaRII) as well as Smad7 (a TGF-beta-inducible gene) were significantly reduced in CD4(+) T cells in patients with HAM/TSP compared with healthy donors, and the expression of TGF-betaRII inversely correlated with the HTLV-I tax proviral load. Importantly, both CD4(+)CD25(+) and CD4(+)CD25(-) T cells from HAM/TSP patients exhibited reduced TGF-betaRII expression compared with healthy donors, which was associated with functional deficits in vitro, including a block in TGF-beta-inducible Foxp3 expression that inversely correlated with the HTLV-I tax proviral load, loss of Treg suppressor function, and escape of effector T cells from Treg-mediated control. This evidence suggests that a virus-induced breakdown of immune tolerance affecting both regulatory and effector T cells contributes to the pathogenesis of HAM/TSP.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Forkhead Transcription Factors; Gene Products, tax; Human T-lymphotropic virus 1; Humans; Immune Tolerance; Liver Neoplasms; Paraparesis, Tropical Spastic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Smad4 Protein; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Hemojuvelin (HJV) is a coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. However, the precise combinations of BMP ligands and receptors used by HJV remain unknown. HJV has also been demonstrated to bind to neogenin, but it is not known whether this interaction has a role in regulating hepcidin expression. In the present study, we show that BMP-2, BMP-4, and BMP-6 are endogenous ligands for HJV in hepatoma-derived cell lines, and that all 3 of these ligands are expressed in human liver. We demonstrate in vitro that HJV selectively uses the BMP type II receptors ActRIIA and BMPRII, but not ActRIIB, and HJV enhances utilization of ActRIIA by BMP-2 and BMP-4. Interestingly, ActRIIA is the predominant BMP type II receptor expressed in human liver. While HJV can use all 3 BMP type I receptors (ALK2, ALK3, and ALK6) in vitro, only ALK2 and ALK3 are detected in human liver. Finally, we show that HJV-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or by inhibition of endogenous neogenin expression. Thus, HJV-mediated BMP signaling and hepcidin regulation occur via a distinct subset of BMP ligands and BMP receptors, independently of neogenin.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Antimicrobial Cationic Peptides; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Gene Expression Regulation; GPI-Linked Proteins; Hemochromatosis Protein; Hepcidins; Humans; Ligands; Liver; Membrane Proteins; Transforming Growth Factor beta

There is limited information on the influence of tumor growth on the expansion of tumor-specific TGF-beta-producing CD4(+) T cells in humans. alpha-Fetoprotein (AFP) is an oncofetal Ag and has intrinsic immunoregulatory properties. In this study, we report the identification and characterization of subsets of CD4(+) T cells that recognize an epitope within the AFP sequence (AFP(46-55)) and develop into TGF-beta-producing CD4(+) T cells. In a peptide-specific and dose-dependent manner, AFP(46-55) CD4(+) T cells produce TGF-beta, GM-CSF, and IL-2 but not Th1-, Th2-, Th17-, or Tr1-type cytokines. These cells express CTLA-4 and glucocorticoid-induced TNR receptor and inhibit T cell proliferation in a contact-dependent manner. In this study, we show that the frequency of AFP(46-55) CD4(+) T cells is significantly higher (p = 001) in patients with hepatocellular carcinoma than in healthy donors, suggesting that these cells are expanded in response to tumor Ag. In contrast, tumor necrosis-inducing treatments that are shown to improve survival rate can shift the Th1/TGF-beta-producing CD4(+) T cell balance in favor of Th1 responses. Our data demonstrate that tumor Ags may contain epitopes which activate the expansion of inducible regulatory T cells, leading to evasion of tumor control.

Topics: alpha-Fetoproteins; Amino Acid Sequence; Antigens, CD; Antigens, Differentiation; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; Cells, Cultured; CTLA-4 Antigen; Epitopes; Forkhead Transcription Factors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Peptide Fragments; Phenotype; Sensitivity and Specificity; Transforming Growth Factor beta

The objective of this study was to identify genes regulated by thyroid hormone (T(3)) and associated with tumor invasion. The gene encoding furin, as previously identified by cDNA microarray, is known to be up-regulated by T(3) treatment, and stimulated furin production occurs in thyroidectomized rats after administration of T(3). Presently, by using serial deletion of the promoter and EMSAs, the T(3) response element on the furin promoter was localized to the -6317/-6302 region. T(3)-mediated furin up-regulation was cooperative with TGF-beta because T(3) induction increased after Smad3/4 addition. Furthermore, the invasiveness of HepG2-thyroid hormone receptor (TR) cells was significantly increased by T(3) treatment, perhaps due to furin processing of matrix metalloproteinase-2 and -9. In addition, furin up-regulation either by stable overexpression or T(3) and/or TGF-beta induction was evident in severe-combined immune-deficient mice inoculated with HepG2-TRalpha1 cells. The HepG2-furin mice displayed a higher metastasis index and tumor size than HepG2-neo mice. Notably, the increased liver and lung tumor number or size in the hyperthyroid severe-combined immune-deficient mice as well as TGF-beta mice was attributed specifically to furin overexpression in the HepG2-TRalpha1 cells. Furthermore, this study demonstrated that furin overexpression in some types of hepatocellular carcinomas is TR dependent and might play a crucial role in the development of hepatocellular carcinoma. Thus, T(3) regulates furin gene expression via a novel mechanism or in cooperation with TGF-beta to enhance tumor metastasis in vitro and in vivo.

Topics: Animals; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cells, Cultured; Chlorocebus aethiops; Furin; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, SCID; Models, Biological; Neoplasm Invasiveness; Rats; Rats, Sprague-Dawley; Receptors, Thyroid Hormone; Thyroid Hormones; Transforming Growth Factor beta

The transforming growth factor-beta (TGF-beta) regulates hepatocyte growth, inhibiting proliferation and inducing apoptosis. Indeed, escaping from the TGF-beta suppressor actions might be a prerequisite for liver tumour progression. In this work we show that TGF-beta plays a dual role in regulating apoptosis in FaO rat hepatoma cells, since, in addition to its pro-apoptotic effect, TGF-beta also activates survival signals, such as AKT, the epidermal growth factor receptor (EGFR) being required for its activation. TGF-beta induces the expression of the EGFR ligands transforming growth factor-alpha (TGF-alpha) and heparin-binding EGF-like growth factor (HB-EGF) and induces intracellular re-localization of the EGFR. Cells that overcome the apoptotic effects of TGF-beta undergo morphological changes reminiscent of an epithelial-mesenchymal transition (EMT) process. In contrast, TGF-beta does not activate AKT in adult hepatocytes, which correlates with lack of EGFR transactivation and no response to EGFR inhibitors. Although TGF-beta induces TGF-alpha and HB-EGF in adult hepatocytes, these cells show very low expression of TACE/ADAM 17 (TNF-alpha converting enzyme), which is required for EGFR ligand proteolysis and activation. Furthermore, adult hepatocytes do not undergo EMT processes in response to TGF-beta, which might be due, at least in part, to the fact that F-actin re-organization induced by TGF-beta in FaO cells require the EGFR pathway. Finally, results indicate that EGFR transactivation does not block TGF-beta-induced cell cycle arrest in FaO cells, but must be interfering with the pro-apoptotic signalling. In conclusion, TGF-beta is a suppressor factor for adult quiescent hepatocytes, but not for hepatoma cells, where it plays a dual role, both suppressing and promoting carcinogenesis.

Topics: Actins; ADAM Proteins; ADAM17 Protein; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Enzyme Activation; Epithelial Cells; ErbB Receptors; Hepatocytes; Humans; Male; Mesoderm; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; src-Family Kinases; Transcriptional Activation; Transforming Growth Factor beta

Activation-induced cytidine deaminase (AID) plays a role as a genome mutator in activated B cells, and inappropriate expression of AID has been implicated in the immunopathological phenotype of human B-cell malignancies. Notably, we found that the transgenic mice overexpressing AID developed lung adenocarcinoma and hepatocellular carcinoma (HCC), suggesting that ectopic expression of AID can lead to tumorigenesis in epithelial tissues as well. To examine the involvement of AID in the development of human HCC, we analyzed the AID expression and its correlation with mutation frequencies of the p53 gene in liver tissues from 51 patients who underwent resection of primary HCCs. The specific expression, inducibility by cytokine stimulation and mutagenic activity of AID were investigated in cultured human hepatocytes. Only trace amounts of AID transcripts were detected in the normal liver; however, endogenous AID was significantly upregulated in both HCC and surrounding noncancerous liver tissues with underlying chronic hepatitis or liver cirrhosis (p < 0.05). Most liver tissues with underlying chronic inflammation with endogenous AID upregulation already contained multiple genetic changes in the p53 gene. In both hepatoma cell lines and cultured human primary hepatocytes, the expression of AID was substantially induced by TGF-beta stimulation. Aberrant activation of AID in hepatocytes resulted in accumulation of multiple genetic alterations in the p53 gene. Our findings suggest that the aberrant expression of AID is observed in human hepatocytes with several pathological settings, including chronic liver disease and HCC, which might enhance the genetic susceptibility to mutagenesis leading to hepatocarcinogenesis.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Cytidine Deaminase; Enzyme Activation; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hepatitis, Viral, Human; Hepatocytes; Humans; Immunoblotting; Liver Neoplasms; Male; Middle Aged; Mutation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The cooperation of Ras - extracellular signal-regulated kinase/mitogen-activated protein kinase and transforming growth factor (TGF)-beta signaling provokes an epithelial to mesenchymal transition (EMT) of differentiated p19(ARF) null hepatocytes, which is accompanied by a shift in malignancy and gain of metastatic properties. Upon EMT, TGF-beta induces the secretion and autocrine regulation of platelet-derived growth factor (PDGF) by upregulation of PDGF-A and both PDGF receptors. Here, we demonstrate by loss-of-function analyses that PDGF provides adhesive and migratory properties in vitro as well as proliferative stimuli during tumor formation. PDGF signaling resulted in the activation of phosphatidylinositol-3 kinase, and furthermore associated with nuclear beta-catenin accumulation upon EMT. Hepatocytes expressing constitutively active beta-catenin or its negative regulator Axin were employed to study the impact of nuclear beta-catenin. Unexpectedly, active beta-catenin failed to accelerate proliferation during tumor formation, but in contrast, correlated with growth arrest. Nuclear localization of beta-catenin was accompanied by strong expression of the Cdk inhibitor p16(INK4A) and the concomitant induction of the beta-catenin target genes cyclin D1 and c-myc. In addition, active beta-catenin revealed protection of malignant hepatocytes against anoikis, which provides a prerequisite for the dissemination of carcinoma. From these data, we conclude that TGF-beta acts tumor progressive by induction of PDGF signaling and subsequent activation of beta-catenin, which endows a subpopulation of neoplastic hepatocytes with features of cancer stem cells..

Topics: Anoikis; beta Catenin; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Disease Progression; Epithelial Cells; Humans; Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta

HAb18G/CD147, a new hepatoma-associated antigen cloned and screened from human hepatocellular carcinoma cDNA library, is closely correlated with metastasis process in human hepatoma cells. In the present study we aimed to identify the pivotal molecules of the HAb18G/CD147 signal transduction pathway. The investigation showed that betaig-h3, a secretory extracellular matrix (ECM) protein, was upregulated in HAb18G/CD147-expressing human hepatoma T7721 cells and was downregulated by depressing HAb18G/CD147 expression. The expression of betaig-h3, upregulated in human hepatoma cells, was positively relative to the expression of HAb18G/CD147 in different human hepatoma cell lines. By overexpressing betaig-h3 in human SMMC-7721 hepatoma cells, we discovered that betaig-h3 promoted cell adhesion, invasion, and matrix metalloproteinase (MMP) secretion potential. HAb18G/CD147-induced invasion and metastasis potential of human hepatoma cells can be attenuated by antibodies specific for betaig-h3, and no significant differences on inhibitory effects were observed among T7721 cells incubated with antibodies for betaig-h3 or HAb18G/CD147 or both types together. Taken together, our study suggests that betaig-h3, regulated by the expression of HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates the HAb18G/CD147-induced invasion and metastasis process of human hepatoma cells.

Topics: Antibodies; Basigin; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Line, Tumor; Down-Regulation; Extracellular Matrix Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; RNA, Small Interfering; Transfection; Transforming Growth Factor beta; Up-Regulation

Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Whereas the TbetaRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21(WAF1) transcription, JNK/pSmad3L-mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI-1). We studied the domain-specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV-infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI-1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21(WAF1) decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro-inflammatory cytokine interleukin-1beta stimulated the pSmad3L/PAI-1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF-beta-dependent tumor-suppressive activity by the pSmad3C/p21(WAF1) pathway.. These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF-beta signaling from tumor-suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC.

Topics: alpha-Fetoproteins; Biopsy; Carcinoma, Hepatocellular; Disease Progression; DNA Replication; Hepatitis C, Chronic; Humans; Inflammation; Liver; Liver Cirrhosis; Liver Neoplasms; Prevalence; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Signal Transduction; Smad3 Protein; Thymidine; Transforming Growth Factor beta

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.

Topics: Apoptosis; bcl-Associated Death Protein; Carbazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Indoles; Insulin Receptor Substrate Proteins; Interleukin-4; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoproteins; Protein Kinase C; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Transforming Growth Factor beta

The status of the patient's associated disease can generally affect the onset or healing of acquired reactive perforating collagenosis (ARPC). We treated eight cases of ARPC and noted that the patients had similar findings. However, it was not clear why ARPC developed in the patients with these diseases. Nevertheless, several factors related to the diseases associated with ARPC could affect the degeneration of collagen fibers or the production of dermal products. Some patients had diseases that were characterized by fibrosis and an increased amount of reticular fibers. Factors related to tissue remodeling might act not only in diseases associated with ARPC but also in ARPC itself.

Topics: Adolescent; Adult; Aged; Carcinoma, Hepatocellular; Collagen; Collagen Diseases; Diabetes Mellitus, Type 2; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Pulmonary Fibrosis; Reticulin; Skin Diseases; Transforming Growth Factor beta

Importance of androgen for promotion of hepatocelullar carcinoma (HCC) has long been supported by clinical and experimental evidences. However, mechanisms involved in the carcinogenesis have not yet been fully elucidated. Moreover, unbalanced expression of TGF-beta1 during tumor progression results in prooncogenic rather than growth inhibition. To investigate the effect of androgen on transcriptional regulation of TGF-beta1, we isolated rat TGF-beta1 promoter, based on our previous report (GenBank AF249327), and examined regulation of its promoter activity by dihydrotestosterone in Huh7, LNCaP, and PC3 cells. Several putative transcription factor-binding sites were found, but no TATA box. When the full-length (-4784 to +68) and variously deleted promoter DNAs were evaluated, the promoter region spanning from -2732 to -1203 showed the highest activity towards dihydrotestosterone in a dose-dependent manner in both Huh7 and PC3 cells with androgen receptor (AR) expression. Putative androgen response sequence half site (5'-TGTCCT-3') was identified to be located within -1932 to -1927, proved by mutant (5'-AGACCT-3') analysis and chromatin immunoprecipitation (ChIP) assay. AR mediated upregulation of TGF-beta1 expression was confirmed by HCC developed in nude mice with AR-overexpressed Huh7-cells. This work presents in vivo and in vitro evidences of activation of TGF-beta1 expression by androgen and AR, and implicates the modulation of hepatocarcinogenesis by AR through the regulation of TGF-beta1 expression.

Topics: Androgens; Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cloning, Molecular; Consensus Sequence; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Promoter Regions, Genetic; Receptors, Androgen; Sequence Homology; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

ABCC6, a member of the adenosine 5'-triphosphate-binding cassette family of genes, encodes multidrug resistance-associated protein 6, a putative transmembrane transporter expressed primarily in the liver and to a significantly lower extent in other tissues. Mutations in ABCC6 result in pseudoxanthoma elasticum, a multi-system heritable connective tissue disorder with variable phenotypic expression. To examine the transcriptional regulation and tissue-specific expression of this gene, we cloned 2.6 kb of human ABCC6 promoter and developed a series of 5'-deletion constructs linked to luciferase reporter gene. Transient transfections in a number of cultured cell lines of diverse origin identified a specific NF-kappaB-like sequence (-235/-226), which conferred high level of expression in HepG2 hepatoma cells, inferring liver specificity. The functionality of the promoter fragments was confirmed in vivo by tail vein injection followed by luciferase reporter assay. Testing of selected cytokines revealed that transforming growth factor (TGF)-beta upregulated, while tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma downregulated the promoter activity in HepG2 cells. The responsiveness to TGF-beta was shown to reside primarily within an Sp1/Sp3 cognate-binding site at -58 to -49. The expression of the ABCC6 promoter was also shown to be markedly enhanced by Sp1 protein, as demonstrated by cotransfection of ABCC6 promoter-luciferase constructs and an Sp1 expression vector in Drosophila SL2 cells, which are devoid of endogenous Sp1. Furthermore, four additional transcription factors, with their cognate-binding sequences present in DNA, were shown to bind the 2.6-kb promoter fragment by protein/DNA array. Collectively, the results indicate that human ABCC6 displays tissue-specific gene expression, which can be modulated by proinflammatory cytokines. These findings may have implications for phenotypic expression of heritable and acquired diseases involving abnormality in the ABCC6 gene.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cytokines; Drosophila; Gene Expression Regulation; Genes, Reporter; Humans; Liver; Luciferases; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; NF-kappa B; Promoter Regions, Genetic; Sequence Deletion; Sp1 Transcription Factor; Sp3 Transcription Factor; Tissue Distribution; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta 1 (TGF-beta1) is a proposed regulator of Ids (inhibitors of DNA binding/differentiation) gene expression in epithelial cells. We previously reported that Id proteins are variously expressed in human hepatocellular carcinomas (HCC). However, the mechanism of regulation of Ids in HCC remains obscure. Here, we examined the relationship between Id1 and TGF-beta1 in four HCC cell lines, and studied the changes in cell proliferation, cell cycle and differentiation. The four HCC cell lines expressed Id1, TGF-beta1 and their receptors at various levels. TGF-beta1 strongly inhibited the growth of HuH7 cells, while the growth inhibition was moderate in PLC/PRF/5, and was not observed in HLE and HLF cell lines. TGF-beta1-induced growth inhibition in HuH7 cells was associated with cell accumulation in the G1 phase and partial induction of differentiation (with reduction of AFP and AFP-L3). Induction by TGF-beta1 dose-dependently suppressed Id1 expression in HuH7 cells; 1 ng/ml TGF-beta1 inhibited Id1 by 84.0 and 78.6% that of the untreated control at transcriptional and protein levels, respectively. HLE and HLF cells, which did not exhibit a TGF-beta1 growth inhibitory effect, lacked TGF-beta receptors and Id1 expression was not altered. In PLC/PRF/5 cells, Id1 augmentation was not observed in response to TGF-beta1, indicating that TGF-beta1-induced growth inhibition was not related to Id1 in this cell line. Our results suggest that, in some HCC cells, the pathway of suppression of Id1 by TGF-beta1 may be important in TGF-beta1-induced growth inhibition and partial differentiation.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Inhibitor of Differentiation Protein 1; Liver Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor beta

Thyroid hormone (triiodothyronine, T3) regulates growth, development and differentiation. To examine the influence of T3 on hepatoma cell growth, thyroid receptor (TR)alpha1 or TRbeta1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-beta neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-beta was studied to delineate the repression mechanism. TGF-beta was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-beta repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-beta neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.

Topics: Blotting, Northern; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cloning, Molecular; Cyclin E; Cyclin-Dependent Kinase 2; Humans; Liver Neoplasms; Neutralization Tests; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Triiodothyronine

Recently, DNA methylation and reduced expression of the suppressor of the cytokine signaling-3 (SOCS3) gene in human hepatocellular carcinoma (HCC) patients have been reported. However, the roles of SOCS3 in HCC development in vivo have not been clarified. Using RT-PCR analysis and Western blotting, we confirmed that SOCS3 expression was reduced in HCC patients. However, reduced expression of SOCS3 occurred not only in HCC but also in nontumor regions, and this reduction was stronger as the fibrosis grade increased. Furthermore, SOCS3 levels were inversely correlated with signal transducers and activators of transcription-3 (STAT3) activation as well as transforming growth factor (TGF)-beta1 levels in the non-HCC region. To define the molecular consequences of SOCS3 silencing/STAT3 hyperactivation and liver fibrosis, we examined liver-specific SOCS3-deficient mice. We demonstrated that SOCS3 deletion in the liver resulted in hyperactivation of STAT3 and promoted ConA- and chemical-induced liver fibrosis. The expression of TGF-beta1, a mediator of fibrosis, was enhanced by SOCS3 gene deletion, but suppressed by the overexpression of a dominant-negative STAT3 or SOCS3 both in vivo and in vitro. These data suggest that TGF-beta1 is a target gene of STAT3 and could be one of the mechanisms for enhanced fibrosis in SOCS3-deficient mice. Thus, our present study provides a novel role of SOCS3 and STAT3 in HCC development: in addition to the previously characterized oncogenic potentials, STAT3 enhances hepatic fibrosis through the upregulation of TGF-beta1 expression, and SOCS3 prevents this process.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Gene Expression Regulation; Gene Silencing; Genes, Dominant; Humans; Integrases; Liver; Liver Cirrhosis; Liver Neoplasms; Mice; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

To explore the role of transforming growth factor-beta1 (TGF-beta1)-smad signal transduction pathway in patients with hepatocellular carcinoma.. Thirty-six hepatocellular carcinoma specimens were obtained from Qidong Liver Cancer Institute and Department of Pathology of the Second Affiliated Hospital of Nanjing Medical University. All primary antibodies (polyclonal antibodies) to TGF-beta1, type II Transforming growth factor-beta receptor (TbetaR-II), nuclear factor-kappaB (NF-kappaB), CD34, smad4 and smad7,secondary antibodies and immunohistochemical kit were purchased from Zhongshan Biotechnology Limited Company (Beijing, China). The expressions of TGF-beta1, TbetaR-II, NF-kappaB, smad4 and smad7 proteins in 36 specimens of hepatocellular carcinoma (HCC) and its adjacent tissue were separately detected by immunohistochemistry to observe the relationship between TGF-beta1 and TbetaR-II, between NF-kappaB and TGF-beta1, between smad4 and smad7 and between TGF-beta1 or TbetaR-IIand microvessel density (MVD). MVD was determined by labelling the vessel endothelial cells with CD34.. The expression of TGF-beta1, smad7 and MVD was higher in HCC tissue than in adjacent HCC tissue (P<0.01, P<0.05, P<0.01 respectively). The expression of TbetaR-IIand smad4 was lower in HCC tissue than in its adjacent tissue (P<0.01, P<0.05 respectively). The expression of TGF-beta1 protein and NF-kappaB protein was consistent in HCC tissue. The expression of TGF-beta1 and MVD was also consistent in HCC tissue. The expression of TbetaR-IIwas negatively correlated with that of MVD in HCC tissue.. The expressions of TGF-beta1, TbetaR-II, NF-kappaB, smad4 and smad7 in HCC tissue, which are major up and down stream factors of TGF-beta1-smad signal transduction pathway , are abnormal. These factors are closely related with MVD and may play an important role in HCC angiogenesis. The inhibitory action of TGF-beta1 is weakened in hepatic carcinoma cells because of abnormality of TGF-beta1 receptors (such as TbetaR-II) and postreceptors (such as smad4 and smad7). NF-kappaB may cause activation and production of TGF-beta1.

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; NF-kappa B; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Smad7 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-beta. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-beta-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-beta, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-beta superfamily of cytokines, TGF-beta1, -beta2 and -beta3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-alpha receptor revealed decreased TGF-beta-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-beta-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p16; Disease Progression; Hepatocytes; Humans; Liver Neoplasms; Mesoderm; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, SCID; Phosphorylation; Platelet-Derived Growth Factor; Rats; Receptor, Platelet-Derived Growth Factor alpha; Receptors, Platelet-Derived Growth Factor; Smad Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p14ARF

The programmed cell death 4 (PDCD4) gene was originally identified as a tumor-related gene in humans and acts as a tumor-suppressor in mouse epidermal carcinoma cells. However, its function and regulatory mechanisms of expression in human cancer remain to be elucidated. We therefore investigated the expression of PDCD4 in human hepatocellular carcinoma (HCC) and the role of PDCD4 in human HCC cells. Downregulation of PDCD4 protein was observed in all HCC tissues tested compared with corresponding noncancerous liver, as revealed by Western blotting or immunohistochemical staining. Human HCC cell line, Huh7, transfected with PDCD4 cDNA showed nuclear fragmentation and DNA laddering characteristic of apoptotic cells associated with mitochondrial changes and caspase activation. Transforming growth factor-beta1 (TGF-beta1) treatment of Huh7 cells resulted in increased PDCD4 expression and occurrence of apoptosis, also concomitant with mitochondrial events and caspase activation. Transfection of Smad7, a known antagonist to TGF-beta1 signaling, protected cells from TGF-beta1-mediated apoptosis and suppressed TGF-beta1-induced PDCD4 expression. Moreover, antisense PDCD4 transfectants were resistant to apoptosis induced by TGF-beta1. In conclusion, these data suggest that PDCD4 is a proapoptotic molecule involved in TGF-beta1-induced apoptosis in human HCC cells, and a possible tumor suppressor in hepatocarcinogenesis.

Topics: Aged; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Down-Regulation; Female; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Liver Neoplasms; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

In the liver, derangement of TGF-beta signaling is associated with an increased incidence of hepatocellular carcinoma (HCC), but the mechanism is not clear. We report here that forced expression of a major TGF-beta signaling transducer, Smad3, reduces susceptibility to HCC in a chemically induced murine model. This protection is conferred by Smad3's ability to promote apoptosis by repressing Bcl-2 transcription in vivo through a GC-rich element in the Bcl-2 promoter. We also show that the proapoptotic activity of Smad3 requires both input from TGF-beta signaling and activation of p38 MAPK, which occurs selectively in the liver tumor cells. Thus, Smad3 enables the tumor suppression function of TGF-beta by serving as a physiological mediator of TGF-beta-induced apoptosis.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cells, Cultured; Disease Susceptibility; Down-Regulation; Hepatocytes; Liver Neoplasms, Experimental; Mice; Mice, Transgenic; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta

We investigated the expression levels of several genes related to cell proliferation in human mesenchymal stem cells (hMSCs) during in vitro culture for use in clinical applications. In this study, we focused on the relationship between hMSC proliferation and transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and marked changes in hMSC morphology were not observed in 3 months of in vitro culture. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. There had been no change in the TGFbeta3, TGFbetaRII, and TGFbetaRIII mRNA expressions by the 12th passage from the primary culture (at about 3 months). The mRNA expression of Smad3 increased, but those of c-myc and nucleostemin decreased with the length of hMSC in vitro culture. In addition, the expression profiles of the genes that regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months of in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.

Topics: Activin Receptors, Type I; Carcinoma, Hepatocellular; Cell Proliferation; Follow-Up Studies; Gene Expression; HeLa Cells; Humans; Liver Neoplasms; Mesenchymal Stem Cells; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Tumor Cells, Cultured

This study analyzed gene expression patterns and global genomic alterations in hepatocellular carcinomas (HCC), hepatoblastomas (HPBL), tissue adjacent to HCC and normal liver tissue derived from normal livers and hepatic resections. We found that HCC and adjacent non-neoplastic cirrhotic tissue have considerable overlap in gene expression patterns compared to normal liver. Several genes including Glypican 3, spondin-2, PEG10, EDIL3 and Osteopontin are over-expressed in HCC vs. adjacent tissue whereas Ficolin 3 is the most consistently under-expressed gene. HCC can be subdivided into three clusters based on gene expression patterns. HCC and HPBL have clearly different patterns of gene expression, with genes IGF2, Fibronectin, DLK1, TGFb1, MALAT1 and MIG6 being over-expressed in HPBL versus HCC. In addition, specific areas of the genome appear unstable in HCC, with the same regions undergoing either deletion or increased gene dosage in all HCC. In conclusion, a set of specific genes and areas of genomic instability are found across the board in liver neoplasia.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Carrier Proteins; Cell Adhesion Molecules; Cluster Analysis; DNA-Binding Proteins; Fibronectins; Fibrosis; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome; Glycoproteins; Glypicans; Heparan Sulfate Proteoglycans; Hepatoblastoma; Humans; Insulin-Like Growth Factor II; Intercellular Signaling Peptides and Proteins; Lectins; Liver Neoplasms; Membrane Proteins; Osteopontin; Proteins; Repressor Proteins; RNA-Binding Proteins; Sialoglycoproteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

The molecular mechanisms underlying the progression of cirrhosis toward hepatocellular carcinoma were investigated by a combination of DNA microarray analysis and literature data mining. By using a microarray screening of suppression subtractive hybridization cDNA libraries, we first analyzed genes differentially expressed in tumor and nontumor livers with cirrhosis from 15 patients with hepatocellular carcinomas. Seventy-four genes were similarly recovered in tumor (57.8% of differentially expressed genes) and adjacent nontumor tissues (64% of differentially expressed genes) compared with histologically normal livers. Gene ontology analyses revealed that downregulated genes (n = 35) were mostly associated with hepatic functions. Upregulated genes (n = 39) included both known genes associated with extracellular matrix remodeling, cell communication, metabolism, and post-transcriptional regulation gene (e.g., ZFP36L1), as well as the tumor suppressor gene menin (multiple endocrine neoplasia type 1; MEN1). MEN1 was further identified as an important node of a regulatory network graph that integrated array data with array-independent literature mining. Upregulation of MEN1 in tumor was confirmed in an independent set of samples and associated with tumor size (P = .016). In the underlying liver with cirrhosis, increased steady-state MEN1 mRNA levels were correlated with those of collagen alpha2(I) mRNA (P < .01). In addition, MEN1 expression was associated with hepatic stellate cell activation during fibrogenesis and involved in transforming growth factor beta (TGF-beta)-dependent collagen alpha2(I) regulation. In conclusion, menin is a key regulator of gene networks that are activated in fibrogenesis associated with hepatocellular carcinoma through the modulation of TGF-beta response.

Topics: Carcinoma, Hepatocellular; Collagen Type I; Gene Library; Genes, Tumor Suppressor; Hepatocytes; Humans; Liver Cirrhosis; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins; RNA, Messenger; Transfection; Transforming Growth Factor beta; Up-Regulation

To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells.. Fifty-four adult male Wistar rats were randomly divided into three groups: A normal control (NC) group, a partial hepatectomized (PH) group and a sham operated (SO) group. To study the effect of liver regeneration on BMP-2 expression, rats were sacrificed before and at different time points after PH or the sham intervention (6, 12, 24 and 48 h). For each time point, six rats were used in parallel. Expression and distribution of BMP-2 protein were determined in regenerating liver tissue by Western blot analysis and immunohistochemistry. Effects of BMP-2 on cell proliferation of human Huh7 hepatoma cell line were assessed using an MTT assay.. In the normal liver strong BMP-2 expression was observed around the central and portal veins. The expression of BMP-2 decreased rapidly as measured by both immunohistochemistry and Western blot analysis. This decrease was at a maximum of 3.22 fold after 12 h and returned to normal levels at 48 h after PH. No significant changes in BMP-2 immunoreactivity were observed in the SO group. BMP-2 inhibited serum induced Huh7 cell proliferation.. BMP-2 is expressed in normal adult rat liver and negatively regulates hepatocyte proliferation. The observed down regulation of BMP-2 following partial hepatectomy suggests that such down regulation may be necessary for hepatocyte proliferation.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Down-Regulation; Hepatocytes; Humans; Liver Neoplasms; Liver Regeneration; Male; Rats; Rats, Wistar; Transforming Growth Factor beta

Cell-matrix adhesive interaction has an important role in the invasive process of tumor cells, and integrins are the major receptors mediating cell-matrix adhesion. The current study is to investigate the modulation of basement membrane (BM) proteins, especially collagen IV (C IV), laminin (LN), and fibronectin (FN) in the invasive processes of human hepatocellular carcinoma (HCC) cells in vitro, and to reveal the roles of beta1 integrins and RGD-containing oligopeptide in the cell-matrix interaction.. Static adhesion assay was performed to study the rates of adhesion of MHCC97-H cells, treated or untreated with anti-beta1 (2 microg ml(-1)) and GRGDS, to C IV (50 microg ml(-1)), LN (50 microg ml(-1)) or FN (50 microg ml(-1)). Gelatin zymography was used to detect the secretion of MMPs in the conditioned medium of MHCC97-H cells incubated 24 h by C IV, LN or FN, and interactions with anti-beta1 and GRGDS. Transwell chamber assay was used to investigate the influence of C IV, LN or FN, interacting with anti-beta1 and GRGDS, on the cellular mobility of MHCC97-H cells.. Compared with blank control group, MHCC97-H cells showed significantly higher rates of adhesion to C IV, LN, and FN. Pretreatment with anti-beta1 could suppress adhesion to C IV, LN or FN, but GRGDS inhibited adhesion to FN (P<0.05) only. LN and FN could stimulate the secretion of MMPs by MHCC97-H cells cultured in vitro, especially MMP-9 and its activated type. Treatment with anti-beta1 could partly counteract the effects of LN and FN. GRGDS could prominently induce the secretion of MMPs, but the effect could be inhibited by pretreatment of anti-beta1. The results of Transwell chamber assay showed that LN, FN, and GRGDS could increase the number of tumor cells penetrating the microporous membrane, but the data of C IV did not reach significance. The effects were partly counteracted by anti-beta1.. BM proteins play an active role in the invasive process of human hepatocellular carcinoma cells. Integrin beta1 is an important molecule which mediates the cell-matrix adhesive interaction of tumor cells. RGD-containing peptides competitively combine with the binding site of integrin beta1, and the effects of FN are RGD sequence-dependent.

Topics: Basement Membrane; Carcinoma, Hepatocellular; Cell Adhesion; Collagen Type IV; Extracellular Matrix Proteins; Fibronectins; Humans; Integrin beta Chains; Laminin; Liver Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Aged; Aged, 80 and over; Aortic Aneurysm; Aortic Dissection; Biomarkers, Tumor; Carcinoma, Hepatocellular; Fibrillin-1; Fibrillins; Humans; Liver Neoplasms; Male; Microfilament Proteins; Tomography, X-Ray Computed; Transforming Growth Factor beta

Immortalized p19(ARF) null hepatocytes (MIM) feature a high degree of functional differentiation and are susceptible to transforming growth factor (TGF)-beta driven growth arrest and apoptosis. In contrast, polarized MIM hepatocytes expressing hyperactive Ha-Ras continue proliferation in cooperation with TGF-beta, and adopt an invasive phenotype by executing an epithelial to mesenchymal transition (EMT). In this study, we analyzed the involvement of Ras subeffectors in TGF-beta mediated hepatocellular EMT by employing MIM hepatocytes, which express Ras mutants allowing selective activation of either mitogen-activated protein kinase (MAPK) signaling (V12-S35) or phosphoinositide 3-OH (PI3)3 kinase (PI3K) signaling (V12-C40). We found that MAPK signaling in MIM-S35 hepatocytes was necessary and sufficient to promote resistance to TGF-beta mediated inhibition of proliferation in vitro and in vivo. MIM-S35 hepatocytes showed also PI3K activation during EMT, however, MAPK signaling on its own protected hepatocytes from apoptosis. Yet, MIM-C40 hepatocytes failed to form tumors and required additional MAPK stimulation to overcome TGF-beta mediated growth arrest. In vivo, the collaboration of MAPK signaling and TGF-beta activity drastically accelerated the cell-cycle progression of the hepatocytes, leading to vast tumor formation. From these data we conclude that MAPK is crucial for the cooperation with TGF-beta to regulate the proliferation as well as the survival of hepatocytes during EMT, and causes the fatal increase in hepatocellular tumor progression.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Humans; Mice; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; ras Proteins; Signal Transduction; Transforming Growth Factor beta

We have previously shown that transforming growth factor-beta1 (TGF-beta1) markedly stimulates the invasive capacity of rat ascites hepatoma AH130 W1 cells in vitro and in vivo. A differential hybridization procedure was used to isolate genes that were specifically up-regulated in TGF-beta1 treated W1 cells. Among ten independent cDNA clones, we focused on LMO7 and a variant isoform, LMO7S, that was generated by alternative splicing. LMO7 had PDZ and LIM domains, while LMO7S had only PDZ domain. TGF-beta1 up-regulated expression levels of LMO7 and LMO7S. LMO7 expression was up-regulated in the highly metastatic clone MM1.

Topics: Animals; Ascites; Carcinoma, Hepatocellular; Homeodomain Proteins; Liver Neoplasms; Neoplasm Invasiveness; Rats; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Up-Regulation

Transforming growth factor beta (TGF-beta) stimulation results in the assembly of Smad-containing protein complexes that mediate activation or repression of TGF-beta responsive genes. To determine if disruption of specific Smad protein-protein interactions would selectively inhibit responses to TGF-beta or generally interfere with Smad-dependent signaling, we developed three Smad-binding peptide aptamers by introducing Smad interaction motifs from Smad-binding proteins CBP, FoxH1 and Lef1 into the scaffold protein E. coli thioredoxin A (Trx). All three classes of aptamers bound to Smads by GST pulldown assays and co-immunoprecipitation from mammalian cells. Expression of the aptamers in HepG2 cells did not generally inhibit Smad-dependent signaling as evaluated using seven TGF-beta responsive luciferase reporter genes. The Trx-xFoxH1b aptamer inhibited TGF-beta-induced expression from a reporter dependent on the Smad-FoxH1 interaction, A3-lux, by 50%. Trx-xFoxH1b also partially inhibited two reporters not dependent on a Smad-FoxH1 interaction, 3TP-lux and Twntop, and endogenous PAI-1 expression. Trx-Lef1 aptamer only inhibited expression of the Smad-Lef1 responsive reporter gene TwnTop. The Trx-CBP aptamer had no significant effect on reporter gene expression. The results suggest that Smad-binding peptide aptamers can be developed to selectively inhibit TGF-beta-induced gene expression.

Topics: Amino Acid Sequence; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Corticosterone; DNA-Binding Proteins; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glutathione Transferase; Humans; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Open Reading Frames; Polymerase Chain Reaction; Smad3 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Xenopus

In this study, we determined the frequencies of the genotypes associated with the polymorphism of the cytokines genes, and investigated their association with the risk of hepatocellular carcinoma (HCC) in hepatitis B virus (HBV) carriers.. Genetic polymorphism in the cytokines TNF-alpha, IFN-gamma, TGF-beta1, IL-6, and IL-10 were studied in 236 Japanese patients with HBV infection. The genetic polymorphisms of these cytokines were analyzed by polymerase chain reaction-sequence-specific primer (SSP).. There was no statistically significant difference in the genetic polymorphisms of TNF-alpha, IFN-gamma, and IL-10 genes between HBV carriers with HCC and those without HCC. However, the TGF-beta1+29 (codon 10) C/C genotype was lower in HBV carriers with HCC than in those without HCC (HCC 14.6% vs non-HCC 31.9%). The association of HCC was significantly lower in HBV carriers with C/C genotype than in those with T/C or T/T genotype in position +29 of the TGF-beta1 gene.. Our findings suggest that the genetic polymorphism in codon 10 of the TGF-beta1 gene may play a role in HCC development in patients with chronic HBV infection.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Codon; Female; Genotype; Hepatitis B, Chronic; Humans; Interferon-gamma; Interleukin-10; Interleukin-6; Japan; Liver Neoplasms; Male; Middle Aged; Polymorphism, Genetic; Promoter Regions, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Hepatocellular carcinomas (HCC) often show resistance to the effects of transforming growth factor-beta (TGF-beta). This study focuses on molecular mechanisms of this resistance to explore ways to overcome it.. Transcription and protein expression of TGF-beta type I and type II receptors (TGF-betaRI/RII) were analyzed in clinical HCCs and the human hepatoma cell lines HuH-7 and HepG2. HuH-7 cells were transiently and stably transfected with a constitutively active TGF-betaRI mutant (CA TGF-betaRI). Resulting growth kinetics, integrin expression, invasiveness, TGF-beta-mediated activation of human plasminogen activator inhibitor type-1 (PAI-1) promoter and Smad expression were determined.. In clinical HCCs, there was less TGF-betaRII (6/10 cases) and more TGF-betaRI (8/10 cases) protein expression detectable in tumor compared to adjacent liver tissue. In HuH-7 cells, TGF-betaRII expression was likewise decreased. Cells transiently transfected with CA TGF-betaRI exhibited strong TGF-beta-related PAI-1 promoter activation. Stably transfected cells showed an attenuated response of the PAI-1 promoter, but increased Smad7 expression. Proliferation of stable clones was decreased. There was no change in integrin expression or invasiveness.. Decreased TGF-betaRII protein expression might cause TGF-beta resistance in a subset of clinical HCCs. Stable transfection with CA TGF-betaRI reverses this in HuH-7 cells without increasing invasiveness.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Profiling; Genetic Therapy; Humans; Mutation; Neoplasm Invasiveness; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Integrin-mediated cell adhesion transduces signals to regulate actin cytoskeleton and cell proliferation. While understanding how integrin signals cross-talk with the TGF-beta1 pathways, we observed lamellipodia formation and cyclin regulation in Hep3B cells, following TGF-beta1 treatment. To answer if integrin signaling via actin organization might regulate cell cycle progression after TGF-beta1 treatment, we analyzed cross-talk between the two receptor-mediated pathways in hepatoma cells on specific ECMs. We found that basal and TGF-beta1-mediated activation of c-Src and Rac1, expression of cyclins E and A, and suppression of p27Kip1 were significant in cells replated on fibronectin, but not in cells on collagen I, indicating a different integrin-mediated cellular response to TGF-beta1 treatment. Levels of tyrosine phosphorylation and actin-enriched lamellipodia on fibronectin were also more prominent than in cells on collagen I. Studies using pharmacological inhibitors or transient transfections revealed that the preferential TGF-beta1 effects in cells on fibronectin required c-Src family kinase activity. These observations suggest that a specific cross-talk between TGF-beta1 and fibronectin-binding integrin signal pathways leads to the activation of c-Src/Rac1/actin-organization, leading to changes in cell cycle regulator levels in hepatoma cells. Therefore, this study represents another mechanism to regulate cell cycle regulators when integrin signaling is collaborative with TGF-beta1 pathways.

Topics: Actins; Blotting, Western; Bromodeoxyuridine; Carcinoma, Hepatocellular; Cell Adhesion; Cell Cycle; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cell Proliferation; Collagen Type I; CSK Tyrosine-Protein Kinase; Cyclin A; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Fibronectins; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Humans; Integrin alpha2; Integrins; Microscopy, Phase-Contrast; Mutation; Phosphotransferases; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; rac1 GTP-Binding Protein; Signal Transduction; src-Family Kinases; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins

Topics: Carcinoma, Hepatocellular; Cytokines; Humans; Liver Neoplasms; NF-kappa B; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. However, the exact mechanism through which TGF-beta induces cell death is still unknown. We examined a potential role of various death receptor/ligand systems in TGF-beta-induced apoptosis and identified the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a mediator of TGF-beta-induced apoptosis in hepatoma cells. TGF-beta-induced apoptosis is significantly impaired upon blockage of TRAIL. We show that TRAIL is upregulated in hepatoma cells upon treatment with TGF-beta, whereas TRAIL receptor levels remain unchanged. In conclusion, our results provide evidence that the TRAIL system is critically involved in TGF-beta-induced cell death in liver pathology.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Hepatocytes; Humans; Liver Neoplasms; Membrane Glycoproteins; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Hepatitis C virus (HCV) is a major risk factor for human hepatocellular carcinoma (HCC) but the mechanisms underlying HCV-induced carcinogenesis are still poorly understood. We have hypothesized that viral variants, selected during long-term infection, might contribute to cellular transformation. To address this issue, we have investigated the effect of natural HCV core variants isolated from liver tumors (T), or their non-tumor (NT) counterparts, on the tumor growth factor-beta (TGF-beta) pathway, a major regulator of cellular proliferation, differentiation and apoptosis. We have found a significant reduction in TGF-beta reporter gene activity with the expression of core sequences isolated from liver tumors. In contrast, moderate or no effects were observed with non-tumor mutants or a core reference sequence. The molecular mechanisms have been characterized and involved the inhibition, by tumor-derived cores, of the DNA-binding activity of the Smad3/4 transcription factors complex. This inhibition occurs through a direct interaction between the central domain (amino acids 59-126) of tumor-derived core and the MH1 DNA-binding domain of Smad3, thus preventing its binding to DNA. We have therefore identified a new cell-signaling pathway targeted by HCV core and inhibited by tumor-derived core sequences. These results suggest that during chronic infection, there is selection of viral variants that may promote cell transformation by providing, to clonally expanding cells, resistance to TGF-beta antiproliferative effects.

Topics: Amino Acid Sequence; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; DNA-Binding Proteins; Hepacivirus; Humans; Liver Neoplasms; Molecular Sequence Data; Mutation; Protein Binding; Protein Structure, Tertiary; Signal Transduction; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured; Viral Core Proteins

The mechanism by which hepatitis C virus induces liver fibrosis remains largely obscure. To characterize the profibrogenic potential of hepatitis C virus, we used the hepatitis C virus replicon cell line Huh-7 5-15, which stably expresses the nonstructural hepatitis C virus genes NS3 through NS5B, and hepatic stellate cells as fibrogenic effector cells.. Rat and human hepatic stellate cells were incubated with conditioned media from replicon cells, and expression of fibrosis-related genes was quantified by using real-time polymerase chain reaction, protein, and functional assays. Transforming growth factor beta1 activity was determined by bioassay.. Hepatitis C virus replicon cells release factors that differentially modulate hepatic stellate cell expression of key genes involved in liver fibrosis in a clearly profibrogenic way, up-regulating procollagen alpha1(I) and procollagen alpha1(III) and down-regulating fibrolytic matrix metalloproteinases. Transforming growth factor beta1 expression and bioactivity were increased severalfold in hepatitis C virus-replicating vs mock-transfected hepatoma cells. However, transforming growth factor beta1 activity was responsible for only 50% of the profibrogenic activity.. Hepatitis C virus nonstructural genes induce an increased expression of transforming growth factor beta1 and other profibrogenic factors in infected hepatocytes. The direct induction of profibrogenic mediators by hepatitis C virus in infected hepatocytes explains the frequent observation of progressive liver fibrosis despite a low level of inflammation and suggests novel targets for antifibrotic therapies in chronic hepatitis C.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Culture Media, Conditioned; Gene Expression Regulation, Viral; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Liver Cirrhosis; Liver Neoplasms; Rats; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Active NF-kappaB renders malignant hepatocytes refractory to the growth inhibitory and pro-apoptotic properties of transforming growth factorbeta1 (TGF-beta1). NF-kappaB counteracts TGF-beta1-induced apoptosis through up-regulation of downstream target genes, such as XIAP and Bcl-X(L), which in turn inhibit the intrinsic pathway of apoptosis. In addition, induction of NF-kappaB by TGF-beta1 inhibits JNK signaling, thereby attenuating TGF-beta1-induced cell death of normal hepatocytes. However, the mechanism involved in the negative cross-talk between the NF-kappaB and JNK pathways during TGF-beta1 signaling has not been determined. In this study, we have identified the XIAP gene as one of the critical mediators of NF-kappaB-mediated suppression of JNK signaling. We show that NF-kappaB plays a role in the up-regulation of XIAP gene expression in response to TGF-beta1 treatment and forms a TGF-beta1-inducible complex with TAK1. Furthermore, we show that the RING domain of XIAP mediates TAK1 polyubiquitination, which then targets this molecule for proteosomal degradation. Down-regulation of TAK1 protein expression inhibits TGF-beta1-mediated activation of JNK and apoptosis. Conversely, silencing of XIAP promotes persistent JNK activation and potentiates TGF-beta1-induced apoptosis. Collectively, our findings identify a novel mechanism for the regulation of JNK activity by NF-kappaB during TGF-beta1 signaling and raise the possibility that pharmacologic inhibition of the NF-kappaB/XIAP signaling pathway might selectively abolish the pro-oncogenic activity of TGF-beta1 in advanced hepatocellular carcinomas (HCCs) without affecting the pro-apoptotic effects of TGF-beta1 involved in normal liver homeostasis.

Topics: Animals; Anthracenes; Apoptosis; bcl-X Protein; Carcinoma, Hepatocellular; Cell Death; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Gene Silencing; Glutathione Transferase; Hepatocytes; Humans; Immunoblotting; Luciferases; Male; MAP Kinase Kinase 4; MAP Kinase Kinase Kinases; Mice; Mitogen-Activated Protein Kinase 8; Models, Biological; NF-kappa B; Proteasome Endopeptidase Complex; Protein Binding; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors; Transcription Factor AP-1; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ubiquitin; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

Insulin-like growth factor binding protein-3 (IGFBP-3) is a mediator of growth suppression signals and a putative tumor suppressor gene. The growth suppression mechanisms of IGFBP-3 have not been well clarified. We examined the expression of IGFBP-3 transcripts in human hepatocellular carcinoma (HCC) and the relationship between IGFBP-3 expression and the transforming growth factor-beta (TGF-beta) and/or retinoblastoma (Rb) signaling pathways. In situ hybridization revealed IGFBP-3 transcripts in cancer cells in 6 of 57 (10%) HCCs, including moderately and poorly differentiated HCCs with intrahepatic metastasis. In contrast, all lung metastatic nodules of 4 HCCs showed IGFBP-3 transcripts in cancer cells. The cDNA microarray showed that genes for the TGF-beta pathway and Rb were up-regulated in IGFBP-3-expressing HCCs. In 6 HCCs presenting IGFBP-3, immunohistochemical analyses showed abnormalities in the TGF-beta and/or Rb pathways; the loss of phosphorylated-Smad2 was observed in 2, and overexpression of phosphorylated-Rb was observed in the remaining 4 HCCs. The present study suggests that IGFBP-3 mediates growth suppression signals via the TGF-beta and/or Rb pathways in HCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Case-Control Studies; Cell Differentiation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Insulin-Like Growth Factor Binding Protein 3; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Retinoblastoma Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

How hepatocellular carcinoma (HCC) cells acquire the ability to invade surrounding tissue is unknown, but epithelial mesenchymal transition (EMT) likely plays a role. We investigate how transforming growth factor (TGF)-beta1 and extracellular matrix protein Laminin-5 (Ln-5) induce EMT and cancer invasion.. Snail, Slug, E-cadherin, beta-catenin and Ln-5 were investigated on HCC tissues and on HCC cell lines.. We show that in HCC but not in peritumoral tissue of the same HCC patients, Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated and beta-catenin is translocated into the nuclei. In vitro, HCC "invasive" cells, partially EMT-transformed, show low levels of E-cadherin. In presence of Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated, beta-catenin is translocated into the nuclei, and cells undergo a dramatic morphological change, becoming scattered and undergoing a complete EMT. This effect is reversed by anti-alpha3 but not by anti-alpha6 integrin blocking antibody. HCC "noninvasive" cells are not EMT-transformed, and have constitutively high levels of E-cadherin. In presence of Ln-5, cells undergo partial EMT, Snail, and Slug are up-regulated, E-cadherin is down-regulated but cells do not scatter. However, the presence of both Ln-5 and TGF-beta1 completes the EMT process, beta-catenin is translocated into the nuclei, cells scatter and become invasive, recalling the "invasive" cells. In this case, too, the effect is reversed by anti-alpha3 integrin blocking antibody.. Our study shows that Ln-5 and TGF-beta1 cooperatively induce EMT in HCC, suggesting the microenvironment as a potential target for new biological therapies.

Topics: beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Kalinin; Liver Neoplasms; Mesoderm; Neoplasm Invasiveness; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; DNA Primers; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Integrin alpha3; Liver Neoplasms; Molecular Sequence Data; Neoplasm Invasiveness; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Proto-Oncogene Protein c-ets-1; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

The Wnt signaling pathway is activated in most human colorectal tumors. Mutational inactivation in the tumor suppressor adenomatous polyposis coli (APC), as well as activation of beta-catenin, causes the accumulation of beta-catenin, which in turn associates with the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors and activates transcription of their target genes. Here we show that beta-catenin activates transcription of the BMP and activin membrane-bound inhibitor (BAMBI)/NMA gene. The expression level of BAMBI was found to be aberrantly elevated in most colorectal and hepatocellular carcinomas relative to the corresponding non-cancerous tissues. Expression of BAMBI in colorectal tumor cell lines was repressed by a dominant-negative mutant of TCF-4 or by an inhibitor of beta-catenin-TCF interaction, suggesting that beta-catenin is responsible for the aberrant expression of BAMBI in colorectal tumor cells. Furthermore, overexpression of BAMBI inhibited the response of tumor cells to transforming growth factor-beta signaling. These results suggest that beta-catenin interferes with transforming growth factor-beta-mediated growth arrest by inducing the expression of BAMBI, and this may contribute to colorectal and hepatocellular tumorigenesis.

Topics: Adenoviridae; Animals; beta Catenin; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; Colorectal Neoplasms; COS Cells; Cytoskeletal Proteins; Dimerization; DNA Mutational Analysis; DNA-Binding Proteins; Genes, Reporter; Humans; Immunohistochemistry; Liver; Luciferases; Lymphoid Enhancer-Binding Factor 1; Membrane Proteins; Oligonucleotide Array Sequence Analysis; Plasmids; Promoter Regions, Genetic; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Hepatitis B virus (HBV) X antigen (HBxAg) may contribute to the development of hepatocellular carcinoma (HCC) by activation of signalling pathways such as NF-kappaB. To identify NF-kappaB target genes differentially expressed in HBxAg-positive compared to -negative cells, HepG2 cells consistently expressing HBxAg (HepG2X cells) were stably transfected with pZeoSV2 or pZeoSV2-IkappaBalpha. mRNA from each culture was isolated and compared by PCR select cDNA subtraction. The results showed lower levels of alpha(2)-macroglobulin (alpha(2)-M) in HepG2X-pZeoSV2 compared to HepG2X-pZeoSV2-IkappaBalpha cells. This was confirmed by Northern and Western blotting, and by measurement of extracellular alpha(2)-M levels. Elevated transforming growth factor-beta1 (TGF-beta1) levels were also seen in HepG2X compared to control cells. Serum-free conditioned medium (SFCM) from HepG2X cells suppressed DNA synthesis in a TGF-beta-sensitive cell line, Mv1Lu. The latter was reversed when the SFCM was pretreated with exogenous, activated alpha(2)-M or with anti-TGF-beta. Since elevated TGF-beta1 promotes the development of many tumour types, these observations suggest that the HBxAg-mediated alteration in TGF-beta1 and alpha(2)-M production may contribute importantly to the pathogenesis of HCC.

Topics: alpha-Macroglobulins; Carcinoma, Hepatocellular; Cell Line; Gene Expression Regulation; Humans; I-kappa B Proteins; Liver Neoplasms; NF-KappaB Inhibitor alpha; RNA, Messenger; Trans-Activators; Transforming Growth Factor beta; Viral Regulatory and Accessory Proteins

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor beta1 (TGF-beta1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-beta1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-beta1. The results indicated that greater than 90% of the TGF-beta1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21(Cip1/Waf1) expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21(Cip1/Waf1) and p27(Kip1) into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-beta1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-beta1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21(Cip1/Waf1) and p27(Kip1), resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-beta1-induced apoptosis downstream of the pRb/E2F-1 pathway.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p21; Humans; Liver Neoplasms; Protein Transport; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Although transforming growth factor beta1 (TGF-beta1) acts via the Smad signaling pathway to initiate de novo gene transcription, the TGF-beta1-induced MAPK kinase activation that is involved in the regulation of apoptosis is less well understood. Even though the p38 MAP kinase and c-Jun NH(2)-terminal kinases (JNKs) are involved in TGF-beta1-induced cell death in hepatoma cells, the upstream mediators of these kinases remain to be defined. We show here that the members of the mixed lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper-bearing kinase (DLK)) are expressed in FaO rat hepatoma cells and are likely to act between p38 and TGF-beta receptor kinase in death signaling. TGF-beta1 treatment leads to an increase in MLK3 activity. Overexpression of MLK3 enhances TGF-beta1-induced apoptotic death in FaO cells and Hep3B human hepatoma cells, whereas expression of the dominant-negative forms of MLK3 suppresses cell death induced by TGF-beta1. The dominant-negative forms of MLK1 and -2 also suppress TGF-beta1-induced cell death. In MLK3-overexpressing cells, ERK, JNKs, and p38 MAP kinases were further activated in response to TGF-beta1 compared with the control cells. In contrast, overexpression of the dominant-negative MLK3 resulted in suppression of TGF-beta1-induced MAP kinase activation and TGF-beta1-induced caspase-3 activation. We also show that only the inhibition of the p38 pathway suppressed TGF-beta1-induced apoptosis. These observations support a role for MLKs in the TGF-beta1-induced cell death mechanism.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Humans; Liver Neoplasms; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinase Kinase 11; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proteins; Rats; Recombinant Fusion Proteins; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta1 (TGF-beta1) has been implicated in tumor progression. The relationship of this cytokine as measured in plasma to anti-tumor immunity and prognosis was investigated. This study consisted of 70 consecutive patients with unresectable hepatocellular carcinoma (HCC) (median age, 65 years). Forty-four healthy age-matched subjects and 32 patients with cirrhosis but no carcinoma served as controls. Patients with HCC were divided into those with plasma TGF-beta1 concentrations above (group A, n=21) or below (group B, n=49) 10 ng/ml (the mean concentration+2SD in the concentrations of the controls with cirrhosis was 8.7 ng/ml). Age, gender, Child-Pugh grade, and tumor stage distributions were similar in groups A and B. Considering all tumor stages together and individually, group A had a significantly shorter survival (median for all stages, 2 months) than group B (median for all stages, 10 months; P<0.01, generalized Wilcoxon's test). Groups A and B had significantly shorter survival than controls with cirrhosis (P<0.001 for each). Lymphokine-activated killer (LAK) activity in group A was significantly lower than that in group B (P<0.001). Natural killer (NK) activity in group A was also significantly lower than that in group B (P<0.05). Plasma TGF-beta1 concentration was a significant predictor of survival by Cox's proportional-hazards regression analysis (multivariate analysis, P<0.01). LAK and NK activities were also weak but significant predictors (P<0.05 and <0.05, respectively). These data suggest that plasma TGF-beta1 concentration is a predictor of outcome of patients with unresectable HCC. Circulating TGF-beta1 supposedly contributes to the suppression of anti-tumor immunity in the advanced disease.

Topics: Aged; Carcinoma, Hepatocellular; Female; Humans; Immunity; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Liver Neoplasms; Male; Middle Aged; Outcome Assessment, Health Care; Predictive Value of Tests; Prognosis; Survival Analysis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Activin receptor-like kinase (ALK)7 is a type I serine/threonine kinase receptor of the transforming growth factor (TGF)-beta family of proteins that has similar properties to other type I receptors when activated. To see whether ALK7 can induce apoptosis as can some of the other ALK proteins, we infected the FaO rat hepatoma cell line with adenovirus expressing a constitutively active form of the ALK7. Cells infected with active ALK7 adenovirus showed an apoptotic-positive phenotype, as opposed to those that were infected with a control protein. DNA fragmentation assays and fluorescence-activated cell sorter analysis also indicated that ALK7 infection induced apoptosis in FaO cells. We also confirmed this finding in Hep3B human hepatoma cells by transiently transfecting the constitutively active form of ALK7, ALK7(T194D). Investigation into the downstream targets and mechanisms involved in ALK7-induced apoptosis revealed that the TGF-beta signaling intermediates, Smad2 and -3, were activated, as well as the MAPKs JNK and p38. In addition, caspase-3 and -9 were also activated, and cytochrome c release from the mitochondria was observed. Short interfering RNA-mediated inhibition of Smad3 markedly suppressed ALK7-induced caspase-3 activation. Treatment with protein synthesis inhibitors or the expression of the dominant-negative form of the stress-activated protein/extracellular signal-regulated kinase 1 abolished not only JNK activation but apoptosis as well. Taken together, these results suggest that ALK7 induces apoptosis through activation of the traditional TGF-beta pathway components, thus resulting in new gene transcription and JNK and p38 activation that initiates cross-talk with the cellular stress death pathway and ultimately leads to apoptosis.

Topics: Activin Receptors, Type I; Adenoviridae; Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cell Line; Cell Line, Tumor; Cell Separation; Cell Survival; Cytochromes c; DNA Fragmentation; DNA-Binding Proteins; Dose-Response Relationship, Drug; Flow Cytometry; Genes, Dominant; Genetic Vectors; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Plasmids; Rats; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death in the world. The present study aimed to investigate the genes involved in viral carcinogenesis and tumor progression in liver transplant recipients with hepatitis C virus (HCV) and HCC. To accomplish this, we performed the analysis of hepatic gene expression in HCV-infected liver recipient patients with stages of disease ranging from early cirrhosis with preserved volume and function to late cirrhosis with diminished volume and function with and without HCC. We found consistent differences between the gene expression patterns in HCV-HCC and those of early HCV-cirrhosis, late HCV cirrhosis, and normal control livers. The expression patterns in HCC were also readily distinguished between early and advanced HCC tumor stages. Moreover, we found different gene expression patterns between early cirrhosis and late cirrhosis. In conclusion, these findings confirm the presence of multiple molecular alterations during HCV-HCC hepatocarcinogenesis and, clinically, indicate the possibility for identifying prognostic factors associated with HCC progression in liver transplant patients waiting for a donor and/or posttransplantation recurrence.

Topics: Adult; Carcinoma, Hepatocellular; Disease Progression; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycoproteins; Hepatitis C; Humans; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Protein Folding; Transforming Growth Factor beta; Up-Regulation

Bone morphogenetic proteins (BMPs) play central roles in differentiation, development, and physiologic tissue remodeling. Recently, we have demonstrated that a protein inhibitor of activated STAT, PIASy, suppresses TGF-beta signaling by interacting with Sma and MAD-related protein 3 (Smad3). In this study, we examined a PIASy-dependent inhibitory effect on BMP signaling. PIASy expression was induced by BMP-2 stimulation and suppressed BMP-2-dependent Smad activity in hepatoma cells. Furthermore, BMP-2-regulated Smads directly bound to PIASy. We also demonstrated that the RING domain of PIASy played an important role in PIASy-mediated suppression of Smad activity. We here provide evidence that the inhibitory action of PIASy on BMP-regulated Smad activity was due to direct physical interactions between Smads and PIASy through its RING domain.

Topics: Blotting, Northern; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line; DNA-Binding Proteins; Gene Expression Regulation; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Luciferases; Poly-ADP-Ribose Binding Proteins; Precipitin Tests; Protein Binding; Protein Inhibitors of Activated STAT; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Time Factors; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta

Growth inhibition by transforming growth factor (TGF)-beta 1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-beta 1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-beta 1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-beta 1 and partially released the cells from the growth arrest by TGF-beta 1. Expression of S100C/A11, a member of the Ca(2+)-binding S100 protein family, also markedly increased after addition of TGF-beta 1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-beta 1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-beta 1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-beta 1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-beta 1 via induction of p21/Waf1.

Topics: Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Humans; Interferon Regulatory Factor-1; Liver Neoplasms; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S100 Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Protein p53

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line; Cell Line, Tumor; Computational Biology; Databases, Protein; Gene Library; Homeodomain Proteins; Humans; Kidney; LIM Domain Proteins; Liver Neoplasms; Membrane Proteins; Placenta; Protein Interaction Mapping; Proteomics; Signal Transduction; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Two-Hybrid System Techniques

Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine implicated as a pathogenic mediator in various liver diseases. Enhanced TGF-beta production and lack of TGF-beta responses are often observed during hepatitis C virus (HCV) infection. In this study, we demonstrate that TGF-beta-mediated transactivation is decreased in cells exogenously expressing the intact HCV polyprotein. Among 10 viral products of HCV, only core and nonstructural protein 3 (NS3) physically interact with the MH1 (Mad homology 1) region of the Smad3 and block TGF-beta/Smad3-mediated transcriptional activation through interference with the DNA-binding ability of Smad3, not the nuclear translocation. However, the interactive domain of NS3 extends to the MH2 (Mad homology 2) region of Smad3 and a distinction is found between effects mediated, respectively, by these two viral proteins. HCV core, in the presence or absence of TGF-beta, has a stronger suppressive effect on the DNA-binding and transactivation ability of Smad3 than NS3. Although HCV core, NS3, and the HCV subgenomic replicon all attenuate TGF-beta/Smad3-mediated apoptosis, only HCV core represses TGF-beta-induced G1 phase arrest through downregulation of the TGF-beta-induced p21 promoter activation. Along with this, HCV core, rather than NS3, exhibits a significant inhibitory effect on the binding of Smad3/Sp1 complex to the proximal p21 promoter in response to TGF-beta. In conclusion, HCV viral proteins interact with the TGF-beta signaling mediator Smad3 and differentially impair TGF-beta/Smad3-mediated transactivation and growth inhibition. This functional counteraction of TGF-beta responses provides insights into possible mechanisms, whereby the HCV oncogenic proteins antagonize the host defenses during hepatocarcinogenesis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA-Binding Proteins; G1 Phase; Hepacivirus; Humans; Liver Neoplasms; Plasmids; Polymerase Chain Reaction; Recombinant Fusion Proteins; Smad3 Protein; Trans-Activators; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Viral Core Proteins; Viral Nonstructural Proteins; Viral Proteins

Most individuals exposed to hepatitis C virus (HCV) become chronically infected and are predisposed to liver disease. The mechanisms underlying viral persistence and disease progression are unknown. A role for the HCV NS5A protein in viral replication and interferon resistance has been demonstrated. To identify mechanisms affected by NS5A, we analyzed the gene expression of Huh7 cells expressing NS5A and control cells using oligonucleotide microarrays. A set of 103 genes (43 up-regulated, 60 down-regulated) whose expression was modified by at least twofold was selected. These included genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and genes regulating immune responses. The finding of modulated expression of genes related to the TGF-beta superfamily and liver fibrosis was observed. Interestingly, both the tumor necrosis factor and lymphotoxin beta receptors were down-regulated by NS5A. Similar data were obtained following expression of four NS5A mutants obtained from patients who were not responsive or were sensitive to interferon therapy. Through computational analysis, we determined that 39 of the 43 genes up-regulated by NS5A contained one or more nuclear factor kappaB (NF-kappaB) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF-kappaB consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF-kappaB by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of IkappaBalpha reverted NS5A mediated up-regulation of gene expression. In conclusion, this study suggests a role of NS5A and NF-kappaB in HCV pathogenesis and related liver disease. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Topics: Amino Acid Sequence; Binding Sites; Calcium; Carcinoma, Hepatocellular; Carrier Proteins; Gene Expression Profiling; Gene Expression Regulation, Viral; Hepatitis C; Humans; Interferon-alpha; Interleukin-1; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Lipid Metabolism; Liver Neoplasms; Molecular Sequence Data; NF-kappa B; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Viral Nonstructural Proteins

Insulin and dexamethasone are potent inhibitors of apoptosis induced by transforming growth factor-beta1 (TGF-beta) in hepatoma cells. Using FTO-2B rat hepatoma cells, we determined whether the anti-apoptotic effects of these agents result from interference within or upstream of the TGF-beta-induced caspase cascade. Activation of different initiator and effector caspases, Bax and Bcl-xL expression, mitochondrial cytochrome c release and activation of PKB/Akt were analyzed by use of synthetic caspase substrates and Western blotting, respectively. TGF-beta-induced apoptosis was characterized by release of cytochrome c from mitochondria and activation of caspases-3, -7, -8 and -9. These effects were observable as early as 8-12 h after start of treatment and increased with time of observation. Inhibition of TGF-beta-induced apoptosis by insulin and dexamethasone was paralleled by a strong reduction of caspase-3-like activity. Caspase-8 activation was almost completely suppressed by these agents, and caspase-9 activity was decreased to levels within or slightly above unstimulated control cells. In addition, cytochrome c release from mitochondria was efficiently repressed, which was associated with upregulation of Bcl-xL by dexamethasone and activation of PKB/Akt by insulin. Thus, both anti-apoptotic compounds exert their inhibitory effects through modulation of anti-apoptotic signalling pathways involved in regulation of cytochrome c release and activation of the caspase machinery.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase Inhibitors; Caspases; Cell Line, Tumor; Dexamethasone; Enzyme Activation; Enzyme Inhibitors; Humans; Insulin; Liver Neoplasms, Experimental; Rats; Transforming Growth Factor beta

Transforming growth factor beta (TGFbeta) is a central mitoinhibitory factor for epithelial cells, and alterations of TGFbeta signalling have been demonstrated in many different human cancers. We have analysed human hepatocellular carcinomas (HCCs) for potential pro-tumourigenic alterations in regard to expression of Smad4 and mutations and expression changes of the pro-oncogenic transcriptional co-repressors Ski and SnoN, as well as mRNA levels of matrix metalloproteinase-2 (MMP2), which is transcriptionally regulated by TGFbeta. Smad4 mRNA was detected in all HCCs; while, using immunohistology, loss of Smad4 expression was found in 10% of HCCs. Neither mutations in the transformation-relevant sequences nor significant pro-tumourigenic expression changes of the Ski and SnoN genes were detected. In HCC cell lines, expression of both genes was regulated, potentially involving phosphorylation. Ski showed a distinct nuclear speckled pattern, indicating recruitment to active transcription complexes. MMP2 mRNA levels were increased in 19% of HCCs, whereas MMP2 mRNA was not detectable in HCC cell lines, suggesting that MMP2 was derived only from tumour stroma cells. Transcript levels of Smad4, Ski, SnoN and MMP2 correlated well. These data argue against a significant role of Ski and SnoN in human hepatocarcinogenesis and suggest that, in the majority of HCCs, the analysed factors are co-regulated by an upstream mechanism, potentially by TGFbeta itself.

Topics: Carcinoma, Hepatocellular; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Matrix Metalloproteinase 2; Proto-Oncogene Proteins; RNA, Messenger; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Thyroid hormones regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors and associated pathways. We investigated the triiodothyronine (T3) modulation of gene expression, in human hepatocellular carcinoma cell lines, via a PCR-based cDNA subtraction method. Here we present further data on one of the T3-upregulated genes, fibronectin (FN). We demonstrate that the induction of FN protein expression by T3 in TRalpha1 and TRbeta1 over-expressing cells was time and dose-dependent at the mRNA and protein levels. Blockade of protein synthesis by cycloheximide almost completely inhibited the concomitant induction of FN mRNA by T3, indicating that T3 indirectly regulates FN. Furthermore, nuclear-run on and FN promoter assay clearly can specifically increase the number of FN transcriptional demonstrated that the presence of T3 initiations. In addition, we further confirmed that the up-regulation of FN by T3 was mediated, at least in part, by transforming growth factor-beta (TGF-beta), because the induction of FN was blocked in a dose-dependent manner by the addition of TGF-beta neutralizing antibody. In an effort to elucidate the we demonstrated the involvement of the signaling pathways involved in the activation of FN by T3, mitogen activated protein kinase/c-Jun N-terminal kinase/p38 MAPK (MAPK/JNK/p38) pathway. Although T3 induces the expression of TGF-beta, neither wild-type nor dominant-negative Smad3 or Smad4 over-expression affected the activation of FN by T3. Thus, we demonstrate that T3 regulates FN gene expression indirectly at the transcriptional level, with the participation of the MAPK/JNK/p38 pathway and the TGF-beta signaling pathway but independent of Smad3/4.

Topics: Carcinoma, Hepatocellular; Cycloheximide; DNA-Binding Proteins; Enzyme Inhibitors; Fibronectins; Gene Expression Regulation; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinases; Smad3 Protein; Smad4 Protein; Thyroid Hormone Receptors alpha; Thyroid Hormone Receptors beta; Trans-Activators; Transforming Growth Factor beta; Triiodothyronine; Tumor Cells, Cultured

The tumor suppressor PTEN gene maps to chromosome 10q23.3 and encodes a dual specificity phosphatase. Mutations of this gene had been found in a variety of human tumors. In the present study, we analyzed the structure and expression of the PTEN gene in 34 hepatocellular carcinoma tissues and two hepatoma cell lines. We found neither homozygous nor hemizygous deletions in these samples. We, however, found point mutations in 4 of the 34 tissue samples. Five of ten hepatocellular carcinoma tissues showed reduced PTEN expression at mRNA level. HepG2 and SMMC-7721 hepatoma cells showed decreased PTEN expression at both mRNA and protein levels compared with immortalized L02 hepatic cells. PTEN mRNA in SMMC-7721 hepatoma cells could be reduced by TGF-betaI treatment. We also found that the phosphorylation levels of FAK in both of the hepatoma cell lines were higher than that in L02 hepatic cells. Transient expression of the PTEN gene in SMMC-7721 and HepG2 hepatoma cells resulted in decreased FAK phosphorylation. The level of FAK tyrosine phosphorylation appeared to be inversely correlated with the level of the PTEN protein. In summary, our results indicated that the function of the PTEN gene in hepatocarcinomas may be impaired mainly through point mutations and expression deficiency and that the defect of PTEN in tumor cells could alter the phosphorylation of FAK.

Topics: Base Sequence; Carcinoma, Hepatocellular; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; Phosphoric Monoester Hydrolases; Phosphorylation; Point Mutation; Protein-Tyrosine Kinases; PTEN Phosphohydrolase; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins

Growth factors and extracellular matrices cooperatively regulate cellular behavior. However, the interactions between transforming growth factor-beta 1 (TGF-beta 1) and integrins in hepatic cells are not fully understood. We investigated the effects of beta 1-integrin on TGF-beta 1-regulated growth of hepatoma cells. Human hepatoma cell lines HepG2, Huh7, and Hep3B were stably transfected with beta 1-integrin, and the parental, and mock- and beta 1-integrin-transfected hepatoma cells were treated with TGF-beta 1. Modulation of apoptosis and pathways involved in the process were investigated. TGF-beta 1 suppressed the growth of hepatoma cells, and apoptosis was observed in Hep3B and Huh7. Hepatoma cells transfected with beta 1-integrin were protected from TGF-beta 1-induced apoptosis. Mitogen-activated protein (MAP) kinase inhibitors, PD98059, SB203580, and SP600125, abolished this protective effect of beta 1-integrin, but herbimycin A and wortmannin were ineffective. Hepatoma cells overexpressing beta 1-integrin showed increased activities of MAP kinases, and TGF-beta 1 induced sustained activation of MAP kinases in these cells, but only transient activation in mock-transfected cells. These data suggest that MAP kinases activated by beta 1-integrin provide a strong anti-apoptotic signal during TGF-beta 1-induced apoptosis in human hepatoma cells. Therefore beta 1-integrin-mediated signals may contribute to the development and progression of hepatocellular carcinoma.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Adhesion; Cell Division; Enzyme Activation; Extracellular Matrix Proteins; Humans; Integrin beta1; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Modulation of fibrogenesis, epithelial, and mesenchymal cell fates are prominent effects of transforming growth factor-beta (TGF-beta) signaling by Smad proteins. We have previously shown that Smad2 and Smad3 insufficiency leads to a loss of bile ducts. In addition, Smad3/4 activity is mediated by embryonic liver fodrin (ELF), a beta-Spectrin. In mouse elf(-/-) mutants and in liver explant cultures, loss of ELF function results in T lymphocytic proliferation and absent intrahepatic bile ducts. A similar phenotype is seen in a number of cholestatic diseases with progressive loss of intrahepatic bile ducts and fibrosis. However, the expression patterns of Smads or role of ELF in cholestatic and fibrotic liver diseases are not yet known.. We investigated the role of ELF in primary biliary cirrhosis (PBC), autoimmune hepatitis C, chronic viral hepatitis and in livers from mice deficient in Smad2/Smad3. We generated elf(+/-) mutant mice and analyzed for chronic liver disease and hepatocellular cancer (HCC) from 6 to 12 months. Perturbations in ELF expression were consistently seen only in PBC tissues. ELF expression was similarly aberrant in tissues from Smad2(+/-)/Smad3(+/-) mutant mice. Further studies indicated that ELF mislocalization is correlated with aberrant localization of Smad3 in some PBC tissues. Thirteen of 17 elf(+/-) mutant mice developed steatosis, fibrosis, hepatic dysplasia, with HCC in two mice.. These results suggest that a compromised cytoarchitecture and polarized trafficking of TGF-beta signaling molecules, ELF and Smad3 are involved in the pathogenesis of PBC as well as HCC.

Topics: Animals; Base Sequence; Biomarkers, Tumor; Biopsy, Needle; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Cohort Studies; DNA-Binding Proteins; Ephrin-A2; Female; Humans; Immunohistochemistry; Liver Cirrhosis; Liver Neoplasms; Male; Mice; Mice, Mutant Strains; Microfilament Proteins; Molecular Sequence Data; Polymerase Chain Reaction; Precancerous Conditions; Signal Transduction; Smad2 Protein; Smad3 Protein; Spectrin; Trans-Activators; Transforming Growth Factor beta; Tumor Suppressor Proteins

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. We previously investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. The results suggested that the HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from the retinoblastoma tumor suppressor gene product pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal. In this study, we examined the molecular mechanism of the HGF-induced cell cycle arrest in HepG2 cells. Cyclin A/Cdk2 complexes phosphorylated serine residues on pRb crucial for the G1 to S phase transition in proliferating HepG2 cells, and HGF treatment inhibited the phosphorylation. The expression of cyclin A was decreased and the expression of a Cdk inhibitor p21(Cip1) was increased in HGF-treated HepG2 cells, and these changes were prevented by pretreatment with a low concentration of a MEK inhibitor. These results suggest that the decrease in cyclin A expression and increase in p21(Cip1) expression through a high-intensity ERK signal by HGF lead to suppression of the phosphorylation of pRb by Cdk2, which contributes to the cell cycle arrest at G1 in HepG2 cells by HGF. Furthermore, the expression of E2F-1, a member of the E2F transcription factor family, was decreased in HGF-treated HepG2 cells, suggesting that the decrease in E2F-1 expression may also contribute to the cell cycle arrest at G1.

Topics: Carcinoma, Hepatocellular; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Down-Regulation; E2F Transcription Factors; E2F1 Transcription Factor; Flavonoids; G1 Phase; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Mitogens; Phosphorylation; S Phase; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Relatively little is known about the biochemical mechanisms controlling proliferation and neoplastic transformation of Hepatocellular carcinoma (HCC). The aim of study was to determine the level of the oncoproteins Bcl-2, transforming growth factor-beta1 (TGF-beta1) and alpha fetoprotein (AFP) in serum of patients with chronic hepatitis C (CHC), and liver cirrhosis (LC) as compared to HCC as a biomarkers of malignant transformation and early detection of suspected patients. A total of forty-three patients were included, 30 of them were males and 13 females, their ages ranged from 29-66 years (49.37 +/- 8.35). Increased levels of Bcl-2 were found in liver cirrhosis and HCC groups as compared to CHC and control groups (P < 0.001). The level of Bcl-2 was higher in CHC than control but the difference was insignificant (P > 0.05). Serum TGF-beta1 was significantly increased in CHC and liver cirrhosis groups as compared to HCC and control groups (p < 0.001). However, there was no significant difference between TGF-beta1 in HCC and control group (P > 0.05). The AFP level was significantly increased in HCC than CHC and liver cirrhosis. No significant difference was detected in AFP between CHC and LC patients (P > 0.05) or between CHC and healthy control (P > 0.05). A positive correlation was found between Bcl-2, and AFP in LC and HCC groups. It is concluded that the increased level of Bcl-2 in HCC may be involved in hepatocacingenesis. TGF-beta1 may be the primary marker to start the process of carcinogenesis, however, low level of TGF-beta1 may be needed to the progress of malignancy.

Topics: Adult; Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Female; Hepatitis C, Chronic; Humans; Immunoassay; Liver Cirrhosis; Liver Function Tests; Liver Neoplasms; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Transforming Growth Factor beta1

The Glypican (GPC) family is a prototypical member of the cell-surface heparan sulfate proteoglycans (HSPGs). The HSPGs have been demonstrated to interact with growth factors, act as coreceptors and modulate growth factor activity. Here we show that based on oligonucleotide array analysis, GPC3 was upregulated in hepatocellular carcinoma (HCC). By northern blot analysis, GPC3 mRNA was found to be upregulated in 29 of 52 cases of HCC (55.7%). By Western blot analysis carried out with a monoclonal anti-GPC3 antibody we generated, the GPC3 protein was found to be overexpressed in 6 hepatoma cell lines, HepG2, Hep3B, HT17, HuH6, HuH7 and PLC/PRF/5, as well as 22 tumors (42.3%). To investigate the role of overexpressed GPC3 in liver cancer, we analyzed its effects on cell growth of hepatoblastoma-derived cells. Overexpression of GPC3 modulated cell proliferation by inhibiting fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 7 (BMP-7) activity. An interaction of GPC3 and FGF2 was revealed by co-immunoprecipitation, while GPC3 was found to inhibit BMP-7 signaling through the Smad pathway by reporter gene assay. The modulation of growth factors by GPC3 may help explain its role in liver carcinogenesis. In addition, the ability of HCC cells to express GPC3 at high levels may serve as a new tumor marker for HCC.

Topics: Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Blotting, Northern; Blotting, Western; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Cell Division; DNA-Binding Proteins; DNA, Complementary; Dose-Response Relationship, Drug; Female; Fibroblast Growth Factor 2; Genes, Reporter; Genetic Vectors; Glypicans; Heparan Sulfate Proteoglycans; Humans; Immunohistochemistry; Ligands; Liver Neoplasms; Luciferases; Male; Middle Aged; Plasmids; Protein Binding; RNA, Messenger; Signal Transduction; Smad Proteins; Time Factors; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

We have previously shown that transforming growth factor-beta(1) (TGF-beta(1))-induced apoptosis in FaO hepatoma cells is mediated by cytochrome c release, apoptosome formation, and caspase activation. Although TGF-beta(1) acts via the SMAD signaling pathway to initiate de novo gene transcription, little is known about the downstream gene targets that are involved in the regulation of apoptosis. Therefore, in this study, we used in-house microarrays (approximately 5500 genes) to identify pathway-specific gene clustering in TGF-beta(1)-treated cells. A total of 142 genes showed time-dependent changes in expression during TGF-beta(1)-induced apoptosis. The polycaspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone, which, on its own, had no effect on gene transcription, blocked TGF-beta(1)-induced cell death and significantly altered the expression of 261 genes, including 185 down-regulated genes. Cluster analysis identified up-regulation of early response genes (0-4 h) encoding for the extracellular matrix and cytoskeleton, including the pro-apoptotic CTGF gene, and delayed response genes (8-16 h), including pro-apoptotic genes. A second delayed response cluster (44 genes) was also observed when TGF-beta(1)-induced caspase activation was blocked by benzyloxycarbonyl-VAD-fluoromethyl ketone. This cluster included genes encoding stress-related proteins (e.g. Jun, ATF3, TAB1, and TANK), suggesting that their up-regulation may be in response to secondary necrosis. Finally, we identified an early response set of nine down-regulated genes that are involved in antioxidant defense. We propose that the regulation of these genes by TGF-beta(1) could provide a molecular mechanism for the observed elevation in reactive oxygen species after TGF-beta(1) treatment and may represent the primary mechanism through which TGF-beta(1) initiates apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Multigene Family; Oligodeoxyribonucleotides, Antisense; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

In this study we analyzed by immunohistochemistry the expression of TGF-beta1 protein and TGF-beta receptors I and II in 4 low-grade dysplastic nodules, 2 high-grade dysplastic nodules, 6 early, 22 small, and 62 advanced hepatocellular carcinomas. The expression of TGF-beta1 protein by hepatocytes was decreased in advanced hepatocellular carcinoma compared with small or early hepatocellular carcinoma(P < .05). Frequent and intense staining of TGF-beta1 protein was noted in the sinusoidal endothelium of advanced hepatocellular carcinomas despite of its decreased staining in hepatocellular carcinoma cells. Reduced expression of TGF-beta receptors I and II compared with surrounding nontumorous tissue were noted from the early hepatocellular carcinoma stage suggesting that down-regulation of TGF-beta receptors is correlated with progression from premalignant to malignant phenotype. Reduced expression of both TGF-beta1 and TGF-beta receptor II in neoplastic hepatocytes were also significantly correlated with increased tumor size and increased proliferative activity(P < .05). These findings suggest that during hepatocarcinogenesis, the inhibitory effects of TGF-beta1 protein on hepatocellular carcinoma cells is outweighed by its effects on stromal elements, which, overall, contributes indirectly to a tumor growth stimulatory environment. Also, the growth-inhibitory effects of TGF-beta1 may have been further negated by reduced TGF-beta receptors on hepatocellular carcinoma cells.

Topics: Carcinoma, Hepatocellular; Focal Nodular Hyperplasia; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Liver Neoplasms; Precancerous Conditions; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1

NF-kappaB has been implicated in the regulation of apoptosis, a key mechanism of normal and malignant growth control. Previously, we demonstrated that inhibition of NF-kappaB activity by TGF-beta1 leads directly to induction of apoptosis of murine B-cell lymphomas and hepatocytes. Thus, we were surprised to determine that NF-kappaB is transiently activated in response to TGF-beta1 treatment. Here we elucidate the mechanism of TGF-beta1-mediated regulation of NF-kappaB and induction of apoptosis in epithelial cells. We report that TGF-beta1 activates IKK kinase, which mediates IkappaB-alpha phosphorylation. In turn, the activation of IKK following TGF-beta1 treatment is mediated by the TAK1 kinase. As a result of NF-kappaB activation, IkappaB-alpha mRNA and protein levels are increased leading to postrepression of NF-kappaB and induction of cell death. Inhibition of NF-kappaB following TGF-beta1 treatment increased AP-1 complex transcriptional activity through sustained c-Jun phosphorylation, thereby potentiating AP-1/SMADs-mediated cell killing. Furthermore, TGF-beta1-mediated upregulation of Smad7 appeared independent of NF-kappaB. In hepatocellular carcinomas of TGF-beta1 or TGF-alpha/c-myc transgenic mice, we observed constitutive activation of NF-kappaB that led to inhibition of JNK signaling. Overall, our data illustrate an autocrine mechanism based on the ability of IKK/NF-kappaB/IkappaB-alpha signaling to negatively regulate NF-kappaB levels thereby permitting TGF-beta1-induced apoptosis through AP-1 activity.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cells, Cultured; DNA-Binding Proteins; Enzyme Activation; Hepatocytes; I-kappa B Kinase; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; MAP Kinase Kinase Kinases; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphorylation; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins c-myc; Signal Transduction; Smad7 Protein; Trans-Activators; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

TG-interacting factor (TGIF) is a transcriptional co-repressor that directly associates with Smad (Sma- and Mad-related protein) proteins and inhibits Smad-mediated transcriptional activation. By using Affymetrix (Santa Clara, CA, U.S.A.) oligonucleotide microarray analysis, we found that TGIF mRNA level was elevated by transforming-growth-factor-beta (TGF-beta) treatment in a human T-cell line, HuT78. Subsequent reverse-transcription PCR assays indicated that TGF-beta1 and activin were able to induce a rapid and transient increase in the level of TGIF in both HuT78 and HepG2 hepatoma cells. To analyse whether or not the regulation of TGIF mRNA occurs at the transcriptional level, a 2.4 kb human TGIF promoter was isolated. A primer extension assay was performed to localize the putative transcription initiation site of the promoter. When transiently expressed in HepG2 cells, this promoter was stimulated by TGF-beta1 and activin treatment in a time-dependent manner. A series of deletion mutants of the TGIF promoter were also generated to further characterize the TGF-beta responsive region of the promoter. In addition, expression of TGIF was able to cause a dose-dependent inhibition of TGF-beta and activin signalling. Taken together, these experiments indicated that TGIF is a novel transcriptional target of TGF-beta and activin signalling and is likely involved in a negative feedback loop to desensitize TGF-beta/activin action.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Line; DNA Primers; DNA-Binding Proteins; Gene Expression Regulation; Homeodomain Proteins; Humans; Kidney; Kinetics; Liver Neoplasms; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Smad Proteins; T-Lymphocytes; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Tamoxifen has been tried in patients with hepatocellular carcinoma (HCC), however, its inhibitory mechanism remains unknown. In this study, we evaluated the effects of tamoxifen on HCC cell growth and the expression of transforming growth factor-beta1 (TGF-beta1) which had been known as an important cytokine in growth of HCC.. Hep 3B cells were cultivated in estrogen free media with 0.1 micro M, 0.5 micro M, 1 micro M, 5 micro M, and 10 micro M of tamoxifen for 6 days. Viable cells were counted daily and the TGF-beta1 concentrations in supernatant were measured by ELISA method.. The number of viable HCC cells increased rather significantly after the treatment of tamoxifen of lower concentration (0.1micro M) compared with that of the control (2.59x10(7) vs. 1.97x107; p<0.05). As the concentration of treated tamoxifen was higher, the number of viable HCC cells became gradually less, resulting in the significant decrease of it at the highest concentration (10micro M) compared with that of the control (1.40x10(7) vs. 1.97x10(7); p<0.05). TGF-beta1 concentration in supernatant of tamoxifen-treated samples was significantly decreased compared with those of controls, regardless of the amount of treated tamoxifen.. These results suggest that tamoxifen may suppress TGF-beta1 expression to an extent, although it has different effects on the proliferation of HCC cells, at the various concentrations of this agent in vitro. Such effects of tamoxifen on TGF-beta1 expression may inhibit the growth and progression of HCCs over-expressing TGF-beta1 in vivo.

Topics: Antineoplastic Agents, Hormonal; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Humans; Liver Neoplasms; Tamoxifen; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor beta1 (TGF beta 1)-induced G2 arrest was observed when a proliferation inhibitory function of the retinoblastoma protein (Rb) was compromised, but the mechanism underlying the G2 arrest was poorly characterized compared with that of G1 arrest. In the present study, we characterized G2 arrest induced by TGF beta1 (1 ng/mL) in the Rb-negative hepatoma cell line (Hep3B) and compared with G1 arrest in the Rb-positive hepatoma cell line (Huh7). Activities of cyclin-dependent kinases (CDK) 2 and cell division cycle (CDC) 2 were markedly decreased at 24 h, the time when cell-cycle arrest became apparent in both cell lines. However, considerable amounts of inactive CDC2-cyclinB1 complexes were present in the nucleus of G2-arrested Hep3B but were not present in G1-arrested Huh7. The inhibitory phosphorylation of CDC2 on Tyr-15 was significantly elevated at 12-24 h, and its levels gradually declined during G2 arrest in Hep3B. In particular, augmentation of CDK inhibitors p21cip1 and p27kip1 and Wee1 kinase and diminution of CDC25C phosphatase coincided with induced Tyr-15 phosphorylation and inhibition of CDC2. Wee1 in Hep3B was unstable and was degraded in a proteasome-dependent manner, but it became substantially stabilized within 6 h of TGF beta 1 treatment. Moreover, a Wee1 inhibitor, PD0166285, abrogated the TGF beta 1-induced G2 arrest in Hep3B. These findings suggest that TGF beta 1 induced G2 arrest in Hep3B at least in part through stabilization of Wee1 and subsequent increase in Tyr-15 phosphorylation and inhibition of CDC2.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cell Division; Cysteine Endopeptidases; DNA Primers; G2 Phase; Humans; Kinetics; Liver Neoplasms; Multienzyme Complexes; Nuclear Proteins; Phosphorylation; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Resistance to growth inhibitory effects of transforming growth factor (TGF)-beta is a frequent consequence of malignant transformation. On the other hand, serum concentrations of TGF-beta, plasminogen activator inhibitor type 1 (PAI-1), and vascular endothelial growth factor (VEGF) are elevated as tumor progresses. The molecular mechanism of autocrine TGF-beta signaling and its effects on PAI-1 and VEGF production in human hepatocellular carcinoma (HCC) is unknown. TGF-beta signaling involves TGF-beta type I receptor-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. To investigate the involvement of autocrine TGF-beta signal in cell growth, PAI-1 and VEGF production of HCC, we made stable transfectants of human HCC line (HuH-7 cells) to express a mutant Smad2(3S-A), in which serine residues of SSXS motif were changed to alanine. The transfectants demonstrated an impaired Smad2 signaling. Along with the resistance to growth inhibition by TGF-beta, forced expression of Smad2(3S-A) induced endogenous TGF-beta secretion. Moreover, this increased TGF-beta enhanced ligand-dependent signaling through the activated Smad3 and Smad4 complex, and transcriptional activities of PAI-1 and VEGF genes. In conclusion, distortion of autocrine TGF-beta signals in human HCC accelerates their malignant potential by enhancing cell growth as well as PAI-1 and VEGF production.

Topics: alpha-Fetoproteins; Amino Acid Motifs; Amino Acid Substitution; Autocrine Communication; Carcinoma, Hepatocellular; Disease Progression; DNA Replication; DNA-Binding Proteins; Endothelial Growth Factors; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Lymphokines; Macromolecular Substances; Mutagenesis, Site-Directed; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Recombinant Fusion Proteins; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

EBV-encoded LMP-1 is absolutely required for EBV transformation of cells. Previous studies showed that LMP-1 is responsible for mediating resistance to the anti-proliferative effects of TGF-beta that characterizes EBV-transformed cells. To clarify the mechanisms of resistance to TGF-beta by LMP-1, we examined the effect of expression of LMP-1 on the activity of TGF-beta-responsive promoters. Interestingly, LMP-1 inhibited TGF-beta-responsive promoters activity despite lack of direct interaction of LMP-1 and Smad proteins, intracellular signaling molecules in the TGF-beta signal transduction pathway. Although TGF-beta treatment increased the expression of p15, TGF-beta-induced gene, this effect was counteracted by expression of LMP-1. The repressive effect was mapped to the NF-kappaB activation domains in the cytoplasmic carboxyl terminus of LMP-1. Furthermore, LMP-1-mediated inhibition of TGF-beta-responsive promoter was markedly restored after inhibition of NF-kappaB activity. LMP-1 failed to affect receptor-dependent formation of heteromers containing Smad proteins as well as the DNA-binding activity of Smad proteins. Overexpression of the transcriptional coactivator CBP and p300 abrogated the inhibitory effect of LMP-1 on the TGF-beta-responsive promoter. Our results suggest that LMP-1 represses the TGF-beta signaling through the NF-kappaB signaling pathway at transcriptional level by competing for a limited pool of transcriptional coactivators. These results enhance our understanding of the molecular mechanisms of viral pathogenesis in EBV-associated malignancies.

Topics: Animals; Binding, Competitive; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line; Cell Transformation, Viral; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; DNA-Binding Proteins; Gene Expression Regulation, Viral; Genes, p16; Genes, Reporter; Herpesvirus 4, Human; Humans; I-kappa B Proteins; Liver Neoplasms; Lung; Mink; Models, Genetic; NF-kappa B; NF-KappaB Inhibitor alpha; Nuclear Proteins; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Suppressor Proteins; Viral Matrix Proteins

The regulation of cell growth and differentiation by transforming growth factor-beta (TGF-beta) is mediated by the Smad proteins. In the nucleus, the Smad proteins are negatively regulated by two closely related nuclear proto-oncoproteins, Ski and SnoN. When overexpressed, Ski and SnoN induce oncogenic transformation of chicken embryo fibroblasts. However, the mechanism of transformation by Ski and SnoN has not been defined. We have previously reported that Ski and SnoN interact directly with Smad2, Smad3, and Smad4 and repress their ability to activate TGF-beta target genes through multiple mechanisms. Because Smad proteins are tumor suppressors, we hypothesized that the ability of Ski and SnoN to inactivate Smad function may be responsible for their transforming activity. Here, we show that the receptor regulated Smad proteins (Smad2 and Smad3) and common mediator Smad (Smad4) bind to different regions in Ski and SnoN. Mutation of both regions, but not each region alone, markedly impaired the ability of Ski and SnoN to repress TGF-beta-induced transcriptional activation and cell cycle arrest. Moreover, when expressed in chicken embryo fibroblasts, mutant Ski or SnoN defective in binding to the Smad proteins failed to induce oncogenic transformation. These results suggest that the ability of Ski and SnoN to repress the growth inhibitory function of the Smad proteins is required for their transforming activity. This may account for the resistance to TGF-beta-induced growth arrest in some human cancer cell lines that express high levels of Ski or SnoN.

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Division; Cell Transformation, Neoplastic; Chick Embryo; Chickens; DNA-Binding Proteins; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Mutagenesis; Proto-Oncogene Proteins; Smad2 Protein; Smad3 Protein; Smad4 Protein; Suppression, Genetic; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Unlike other tissues such as breast, colon and renal cell carcinoma, it is not an easy task to single out any representative oncogene or tumor suppressor genes in the development of hepatocellular carcinoma (HCC), which play a pivotal role. To investigate putatively altered main pathways in HCC, F344 male rats were treated with a single injection of N-nitrosodiethylamine (DEN), followed by either twice/week injections of nodularin for 10 weeks or thioacetamide (TAA) in drinking water for 39 weeks. p53 expression was dramatic in both hepatocytes and mesenchymal cells after a single injection of DEN, however, PCR-SSCP assay could not detect any p53 mutation during the development of hepatocellular adenoma (HCA). The data indicate that wtp53 response was mostly for removal of damaged cells during the initiation of carcinogenesis. When treated with DEN-TAA, induction of gankyrin expression during hepatic fibrosis preceded the loss of pRB protein, accompanied with significant expressions of G1phase cyclins and CDKs. Moreover, p16(INK4A) exon 1 was hypermethylated during the development of poorly differentiated HCCs. These changes would result in complete inactivation of the pRB regulatory pathway during hepatocarcinogenesis. Induction of TGF-beta1 expression with loss of its receptor expression occurred rapidly in the altered hepatocytes by DEN-nodularin treatment.. Therefore, escape from TGF-beta1 induced apoptosis and severe degradation of pRB protein during the early stage of carcinogenesis can perform a symphony to proliferate and to transform the altered hepatocytes to tumor cells. Inactivation of p16(INK4A) and p53 genes at the later stage of carcinogenesis would endow HCC with malignancy, which is highly resistant to any therapeutic trials.

Topics: Aging; Alkylating Agents; Animals; Carcinoma, Hepatocellular; Cell Cycle; Cyclin-Dependent Kinase Inhibitor p16; Diethylnitrosamine; Disease Models, Animal; DNA Damage; Liver Cirrhosis; Liver Neoplasms; Male; Peptides, Cyclic; Rats; Rats, Inbred F344; Retinoblastoma Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Protein p53

Transforming growth factor-beta1 (TGF-beta1) can act as both a tumor suppressor and a stimulator of tumor progression. We have examined the relationship between polymorphisms of the TGF-beta1 gene and the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection. A total of 1,237 Korean subjects were prospectively enrolled; 1,046 patients with chronic HBV infection and 191 healthy controls with no evidence of recent or remote HBV infection. The patients were divided into two groups: those without (n = 809) and those with HCC (n = 237). Single nucleotide polymorphisms (SNPs) of TGF-beta1 were searched for and genotyped using the single base extension method. In Korean subjects, only two SNPs were found among the seven known polymorphisms of TGF-beta1, at position -509 and in codon 10. The risk of HCC was significantly lower in patients with the T/T or C/T genotypes than in those with the C/C genotypes at position -509 (P < 0.02), and also lower among those with the Pro/Pro or Leu/Pro genotypes than in those with the Leu/Leu genotypes in codon 10 (P < 0.007). Haplotype analysis revealed that the possession of [-509C > T; L10P] conferred a decreased likelihood of HCC (OR = 0.74; 95% CI, 0.59-0.93; P = 0.008). In conclusion, the presence of the TGF-beta1 -509C > T promoter or of the L10P polymorphism, and the combination of both [-509C > T; L10P] as a haplotype were strongly associated with a reduced risk of HCC in patients with chronic HBV infection.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Female; Genetic Variation; Hepatitis B; Humans; Liver Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Risk Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

The genetic polymorphism of transforming growth factor-beta1 (TGF-beta1) at codons 10 and 25 which influences the production of TGF-beta1 is related to fibrogenesis in the lung and liver. We evaluated the genetic polymorphism at codons 10 and 25 in controls and in patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC).. Blood samples were collected from controls (n=35), patients with LC (n=64), and HCC (n=49). Genomic DNA was isolated and polymerase chain reaction (PCR) was done for a segment including codons 10 and 25. The results of direct sequencing for PCR products were compared between the controls and the patients.. There was no genetic polymorphism at codon 25 and three types of genetic polymorphism at codon 10. The leucine homozygous genotype (CTG/CTG) at codon 10 was more common in patients with LC than the controls (p=0.01) and especially in patients with LC caused by HBV (p=0.004). The polymorphism at codons 10 in patients with HCC was similar to the controls. However, leucine homozygous genotype was more common in patients with HCC of uninodular morphology than those of massive morphology (p=0.007).. The genetic polymorphism of TGF-beta1 at codon 10 might be associated with LC and morphology of HCC. The potential usefulness of TGF-beta1 genotyping needs further studies in large scale.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Codon; Female; Genotype; Humans; Korea; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Polymorphism, Genetic; Sequence Analysis, Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hepatocytes adopt an invasive and metastatic phenotype caused by the cooperation of transforming growth factor (TGF)-beta and oncogenic Ha-Ras. In the initial phase of this process, c-Fos is rapidly induced by TGF-beta, but then decreases to undetectable levels. Here, we investigated the functional implications of c-Fos activation and its contribution to hepatocellular tumorigenesis. By employing conditional c-Fos expression, we observed that continuous activation of c-Fos and consequently AP-1 activity leads to depolarization of differentiated murine epithelial hepatocytes. Most remarkably, this change in morphology was associated with inhibition of proliferation and induction of cell death. Coexpression of antiapoptotic Bcl-XL or scavenging of reactive oxygen species was sufficient to prevent the c-Fos-mediated phenotype. In contrast, the cooperation of c-Fos with oncogenic Ha-Ras or a Ras mutant selectively activating the MAPK pathway even enhanced c-Fos-induced effects. Showing the negative role in hepatocellular tumorigenesis, c-Fos repressed oncogenic Ras-driven anchorage-independent growth in vitro and strongly suppressed tumour formation in vivo. Taken together, we demonstrate that c-Fos modulates plasticity of epithelial hepatocytes and acts tumour suppressive in neoplastic hepatocytes by stimulating cell cycle inhibition and cell death.

Topics: Animals; Apoptosis; bcl-X Protein; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Cell Death; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Epithelial Cells; Flow Cytometry; Genes, ras; Liver Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Phenotype; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-fos; Reactive Oxygen Species; Signal Transduction; Time Factors; Transcription Factor AP-1; Transcription Factor CHOP; Transcription Factors; Transfection; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor and apoptosis inducer for most normal cells. However, tumor cells are commonly nevertheless sensitive to the tumor-suppressing effects of TGF-beta1. In this paper, we focus on the effects of TGF-beta1 on two important anti-apoptotic protein kinases, protein kinase B (PKB), and focal adhesion kinase (FAK), in SMMC-7721 cells. We found that PKB-Ser-473 phosphorylation was apparently up-regulated by TGF-beta1. In the meantime, PKB-Thr-308 phosphorylation was slightly up-regulated by TGF-beta1. TGF-beta1 could also enhance FAK-Tyr phosphorylation. We observed that integrin-linked kinase (ILK) was also up-regulated by TGF-beta1 in good accordance with PKB-Ser-473 phosphorylation. We first found that TGF-beta1 could stimulate PKB-Ser-473 phosphorylation possibly via up-regulating ILK expression. Furthermore, we also failed to detect PKB-Ser-473 and FAK-Tyr phosphorylation with various concentrations of TGF-beta1 treatment when cells were kept in suspension. The above results indicate that PKB-Ser-473 and FAK-Tyr phosphorylation stimulated by TGF-beta1 are both dependent on cell adhesion.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Dose-Response Relationship, Drug; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Liver Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Serine; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tyrosine

Nuclear factor kappaB (NF-kappaB) is an antiapoptotic factor involved in development, regeneration, and neoplastic progression of the liver. Previously, we have shown that stabilization of inhibitor kappaB (IkappaB)-alpha protein following treatment of hepatocytes with transforming growth factor (TGF)-beta1 promoted NF-kappaB repression, which then permitted induction of AP-1/SMAD-mediated liver cell death. Because basal IkappaB-alpha protein turnover is regulated by protein kinase CK2, here we have elucidated the regulation of CK2 kinase activity and its role in control of NF-kappaB levels following treatment with TGF-beta1. We show that both messenger RNA (mRNA) and protein levels of the CK2alpha catalytic subunit are down-regulated following TGF-beta1 stimulation in murine hepatocyte cells. The ensuing inhibition of CK2 kinase activity promotes stabilization of IkappaB protein, which is followed by the shutoff of constitutive NF-kappaB activity and induction of apoptosis. Ectopic expression of CK2alpha inhibits TGF-beta1-induced apoptosis through sustained activation of NF-kappaB. Conversely, expression of a kinase-dead mutant of CK2alpha potentiates TGF-beta1 cell killing. Importantly, we show that hepatocellular carcinomas (HCCs) derived from TGF-beta1 transgenic mice and human HCC cell lines display enhanced CK2 IkappaB kinase activity that contributes in part to an elevated NF-kappaB activity in vivo. In conclusion, inhibition of CK2 expression levels by TGF-beta1 is crucial for the induction of apoptosis of hepatocytes. Circumvention of this process by up-regulation of CK2 activity in transformed cells may contribute to the promotion of TGF-beta1-induced liver carcinogenesis.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Casein Kinase II; Cells, Cultured; Down-Regulation; Hepatocytes; Humans; I-kappa B Proteins; Liver Neoplasms; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Serine-Threonine Kinases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-b1 (TGF-beta1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-beta1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-beta1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-beta1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-beta1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-beta1 enhanced the expression of alpha5beta1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-beta1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-beta1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-beta1 treatment. Furthermore, TGF-beta1 induced the down-regulation of E-cadherin and the nuclear translocation of beta-catenin. These results indicated that TGF-beta1-promoted cell adhesion and TGF-beta1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-beta1-enhanced cell migration.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Movement; Epithelial Cells; Gene Expression; Humans; Integrin alpha5beta1; Laminin; Liver Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase involved in transcriptional regulation and apoptosis. Here we demonstrate that HIPK2 regulates transforming growth factor (TGF) beta-induced c-Jun NH(2)-terminal kinase (JNK) activation and apoptosis. HIPK2 colocalizes with Daxx, a protein acting in TGF-beta-induced JNK activation and apoptosis, in promyelocytic leukemia (PML) nuclear bodies, and triggers PML-nuclear body disruption and release of Daxx. HIPK2 interacts in vitro and in vivo via its kinase domain with Daxx, and a fraction of Daxx coprecipitates with HIPK2 under physiological conditions. Moreover, overexpression of HIPK2 leads to Daxx phosphorylation, and ectopic expression of HIPK2 activates the JNK signaling pathway, which is enhanced by coexpression of Daxx. HIPK2 signals to JNK via a pathway using Daxx and the mitogen-activated protein kinase kinases MKK4/SEK1 and MKK7. Ectopic expression of HIPK2 and Daxx potentiates TGF-beta-induced apoptosis in human p53-deficient hepatocellular carcinoma cells. Finally, we demonstrate that knockdown of endogenous HIPK2 using RNA interference inhibits TGF-beta-induced JNK activation and apoptosis. Taken together, our findings indicate that HIPK2 participates in the TGF-beta signaling pathway leading to JNK activation and apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Co-Repressor Proteins; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Leukemia, Promyelocytic, Acute; Liver Neoplasms; MAP Kinase Kinase 6; MAP Kinase Kinase 7; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular Chaperones; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Signal Transduction; Transforming Growth Factor beta

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD), a ubiquitous environmental pollutant, elicits a variety of toxicities and is a well-known carcinogen. TCDD alters the expression of many genes including CYP1A1/2, CYP1B1, glutathione S-transferase Ya, aldehyde-3-dehydrogenase, NAD(P)H:quinone oxidoreductase, transforming growth factor (TGF)-alpha and TGF-beta. The present study was aimed at characterization of TCDD to induce plasminogen activator inhibitor-1 (PAI-1) in mouse hepatoma cell lines. A Hepa1c1c7 wild-type cell [H1(wt)], an aryl hydrocarbon receptor (AhR)-deficient mutant [H1(AhR(-))] and an AhR nuclear translocator (Arnt)-deficient mutant [H1(Arnt(-))] were used for this study. TCDD induced PAI-1 in H1(wt) cells, but not in H1(AhR(-)) and H1(Arnt(-)) mutants, indicating a functional role of the AhR-Arnt complex in this effect. Cycloheximide (CHX) treatment resulted in increased PAI-1 mRNA induction, indicating that this response to TCDD is a direct effect on transcription and not a secondary effect mediated by other TCDD-induced proteins. Transfection with PAI-1 promoter led to increased PAI-1 promoter activity in H1(wt) cells treated with TCDD, but no such effect occurred in H1(AhR(-)) or H1(Arnt(-)) cells, implying involvement of the AhR and Arnt. In addition, alpha-naphthoflavone and phenanthroline, two AhR antagonists, each blocked the enhancing effect of TCDD on PAI-1 promoter-coupled luciferase activity in H1(wt) cells. PAI-1 promoter deletion analysis indicated that TCDD-induced PAI-1 transcription was distinctly different from TGF-beta-dependent PAI-1 transcription, particularly in the region between -161 to +73. In summary, TCDD induced the PAI-1 gene directly via an AhR- and Arnt-dependent mechanism, which was distinctly different from TGF-beta-driven PAI-1 transcription.

Topics: Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Blotting, Northern; Blotting, Western; Carcinoma, Hepatocellular; DNA-Binding Proteins; Dose-Response Relationship, Drug; Environmental Pollutants; Mice; Plasminogen Activator Inhibitor 1; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Chronic infection with hepatitis C virus leads to liver cirrhosis and hepatocellular carcinoma. Hepatocellular carcinoma is sometimes associated with p53 dysfunction and decreased p21(WAF1) expression. The p21(WAF1) gene is a major target of p53, and p21(WAF1) protein regulates the activities of cyclin/CDK complexes involved in cell cycle control and tumor formation. Because core protein has oncogenic properties, we investigated the expression of p21(WAF1) following core expression.. We analyzed by Western blot, Northern blot and transfection the expression of p21(WAF1) in HepG2 cell line under transient expression of Hepatitis C core protein by recombinant-adenoviral infection.. Infection of HepG2 with core-encoding viruses induced the down-regulation of p21(WAF1) expression. This effect is due to a decrease in the p21(WAF1) gene transcription and of the p21(WAF1) protein half-life. These results support a role for Hepatitis C virus core protein in cell transformation. We also found also that the transforming growth factor beta can counteract the core-induced p21(WAF1) down-regulation. The antagonist effect of TGF beta, or of other molecules, on p21(WAF1) expression may be of particular interest for the treatment of HCV-positive hepatocellular carcinoma.

Topics: Adenoviridae; beta-Galactosidase; Carcinoma, Hepatocellular; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Viral; Humans; Liver Neoplasms; Recombinant Proteins; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Viral Core Proteins

In the normal liver, the transforming growth factor beta (TGF-beta) signaling pathway plays an important role in inhibiting hepatocyte growth. This effect is mediated through Smad4 (or Dpc4), a tumor-suppressor gene that affects gene transcription and controls cell growth. A loss of Smad4 is associated with carcinoma in a number of other organs, including the pancreas and colon. Despite these facts, several recent studies using cDNA microarrays have surprisingly shown overexpression of Smad4 in hepatocellular carcinoma (HCC). Because Smad4 plays a central role in the TGF-beta signaling pathway, we hypothesized that activation of the TGF-beta signaling pathway may explain Smad4 overexpression. To investigate this, 21 surgically resected HCCs were immunostained with antibodies to Smad4 and TGF-beta receptor II. Tumor and normal liver tissues were stained in all cases, and expression in the tumor was scored in comparison to the nonneoplastic liver. Thirteen hepatic adenomas were also immunostained as a control group. The average age at resection was 58 +/- 16 years for the 17 men and 4 women with HCC. TGF-beta receptor II was weakly expressed in the hepatocyte cytoplasm of all normal livers and was overexpressed in 10 of 21 HCCs. Of these 10 HCCs increased Smad4 immunolabeling was also present in 10 of 10 cases. In contrast, of the 11 of HCCs that did not show TGF-beta overexpression, only 1 showed increased Smad4 immunolabeling. Increased TGF-beta receptor II and Smad4 labeling was associated with a worse nuclear grade and increased mitotic activity. For the hepatic adenomas, the 13 women had an average age at resection of 36 +/- 10 years. Whereas 2 adenomas showed over expression of TGF-beta receptor II, there was no Smad4 overexpression in any case. In conclusion, increased Smad4 protein expression in HCC is tightly linked to overexpression of TGF-beta II receptors and is associated with increased mitoses and a worse nuclear grade. Hepatic adenomas only rarely show overexpression of TGF-beta II receptors and did not show increased Smad4 labeling. The results from this study indicate that Smad4 protein overexpression is present in a subset of HCCs and is strongly correlated with immunostaining for TGF-beta II receptor, findings that may represent activation or dysregulation of the TGF-beta signaling pathway.

Topics: Adenoma; Biomarkers, Tumor; Carcinoma, Hepatocellular; DNA-Binding Proteins; Female; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Mitotic Index; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retrospective Studies; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) exerts potent immunosuppressive effects. In this study, we investigated the potential role of TGF-beta1 produced by hepatocellular carcinoma (HCC) cell lines in immunosuppression mechanisms. Using the Mv1Lu cell-growth inhibition assay and an enzyme-linked immunosorbent assay (ELISA), we detected optimal levels of TGF-beta1 in the culture supernatants conditioned by the HCC cell lines PLC/PRF/5, Hep3B, and HepG2. To determine the biological activity of TGF-beta1 in the supernatants, we examined the effects of the culture supernatants on the production of interferon (IFN)-gamma induced during the culture of peripheral blood mononuclear cells (PBMCs) stimulated with interleukin (IL)-12. IFN-gamma production of IL-12-stimulated PBMCs in the 1:1 dilution of the acid-activated conditioned medium of PLC/PRF/5, Hep3B, and HepG2 reduced to 14.7 +/- 0.8, 17.3 +/- 9.0, and 35.9 +/- 14.6%, respectively, compared with the value in the culture with control medium (complete culture medium). These results suggest that HCC cells producing TGF-beta1 may reduce the generation or activation of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, and thus could enhance their ability to escape immune-mediated surveillance.

Topics: Aged; Carcinoma, Hepatocellular; Female; Humans; Immune Tolerance; Interferon-gamma; Interleukin-12; Leukocytes, Mononuclear; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Although alpha-fetoprotein (AFP) is a useful serologic marker of hepatocellular carcinoma (HCC), it has been reported insufficiently sensitive in detecting small HCCs. Plasma transforming growth factor-beta1 (TGFbeta1) has been reported to be elevated in HCC patients compared with liver cirrhosis patients. It has been reported that TGFbeta1 mRNA was overexpressed in HCC, especially in patients with small HCC and well-differentiated HCC compared with patients with liver cirrhosis. The current study investigated the usefulness of TGFbeta1 compared with AFP in the diagnosis of small HCCs.. Thirty-eight patients with small HCC (< or = 3 cm), 31 patients with liver cirrhosis only, and 23 normal volunteers were studied. Using plasma TGFbeta1 and serum AFP levels measured at the time of diagnosis, the sensitivities and specificities were calculated in the diagnosis of small HCCs.. Plasma TGFbeta1 and serum AFP levels were significantly higher in patients with small HCC than in those with liver cirrhosis. In diagnosing small HCCs, the cut-off values of plasma TGFbeta1 and serum AFP were 800 pg/mL and 200 ng/mL, respectively, where the specificities were over 95%. At the cut-off level of plasma TGFbeta1 and serum AFP, the sensitivities were 68% and 24%, respectively.. The current results suggest that TGFbeta1 may be a useful serologic marker in detecting HCCs earlier because it shows higher sensitivity than, with specificity as, AFP in the diagnosis of small HCCs.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Sensitivity and Specificity; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.

Topics: Animals; Apoptosis; bcl-Associated Death Protein; Carcinoma, Hepatocellular; Carrier Proteins; DNA-Binding Proteins; Liver Neoplasms; Proto-Oncogene Proteins c-bcl-2; Rats; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Transforming growth factor-beta 1 (TGF-beta1) exerts an inhibitory effect on DNA synthesis in hepatocytes. Activin, through different mechanisms, also exhibits an apoptotic effect on hepatocytes. Follistatin antagonizes the actions of activin.. Patients with hepatocellular carcinoma (HCC, n = 20), patients with alcoholic cirrhosis (n = 12), patients with cirrhosis due to other causes (n = 5) and normal controls (n = 19) were studied. TGF-beta1, activin and follistatin concentrations in blood and ascites were measured by ELISA.. All three groups of patients had significantly higher serum levels of total TGF-beta1, activin and follistatin compared to those of controls. In patients with HCC, the total TGF-beta1 level correlated negatively with tumour size (r = -0.644, P = 0.001). The activin level correlated with alkaline phosphatase (ALP) level (r = 0.374, P = 0.046). The follistatin level correlated with the ALP level (r = 0.404, P = 0.026), and the glutamyl transpeptidase level (r = 0.457, P = 0.01). In patients with alcoholic cirrhosis, serum activin correlated with the Child-Pugh score (r = 0.601, P = 0.01). The levels of the cytokines in ascites (n = 16) did not correlate with the corresponding levels in serum.. Serum levels of total TGF-beta1, activin and follistatin were elevated in patients with hepatocellular carcinoma and in patients with alcoholic cirrhosis. Apoptosis of tumour cells may be reduced by a subsequent decrease in serum TGF-beta1 levels when the tumours expand in size. Activin and follistatin were associated with tumour activity, as both correlated with ALP and/or GGT levels. Further studies are required to define the exact relationships between these cytokines, the dynamics of tumour growth and their significance in cirrhosis.

Topics: Activins; Apoptosis; Carcinoma, Hepatocellular; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Follistatin; Humans; Liver Cirrhosis, Alcoholic; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) causes growth inhibition in many cell types. Since its role in the outgrowth of human hepatocellular carcinoma (HCC) is not clearly understood, we investigated the growth inhibitory effects of TGF-beta1, the genetic and molecular integrity of TGF-beta receptors, and the expression levels of cell cycle regulating proteins in 11 human HCC cell lines. Of 11 cell lines, 3 (27%) showed growth inhibition to TGF-beta1, whereas the other 8 cell lines did not. We performed Southern and Northern analysis of TGF-beta type I and II receptors and examined poly-adenine track mutation of the TGF-beta type II receptor, but failed to find any genetic mutation. The transcriptional induction of plasminogen activator inhibitor-1 and p21(WAF1/CIP1) by TGF-beta were detected in all HCC cell lines, implying that the molecular integrity of the TGF-beta receptors might be intact. The amplification and overexpression of cyclin D1 gene was detected in 4 (50%) of 8 HCC cells that showed resistance to TGF-beta1. The suppression of cyclin D1 expression with antisense cyclin D1 facilitated the TGF-beta1-triggered growth inhibition in a TGF-beta1 resistant HCC cell line containing amplified cyclin D1 gene. In conclusion, the overexpression of cyclin D1 may be responsible for the attenuation of TGF-beta1 induced growth inhibition in some HCC cells.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cyclin D1; DNA; DNA, Antisense; Gene Amplification; Growth Inhibitors; Humans; Liver Neoplasms; Receptors, Transforming Growth Factor beta; RNA, Neoplasm; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The human serine/threonine kinase hSGK1 is expressed ubiquitously with highest transcript levels in pancreas and liver. This study has been performed to determine the hSGK1 distribution in normal liver and its putative role in fibrosing liver disease. HSGK1-localization was determined by in situ hybridization, regulation of hSGK1-transcription by Northern blotting, fibronectin synthesis and hSGK1 phosphorylation by Western blotting. In normal liver hSGK1 was mainly transcribed by Kupffer cells. In liver tissue from patients with chronic viral hepatitis, hSGK1 transcript levels were excessively high in numerous activated Kupffer cells and inflammatory cells localized within fibrous septum formations. HSGK1 transcripts were also detected in activated hepatic stellate cells. Accordingly, Western blotting revealed that tissue from fibrotic liver expresses excessive hSGK1 protein as compared to normal liver. TGF-beta1 (2 ng/ml) increases hSGK1 transcription in both human U937 macro-phages and HepG2 hepatoma cells. H(2)O(2) (0.3 mM) activated hSGK1 and increased fibronectin formation in HepG2 cells overexpressing hSGK1 but not in HepG2 cells expressing the inactive mutant hSGK1(K127R). In conclusion hSGK1 is upregulated by TGF-beta1 during hepatitis and may contribute to enhanced matrix formation during fibrosing liver disease.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Chronic Disease; Fibronectins; Gene Expression Regulation; Hepatitis B; Hepatitis C; Hepatitis, Viral, Human; Humans; Hydrogen Peroxide; Immediate-Early Proteins; In Situ Hybridization; Kupffer Cells; Liver; Liver Cirrhosis; Macrophages; Nuclear Proteins; Oxidants; Phosphorylation; Protein Serine-Threonine Kinases; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; U937 Cells

Thrombospondin-1 (TSP-1), a multifunctional matrix protein, affects tumor growth through modulation of angiogenesis and other stromal biological functions. In several of nine human cancer cell lines derived from liver, brain, pancreas, and bone, expression of TSP-1 was up-regulated in response to the two most representative growth factors, epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1). Expression of TSP-1 was markedly enhanced in hepatic HuH-7 cells by EGF but not by TGFbeta1. In contrast, expression of TSP-1 was markedly enhanced by TGFbeta1, but not by EGF, in osteosarcoma MG63 cells. EGF induced activation of TSP-1 promoter-driven luciferase activity in HuH-7 cells, and the elements between -267 and -71 on the 5' region of TSP-1 gene containing two GC boxes to which Sp1 bound, were found to be responsible for the promoter activation by EGF. However, EGF did not alter TSP-1 mRNA stability in hepatic cells. On the other hand, no such enhancement of the TSP-1 promoter activity by TGFbeta1 appeared in MG63 cells. Enhanced expression of TSP-1 by TGFbeta1 in MG63 cells was partially blocked by exogenous addition of SB203580, an inhibitor of p38 mitogen-activated protein kinase. TGFbeta was found to induce marked elongation of TSP-1 mRNA longevity in osteosarcoma cells when mRNA degradation was assayed in the presence of alpha-amanitin. The up-regulation of TSP-1 by EGF and TGFbeta might play a critical role in modulation of angiogenesis and formation of matrices in tumor stroma.

Topics: Bone Neoplasms; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation; Humans; Liver Neoplasms; Osteosarcoma; RNA Stability; Thrombospondin 1; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Co-infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is common and is associated with a more severe liver disease and increased frequency in the development of hepatocellular carcinoma (HCC). Here, we demonstrated that HBV X protein (HBx) and HCV core protein additively repress the universal cyclin-dependent kinase inhibitor p21 gene at the transcription level. The transforming growth factor-beta responsive element and Sp1 site of the p21 promoter were responsible for the effect of HCV core and HBx, respectively. Furthermore, cell growth was additively stimulated by them, suggesting that additive repression of the p21 might be important to understand the cooperative development of HCC by HBV and HCV.

Topics: 3T3 Cells; Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Gene Silencing; Kinetics; Liver Neoplasms; Mice; Promoter Regions, Genetic; Response Elements; RNA; Sp1 Transcription Factor; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Viral Core Proteins; Viral Regulatory and Accessory Proteins

Transforming growth factor (TGF) beta1 is a potent inducer of apoptosis in the liver. During TGF-beta1-induced apoptosis, 3 mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38 kinase) showed simultaneously sustained activation in FaO rat hepatoma cells. TGF-beta1-induced apoptosis was markedly enhanced when ERK activation was selectively inhibited by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. In contrast, both interfering with p38 activity by overexpression of the dominant negative (DN) MKK6 mutant and inhibition of the JNK pathway by overexpression of the DN SEK1 mutant resulted in suppression of mitochondrial cytochrome c release, abrogating TGF-beta1-induced apoptosis. In addition, antiapoptotic Bcl-2 blocked mitochondrial cytochrome c release, suppressing TGF-beta1-induced activation of JNK and p38. Inhibition of ERK activity enhanced TGF-beta1-induced p38 and JNK activation. However, inhibition of the JNK pathway suppressed p38 but induced transient ERK activation. Similarly, interfering with the p38 pathway also attenuated JNK activation but generated transient ERK activation in response to TGF-beta1. These results indicate that disrupting one MAP kinase pathway affects the TGF-beta1-induced activation of other MAP kinases, suggesting cross-talk among MAP kinase pathways. In conclusion, we propose that the balance and integration of MAP kinase signaling may regulate commitment to TGF-beta1-induced apoptosis modulating the release of cytochrome c from mitochondria.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cytochrome c Group; Gene Expression; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Rats; Receptor Cross-Talk; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Regulation of liver cell proliferation is a key event to control organ size during development and liver regeneration. Methionine adenosyltransferase (MAT) 2A is expressed in proliferating liver, whereas MAT1A is the form expressed in adult quiescent hepatocytes. Here we show that, in H35 hepatoma cells, growth factors such as hepatocyte growth factor (HGF) and insulin up-regulated MAT2A expression. HGF actions were time- and dose-response dependent and required transcriptional activity. Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-phosphate kinase (PI 3-K) pathways were required for both HGF-induced cell proliferation and MAT2A up-regulation. Furthermore, in H35 cells treated with HGF, the inhibition of these pathways was associated with the switch from the expression of fetal liver MAT2A to the adult liver MAT1A isoform. Fetal liver hepatocytes exhibited an identical response pattern. Treatment of H35 hepatoma cells with MAT2A antisense oligonucleotides decreased cell proliferation induced by HGF; this decrease correlated with the decay in MAT2A messenger RNA (mRNA) levels. Finally, growth inhibitors such as transforming growth factor (TGF) beta blocked HGF-induced MAT2A up-regulation while increasing MAT1A mRNA levels in H35 cells. In conclusion, our results show that MAT2A expression not only correlates with liver cell proliferation but is required for this process.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Hepatocyte Growth Factor; Liver; Liver Neoplasms; Male; Methionine Adenosyltransferase; Rats; Rats, Sprague-Dawley; Rats, Wistar; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Although microwave coagulation therapy (MCT) has been performed for liver cancer, there has been no report examining the influence of this therapy on the growth of possible remnant cancer.. A solid cube of AH-130 cells (ascites hepatoma cell line) was implanted into the left lateral lobe of the rat liver. Five days later, MCT was applied to the middle liver lobe of these rats. Tumor growth and cytokine levels in plasma and the liver were compared between rats that underwent MCT and rats that did not.. The mean tumor weight in the MCT group (222.6+/-51.5 mg, mean+/-SD) was significantly greater than that in the control group (126.7+/-19.7 mg, P<0.01) at postoperative day (POD) 5. Immunohistochemistry for anti-proliferating cell nuclear antigen showed the labeling index in the MCT group (90.4%) to be higher than that in the control group (76.7%, P<0.01). Liver basic fibroblast growth factor and transforming growth factor-beta 1 levels in the MCT group on POD 3 were significantly higher than levels in the control group.. The present study suggests the clinically important finding that MCT accelerates the growth of small residual tumors in the liver.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Electrocoagulation; Fibroblast Growth Factor 2; Hepatocyte Growth Factor; Liver; Liver Neoplasms, Experimental; Male; Microwaves; Rats; Rats, Inbred Strains; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Metastasis occurrence in the course of hepatocellular carcinoma (HCC) severely affects prognosis and survival. We have shown that HCC invasive cells express alpha3beta1-integrin whereas noninvasive cells do not. Here we show that transforming growth factor (TGF)-beta1 stimulates alpha3-integrin expression at a transcriptional level in noninvasive HCC cells, causing transformation into a motile and invasive phenotype. Such activities are inhibited by neutralizing anti-alpha3- but not anti-alpha6-integrin monoclonal antibodies. HCC invasive cells secrete abundant levels of active TGF-beta1 in comparison with noninvasive cells, but in the latter, addition of active matrix metalloproteinases-2 increases the concentration of active TGF-beta1. In this way, the cells express alpha3-integrin at a transcriptional level and acquire motility on Ln-5. By contrast, an anti-TGF-beta1-neutralizing antibody reduces alpha3-integrin expression and the invasive ability of HCC invading cells. In HCC patients, TGF-beta1 serum concentrations and alpha3-integrin expression are strongly correlated. The integrin, absent in normal and peritumoral liver parenchyma, is abundantly expressed in HCC primary and metastatic tissue. In particular, patients with metastasis show higher levels of TGF-beta1 serum concentrations and stronger expression of TGF-beta1 and alpha3-integrin in HCC tissues. In conclusion, TGF-beta1 may play an important role in HCC invasiveness by stimulating alpha3-integrin expression, and could therefore be an important target for new therapies.

Topics: Antibodies; Antigens, CD; Biocompatible Materials; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Line; Cell Movement; Collagen; Drug Combinations; Humans; Integrin alpha3; Integrin alpha3beta1; Integrins; Kalinin; Laminin; Liver Neoplasms; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Proteoglycans; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hepatocarcinogenesis is closely related to hepatic fibrosis. In this study, we investigated the relationship of type II transforming growth factor-beta receptor (T beta RII) to hepatic fibrosis and hepatocellular carcinoma (HCC). In vivo: liver tissues were obtained from 30 patients (10 chronic hepatitis, 7 cirrhosis, 13 HCC). Protein expression and immunolocalization of T beta RII were examined by Western blot analysis and immunohistochemistry. In vitro: T beta RII protein expression in hepatoma cell lines (HepG2, Hep3B, HLE, HLF and Huh7) was examined by Western blot analysis. Next, we transfected T beta RII cDNA to Huh7, and compared the change of cell number and observed the induction of apoptosis after TGF-beta1 treatment using a FACScan flow cytometer. In vivo: T beta RII immunolocalization in liver tissues was significantly decreased in patients with HCC compared with that of patients with chronic hepatitis or liver cirrhosis. In Western blot analysis, T beta RII expression in tissues attenuated in comparison with that in non-tumor tissues in some patients with HCC. In vitro: T beta RII protein expression in HLE, HLF and Huh7 cells was weaker than that in HepG2 and Hep3B cells. In Huh7 cells transfected T beta RII cDNA, cell arrest and apoptosis were obviously induced. These results indicated that human HCC has a reduced expression of T beta RII for TGF-beta1. This may provide a selective growth advantage to HCC to escape the inhibitory growth signals of TGF-beta1, and may be linked with critical steps in the growth of hepatoma cells.

Topics: Aged; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Count; DNA, Complementary; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

To elucidate involvement of the transforming growth factor-beta (TGF-beta) signaling pathway in endogenous and exogenous liver carcinogenesis, we investigated mutations of TGF-beta receptor type II (TGF-betaRII), Smad2 and Smad4 genes, and expression of TGF-betaRII in hepatocellular carcinomas (HCCs) induced by a choline-deficient L-amino acid-defined (CDAA) diet and by N-nitrosodiethylamine (DEN). Male Fischer 344 rats received a CDAA diet continuously and HCCs were sampled after 75 weeks. Administration of DEN was followed by partial hepatectomy (PH), with colchicine to induce cell cycle disturbance and a selection pressure regimen, HCCs being obtained after 42 weeks. Total RNAs were extracted from individual HCCs and mutations in TGF-betaRII, Smad2 and Smad4 were investigated by reverse transcription (RT)-polymerase chain reaction (PCR)-restriction-single-strand conformation polymorphism (SSCP) analysis followed by sequencing analysis. Mutations of Smad2 were detected in 2 out of 12 HCCs (16.7%) induced by the CDAA diet, a GGT-to-GGC transition (Gly to Gly) at codon 30 and a TCT-to-GCT (Ser to Ala) transversion at codon 118, without any TGF-betaRII or Smad4 alterations. No mutations of TGF-betaRII, Smad2 and Smad4 were encountered in eleven HCCs induced by the exogenous carcinogen. Semi-quantitative RT-PCR revealed reduced expression of TGF-betaRII in 2 HCCs (16.7%) without Smad2 mutations out of 12 HCCs induced by the CDAA diet and none of 11 induced by DEN. These results suggest that the TGF-beta signaling pathway may be disturbed in endogenous liver carcinogenesis in rats.

Topics: Alkylating Agents; Animals; Carcinoma, Hepatocellular; Choline; Diethylnitrosamine; DNA-Binding Proteins; Liver Neoplasms; Male; Polymorphism, Single-Stranded Conformational; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Norepinephrine is considered to possess potent anti-apoptotic action in regenerating hepatocytes. To clarify the role of the sympathetic nervous system in apoptosis that occurs in chronic liver damage and following the promotion of liver cirrhosis, we studied a carbon tetrachloride (CCl4)-induced liver injury model, using spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and chemically sympathectomized WKY. At 24 h after CCl4 administration. acute damage, characterized by vacuolated hepatocytes in the centrilobular zone, was greater in SHR than in WKY. This vacuolated change in WKY hepatocytes was significantly reduced by chemical sympathectomy with 6-hydroxydopamine (6-OHDA). After 48 h, the acute damage was dramatically improved in each animal, without significant differences between the three groups. In chronic damage after weekly repetition of CCl4 treatment for 4 weeks, fibrosis was evident in SHR, while in the other groups there was only scant fibrosis in the centrilobular zone. After 8 weeks' repetition of CCl4, liver cirrhosis was seen only in SHR. The incidence of apoptotic cells in areas of both acute and chronic damage in WKY, detected by terminal deoxynucleotidyl transferase-dUTP nick end labeling, was significantly increased in comparison with that in SHR, and was further increased by 6-OHDA pretreatment. In contrast, there was significantly greater enhancement of the growth of hepatocytes in SHR than in WKY in both acute and chronic damage. Moreover. hepatocyte growth kinetics in WKY was significantly inhibited after sympathectomy in acute injury, as evidenced by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In vitro, the amount of hepatocellular apoptosis induced by transforming growth factor-beta1 was significantly decreased by incubation with norepinephrine. These findings suggest that the anti-apoptotic effect of the sympathetic nervous system increases cell growth kinetics and promotes liver cirrhosis in this animal model.

Topics: Animals; Apoptosis; Carbon Tetrachloride; Carcinoma, Hepatocellular; Cell Division; Hepatocytes; Immunohistochemistry; Liver Cirrhosis; Liver Regeneration; Male; Models, Animal; Norepinephrine; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sympathectomy; Sympathetic Nervous System; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Interleukin-6 (IL-6) is a multifunctional cytokine that plays important roles in the immune system, hematopoiesis, and acute phase reactions. Transforming growth factor-beta (TGF-beta) also has pleiotropy including the production of acute phase proteins in hepatocytes. To elucidate the cross-talk between IL-6 and TGF-beta signaling pathways in hepatic cells, we investigated the effects of TGF-beta on IL-6-induced signal transducer and activator of transcription-3 (STAT3) activation in a human hepatoma cell line, Hep3B. IL-6-induced activation of STAT3 activity and STAT3-mediated gene expression were augmented by TGF-beta in Hep3B cells. We provide evidence that these activities were due to physical interactions between STAT3 and Sma- and MAD-related protein-3, bridged by p300. These results demonstrate a molecular mechanism of a cross-talk between STAT3 and TGF-beta signaling pathways in hepatocytes.

Topics: Carcinoma, Hepatocellular; DNA-Binding Proteins; Gene Expression Regulation; Humans; Interleukin-6; Nuclear Proteins; Signal Transduction; Smad3 Protein; STAT3 Transcription Factor; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)

Topics: Activin Receptors, Type I; Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Chlorocebus aethiops; COS Cells; DNA-Binding Proteins; Gene Products, tax; Genes, pX; Humans; Liver Neoplasms; Lung; Macromolecular Substances; Mink; Nerve Growth Factors; Nuclear Proteins; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Smad Proteins; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenopus laevis; Xenopus Proteins

Hepatocellular carcinoma is a common malignancy and a major complication of untreated haemochromatosis. Encapsulation of liver tumours has been associated with a better prognosis and longer disease-free periods following resection. This study investigated the source of the tumour capsule in patients with haemochromatosis and coexisting hepatocellular carcinoma and examined potential factors influencing development.. Five haemochromatosis patients with encapsulated hepatocellular carcinoma were studied. Myofibroblasts were identified using combined immunohistochemistry and in situ hybridisation for alpha-smooth muscle actin and procollagen alpha1(I) mRNA, respectively. Immunohistochemistry was also performed for transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-beta receptor and malondialdehyde.. Procollagen alpha1(I) mRNA co-localised to alpha-smooth muscle actin positive myofibroblasts. The number of myofibroblasts was maximal within the capsule and decreased away from the tumour. TGF-beta1 protein was expressed in iron-loaded cells in non-tumour liver at the interface of tumour capsule. PDGF-beta receptor expression was observed in mesenchymal cells in the tumour capsule and in portal tracts. Malondialdehyde adducts were observed in the tumour, non-tumour tissue and in the capsule.. This study provides evidence that myofibroblasts are the cell type responsible for collagen production within the tumour capsule surrounding hepatocellular carcinoma in haemochromatosis. The production of TGF-beta1 by iron-loaded hepatic cells at the tumour capsule interface may perpetuate the myofibroblastic phenotype, resulting in the formation of the tumour capsule.

Topics: Actins; Aged; Carcinoma, Hepatocellular; Cell Count; Fibroblasts; Hemochromatosis; Humans; Immunohistochemistry; In Situ Hybridization; Liver Neoplasms; Lysine; Male; Malondialdehyde; Middle Aged; Muscle, Smooth; Procollagen; Receptor, Platelet-Derived Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

The potent growth-inhibitory activity of cytokines of the transforming growth factor-beta (TGF-beta) superfamily and their widespread expression in epithelia suggest that they may play an important role in the maintenance of epithelial homeostasis. To analyse TGF-beta mediated tumor suppressor activity in the liver, we generated transgenic mice overexpressing a dominant negative type II TGF-beta receptor in hepatocytes under control of the regulatory elements of the human C-reactive protein gene promoter. Transgenic animals exhibited constitutive and liver-specific transgene expression. The functional inactivation of the TGF-beta signaling pathway in transgenic hepatocytes was shown by reduced TGF-beta induced inhibition of DNA synthesis in primary hepatocyte cultures. Liver morphology and spontaneous tumorigenesis were unchanged in transgenic mice suggesting that interruption of the signaling of all three isoforms of TGF-beta in hepatocytes does not disturb tissue homeostasis in the liver under physiological conditions. However, following initiation with the carcinogen diethylnitrosamine and tumor-promotion with phenobarbital transgenic mice exhibited a moderate albeit significant increase in the incidence, size and multiplicity of both preneoplastic tissue lesions in the liver and of hepatocellular carcinomas. These results give in vivo evidence for a tumor suppressor activity of the endogenous TGF-beta system in the liver during chemical hepatocarcinogenesis.

Topics: Animals; C-Reactive Protein; Carcinoma, Hepatocellular; Cells, Cultured; Hepatocytes; Liver Neoplasms, Experimental; Male; Mice; Mice, Transgenic; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

Topics: Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Endothelial Growth Factors; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Liver Regeneration; Liver Transplantation; Lymphokines; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Selenoprotein P (SeP) is a selenium-rich plasma protein which accounts for more than 50% this study, the effect of TGF-beta(1) on the expression of SeP in the human liver cell line HepG2 was investigated. Western analysis revealed a dose-dependent reduction of SeP content in cell supernatant. RT-PCR analysis of SeP-mRNA expression demonstrated a marked inhibition and a reporter gene under control of the SeP promoter was negatively regulated by TGF-beta(1). Smad proteins are the transcriptional mediators of TGF-beta signaling. A putative Smad-binding element (SBE) is present in the SeP promoter. In electrophoretic-mobility-shift assays, TGF-beta(1) enhanced the binding of nuclear proteins to this SBE. Overexpression of Smad3 and 4 resulted in a downregulation of SeP-promoter activity whereas deletion of the SBE led to a loss of TGF-beta(1) responsiveness. We conclude that SeP expression is modulated by the binding of Smad3/4 complexes to a functional SBE in the SeP promoter.

Topics: Base Sequence; Carcinoma, Hepatocellular; Gene Expression Regulation; Humans; Liver; Liver Neoplasms; Luciferases; Molecular Sequence Data; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Selenium; Selenoprotein P; Selenoproteins; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Fas-expressing cytotoxic T lymphocytes (CTLs) are important antitumor immune effector cells in patients with hepatocellular carcinoma (HCC). The role of transforming growth factor beta 1 (TGF-beta1) in modulating the expression of Fas by CTLs is not known in HCC. The objectives of this study were to characterize the expression of Fas by CTLs and natural killer (NK) cells among peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs) in patients with HCC and to correlate the association, if any, with serum TGF-beta1 levels.. PBLs from 18 patients with HCC and TILs from 5 HCC liver specimens were isolated, and Fas expression was analyzed by three-color flow cytometry. The results were compared with results from normal control volunteers (n = 19 individuals). Serum TGF-beta1 levels in patients with HCC were measured by enzyme-linked immunosorbent assay.. The median percentage of Fas expression by CD3 positive T cells was significantly higher in patients with HCC compared with normal controls (54.37% vs. 32.03%, respectively; P = 0.0036), and this was attributable solely to Fas expression by CD4 positive PBLs (54.46% vs. 34.90%, respectively; P = 0.0234). In contrast, Fas expression was significantly higher in all the subtypes of TILs (CD3 positive, CD4 positive, CD8 positive, NK cells, and natural T cells) compared with controls (all P values were < 0.001). Tumor size was inversely proportional to the TGF-beta1 levels (correlation coefficient [r] = -0.725; P < 0.0001), which were correlated inversely with Fas expression by CD4 positive PBLs (r = -0.516; P = 0.01).. In patients with HCC, TILs exhibit significantly increased expression of Fas compared with PBLs that may enhance their susceptibility to apoptotic mechanisms. Larger tumors were associated with lower serum TGFbeta1 levels, and this was correlated with greater Fas expression by CD4 positive PBLs.

Topics: Adult; Aged; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; fas Receptor; Female; Flow Cytometry; Humans; Killer Cells, Natural; Liver Neoplasms; Male; Middle Aged; Statistics, Nonparametric; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

In order to investigate whether TGF-beta 1 could rapidly regulate integrin induced signaling, we treated SMMC-7721 human hepatocellular carcinoma cells with human recombinant TGF-beta 1 for 10 min, and examined cell adhesion, integrin amount and FAK tyrosine phosphorylation. We used cell adhesion assay to estimate the affinity of alpha 5 beta 1 integrin with fibronectin, and analyzed the amount of integrin alpha 5 and beta 1 subunits by performing FACS analysis. Then western blot analysis was carried out to examine tyrosine phosphorylation level of FAK. Our results showed that TGF-beta 1 could rapidly attenuated cell adhesion onto Fn without changing the expression of alpha 5 beta 1 integrin, and at the meantime dephosphorylated FAK. It suggested that TGF-beta 1 rapidly regulated the activation of integrin, and stimulated FAK dephosphorylation, which might induce depolarization in SMMC-7721 hepatocellular carcinoma cells, then facilitates the detachment of tumor cells at early stages of migration.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Dose-Response Relationship, Drug; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha5beta1; Liver Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Recombinant Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

The influence of anisoosmolarity on NF-kappaB binding activity was studied in H4IIE rat hepatoma cells. Hypoosmolarity induced a sustained NF-kappaB binding activity whereas the hyperosmotic NF-kappaB response was only minor. Hypoosmotic NF-kappaB activation was accompanied by degradation of the inhibitory IkappaB-alpha. Protein kinase C, PI(3)-kinase, reactive oxygen intermediates and the proteasome apparently participate in mediating the hypoosmotic effect on NF-kappaB. Hypoosmolarity plus PMA induced, amplified and prolonged IkappaB-alpha degradation and NF-kappaB binding activity. Transforming growth factor beta-induced apoptosis was diminished by hypoosmolarity. However, this anti-apoptotic effect was probably not related to NF-kappaB activation.

Topics: Animals; Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspases; Enzyme Inhibitors; Liver Neoplasms; NF-kappa B; Osmolar Concentration; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured

Microsatellite instability occurs frequently in hereditary nonpolyposis colorectal carcinoma, in sporadic gastrointestinal carcinoma, and in other tumors. In these tumors, slippage-related frameshift mutations have been detected at coding mononucleotide repeats in genes such as those for transforming growth factor-beta receptor type II (TGFbetaRII), mannose 6-phosphate/insulinlike growth factor II receptor (M6P/IGFIIR), hMSH3, hMSH6, and Bcl-2-associated X protein (BAX). Because these genes regulate cell growth or repair DNA mismatches, loss of their function is thought to promote tumor development. The authors screened for these frameshift mutations and investigated the incidence of microsatellite instability (MI) in hepatocellular carcinoma (HCC) in Japan.. Fifty HCC samples were analyzed in this study. The authors used polymerase chain reactions to screen for frameshift mutation at the TGFbetaRII (A)(10) tract, the M6P/IGFIIR (G)(8) tract, the hMSH3 (A)(8) tract, the hMSH6 (C)(8) tract, and the BAX (G)(8) tract. For MI analysis, matched tumor and nontumor liver DNA were investigated with respect to 10 microsatellite loci.. No frameshift mutation was detected in any case, and only 4% of these cancers exhibited MI in comparisons between tumor and nontumor liver specimens.. This study suggests that frameshift mutation at coding mononucleotide repeats within TGFbetaRII, M6P/IGFIIR, hMSH3, hMSH6, and BAX genes did not seem to be involved in hepatocarcinogenesis in the Japanese population studied.

Topics: Adult; Aged; Aged, 80 and over; Base Pair Mismatch; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Female; Frameshift Mutation; Humans; Japan; Liver Neoplasms; Male; Microsatellite Repeats; Middle Aged; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, IGF Type 2; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The serum concentration of transforming growth factor beta (TGF-beta) is elevated as tumors progress in hepatocellular carcinoma (HCC) patients. In this study, we examined whether modulation of tumor-derived TGF-beta signal transduction contributes to malignant progression. We investigated the production of TGF-beta1, the biological effects of TGF-beta and neutralizing antibody on HCC cells, activation of Smad 2, Smad 3, and Smad 4, induction of antagonistic Smads (Smad 6 and Smad 7), and promoter activities of two target genes, plasminogen activator inhibitor type 1 (PAI-1) and p15INK4B. In human cell lines HCC-M and HCC-T, TGF-beta accelerates their proliferation. Smad 2 was activated constitutively by an autocrine mechanism, because in the absence of exogenous TGF-beta, a high level of Smad 2 phosphorylation, induction of PAI-1 transcripts, and nuclear localization of Smad 2 were observed. This constitutive activation of Smad 2 was, at least in part, attributable to the lack of induction of antagonistic Smads by TGF-beta. However, Smads activated by tumor-derived TGF-beta constantly suppressed p151NK4B expression. In addition, 3 of 10 human HCC tissues showed nuclear localization of Smad 2 and low mRNA levels of p15INK4B and antagonistic Smads but a high level of PAI-1. Our observations suggest that this constant suppression of the p15INK4B gene could be involved in the malignant progression of HCC.

Topics: Autocrine Communication; Carcinoma, Hepatocellular; DNA-Binding Proteins; Genes, Tumor Suppressor; Humans; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated facilitation of invasion by growth factors and chemotactic factors in tumor cell lines, particularly hepatocellular carcinoma. Hepatoma cells (PLC/PRF/5 and Hep G2) showed strong chemotaxis toward their respective conditioned media while metastatic pancreatic cancer cells (SU.86.86) and colon cancer cells (LS 174T) did not migrate toward their respective conditioned media. Based on immunoblotting, PLC/PRF/5 cells secrete fibronectin (an extracellular matrix constituent), transforming growth factor-beta (TGFbeta; a growth factor), and cathepsin D (a protease). Fibronectin induced a migratory response in PLC/PRF/5 cells, and anti-fibronectin antibody abolished the migratory response of these cells to their conditioned medium. Anti-integrin-beta(1) antibody also impeded migration of these cells toward conditioned medium. Polyclonal anti-TGFbeta antibody and protease inhibitors (alpha(2)-macroglobulin and leupeptin) added to culture media-modulated secretion of fibronectin by PLC/PRF/5 cells. Although exogenous TGFbeta suppressed SU.86.86 cells, it enhanced PLC/PRF/5 cell adhesion to substrate, increasing viable cell numbers. These actions indicate that hepatocellular carcinoma may possess a forceful autocrine mechanism enabling cells to survive and proliferate under cirrhotic conditions.

Topics: Antibodies, Neoplasm; Carcinoma, Hepatocellular; Cathepsin D; Cell Division; Chemotaxis; Fibronectins; Humans; Immunoblotting; Liver Neoplasms; Protease Inhibitors; Transforming Growth Factor beta; Tumor Cells, Cultured

Transcription of the alpha2(I) collagen gene (COL1A2) in fibroblasts is potently induced by transforming growth factor-beta (TGF-beta). Smad family proteins function as intracellular signal transducers for TGF-beta that convey information from the cell membrane to the nucleus. In the present study, we establish the functional requirement for endogenous Smad3 and Smad4 in TGF-beta-stimulated COL1A2 transcription in human skin fibroblasts in vitro. Furthermore, using transfections with a series of 5' deletions of the human COL1A2 promoter, we identify a proximal region between -353 and -148 bp, which is required for full stimulation of transcription by a constitutively active TGF-beta type I receptor. This region of the COL1A2 promoter contains a CAGA motif also found in the promoter of the plasminogen activator inhibitor-1. Substitutions disrupting this sequence decreased the binding of nuclear extracts or recombinant Smad3 to the CAGACA oligonucleotide, and markedly reduced the transcriptional response to TGF-beta or overexpressed Smad3 in transient transfection assays. The insertion of tandem repeats of CAGACA conferred TGF-beta stimulation to a heterologous minimal promoter-reporter construct. Inhibition of endogenous Smad expression in fibroblasts by antisense oligonucleotides or cDNA against Smad3 or Smad4, and transfection of COL1A2 promoter constructs into Smad4-deficient breast adenocarcinoma cells, indicated the critical role of Smads for the full TGF-beta response. The importance of Smad binding to the CAGACA box of COL1A2 was further established by transcriptional decoy oligonucleotide competition. Taken together, the results identify a functional Smad-binding element of the COL1A2 promoter harboring a CAGACA consensus sequence that is both necessary and sufficient for stimulation by TGF-beta, and demonstrate that interaction of this Smad-binding element with endogenous Smads is required for the full TGF-beta response in fibroblasts.

Topics: Adenocarcinoma; Binding Sites; Breast Neoplasms; Carcinoma, Hepatocellular; DNA-Binding Proteins; Female; Fibroblasts; Gene Deletion; Humans; Liver Neoplasms; Procollagen; Promoter Regions, Genetic; Skin; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Retinoic acid (RA) induces apoptosis in Hep3B human hepatoma cells. 9-Cis-RA (c-RA) had a similar effect as all-trans-RA (t-RA) in inducing cell death in Hep3B cells. RA-induced Hep3B-cell death was associated with inhibited expression of the hepatocyte nuclear factor 4 (HNF-4) gene. Palmitoyl-CoA ((C16:0)-CoA), the reported HNF-4 ligand, prevented RA-induced apoptosis. The effect of (C16:0)-CoA was specific, since palmitic acid and co-enzyme A had no effect in preventing RA-induced apoptosis. Bovine serum albumin (BSA) also prevented RA-induced apoptosis. However, in contrast to BSA, which induced cell growth, (C16:0)-CoA alone had no effect on cell growth. Investigating the possible role of HNF-4 in apoptosis, the reported HNF-4 antagonist (C18:0)-CoA was employed, and it also prevented RA-induced apoptosis. By transient transfection, overexpression of HNF-4 did not prevent RA-induced apoptosis. The induction and prevention of apoptosis caused by RA and (C16:0)-CoA were associated, respectively with the induction and inhibition of the expression of transforming growth factor beta (TGFbeta), which is known to play a role in apoptosis. Furthermore, RA and (C16:0)-CoA can regulate AP-1, which is a key regulator of the TGFbeta gene. Our data indicate that fatty acyl-CoAs can prevent RA-induced apoptosis and that TGFbeta, rather than HNF-4, may play a role in these regulatory processes. Our data also suggest that (C16:0)-CoA and (C18:0)-CoA are not the agonist and antagonist for HNF4, respectively in the Hep3B cell system.

Topics: Alitretinoin; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Northern; Carcinoma, Hepatocellular; Culture Media, Serum-Free; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Fatty Acids; Hepatocyte Nuclear Factor 4; Humans; Ligands; Liver Neoplasms; Palmitoyl Coenzyme A; Phosphoproteins; RNA, Messenger; Time Factors; Transcription Factors; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

In this study we assessed the usefulness of serum Transforming Growth Factor-beta1 (TGF-beta1) and soluble Fas (sFas) in distinguishing liver cirrhosis (LC) with and without hepatocellular carcinoma (HCC) as compared with alpha-fetoprotein (AFP). Serum TGF-beta1 and sFas levels were measured by ELISA in 51 LC patients, 54 patients with HCC and 30 healthy donors. Considering as a cut-off limit (mean+1SD of controls) 74 pg/ml and 637 pg/ml for TGF-beta1 and sFas, respectively, we computed serum concentrations of TGF-beta1 and sFas as a score (mean+/-SD). The positive frequency of serum TGF-beta1 levels in HCC patients (54%) was greater than in LC patients (26%) and healthy donors (3%). TGF-beta1 levels were higher in HCC (1.6+/-0.5) than in LC (1.1+/-0.2) (P<0.0001) and healthy donors (0.6+/-0.2). Using a cut-off limit of 82 pg/ml (mean+2SD), the positive frequency of TGF-beta1 was 20% in HCC patients. None of the controls and LC patients had TGF-beta1 levels higher than 82 pg/ml. The positive frequency of serum sFas levels was 100% in HCC patients, 98% in LC patients and 3% in healthy controls. Serum sFas levels were higher in HCC (2.5+/-0.7) than in LC (1.9+/-0.5) (P<0. 001) and healthy donors (0.6+/-0.3). No significant change of positive frequency was obtained by setting sFas cut-off at higher levels. sFas values did not correlate with TGF-beta1 levels. No relationship was found between TGF-beta1 amounts and AFP levels. However, in the 23% of HCC patients, with normal AFP values TGF-beta1 levels were higher than the cut off. These findings suggest the potential usefulness for TGF-beta1 assay in AFP-negative HCC.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; fas Receptor; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Regression Analysis; Transforming Growth Factor beta

We examined the effects of various peroxisome proliferators (PPs) such as the hypolipidaemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO) and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on peroxisomal enzyme activities, apoptosis and DNA synthesis in rat FaO and human HepG2 hepatoma cell lines. Both growing and confluent cultures were treated with PPs (250 microM) for 48 or 72 h. In accordance with our previous observations in PP-treated primary hepatocyte cultures of rat and human origin, the various PPs increased peroxisomal enzyme activities in rat FaO cells but not in human HepG2 cells. PPs strongly induced apoptosis in FaO cells. They did not affect TGFbeta-induced apoptosis, with the exception of DEHP and NAFE, respectively blocking and increasing induced apoptosis in confluent cultures. Moreover, PPs produced a minor, but significant, decrease in DNA synthesis in FaO cells. PPs also decreased DNA synthesis in growing HepG2 cells, and CLO, CIPRO and NAFE induced apoptosis in confluent HepG2 cultures. This is in opposition with the effects of PPs on primary hepatocyte cultures, i.e. inhibition of both spontaneous and TGFbeta-induced apoptosis and increases in DNA synthesis in rat hepatocytes, and unchanged mitosis-apoptosis balance in human hepatocytes.

Topics: Acyl-CoA Oxidase; Animals; Apoptosis; Carcinoma, Hepatocellular; Carnitine O-Acetyltransferase; Cell Division; Cell Nucleus; DNA; DNA Replication; Hepatoblastoma; Liver; Liver Neoplasms; Oxidoreductases; Peroxisome Proliferators; Peroxisomes; Rats; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

beta-1,4-galactosyltransferase 1 (beta1,4-GT 1) is localized both in the Golgi complex where it catalyzes the transfer of galactose from UDP-galactose to terminal N-acetylglucosamine forming Galbeta1 --> 4GlcNAc structure, and on the cell surface where it serves as an adhesion molecule. It has previously been reported that the expression of beta1,4-GT 1 was cell-cycle-specific, regulated by cell growth. Transforming growth factor-beta1 (TGF-beta1) could regulate cell G1/S phase transition and modulate cell growth in many types of cells. In this study, we introduced the antisense-TGF-beta1 into SMMC-7721 cell, a human hepatocarcinoma cell line, for blocking its intrinsic TGF-beta1 expression, and changing its cell-cycle, and then analyzed the gene expression of beta1,4-GT 1 together with the beta1,4-GT activity. The result showed that the antisense-TGF-beta1 transfected SMMC-7721 cells (AST/7721) were growth enhanced, with more cells in S phase and less cells in G2/M phase compared with the mock transfected cells (pcDNA3/7721). At the same time, it was found that the gene expression of beta1,4-GT 1 in AST/7721 was decreased to one fifth that of pcDNA3/7721, and the cell surface beta1,4-GT activity was reduced to one fifth of the control, while the total activity of beta1,4-GT was decreased to one half that of the control. The results indicate that suppression of TGF-beta1 expression resulted in change of cell-cycle together with the decreased gene expression of beta1,4-GT 1 and beta1,4-GT activity in human hepatocarcinoma cells.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Galactosyltransferases; Gene Expression Regulation, Enzymologic; Humans; Hydrolysis; Liver Neoplasms; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Dose-Response Relationship, Drug; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression; Humans; Laminin; Liver Neoplasms; Phosphoric Monoester Hydrolases; Phosphorylation; Protein-Tyrosine Kinases; PTEN Phosphohydrolase; Receptors, Cell Surface; Receptors, Fibronectin; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins; Tyrosine

Transforming growth factor beta (TGF-beta) initiates signaling through heteromeric complexes of transmembrane type I and type II serine/threonine kinase receptors. Activated TGF-beta type I receptor phosphorylates receptor-regulated Smads (2 and 3). Antagonistic Smad 7 forms stable association with the activated TGF-beta type I receptor, blocking phosphorylation of receptor-regulated Smads. On the other hand, elevated serum concentration of TGF-beta along with resistance to its growth-inhibitory effect is commonly observed in human hepatocellular carcinoma (HCC) patients. In this study, we investigated the mechanisms of resistance to tumor-derived TGF-beta in human HCC and hepatoblastoma-derived cell lines, focusing on the roles of receptor-regulated Smads and antagonistic Smad 7. HuH-7 and HepG2 cells showed poor response to TGF-beta-mediated growth inhibition. Because neutralization of TGF-beta in the medium or blockage of signal transduction pathway by inductions of dominant negative Smad 2/3 resulted in a stimulation of cell growth, tumor-derived TGF-beta signal acts on cell growth negatively. However, Smad 7 induced by TGF-beta negatively regulated Smad 2 action and rendered most Smad 2 proteins in the cytoplasm. Taken together, these results indicate that endogenous TGF-beta-mediated induction of Smad 7 results in a higher "threshold" for the antiproliferative signals mediated by receptor-regulated Smads, and can be involved in reduced responsiveness to the cytokine in some human HCC cells.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; DNA-Binding Proteins; Humans; Liver Neoplasms; Phosphorylation; Plasminogen Activator Inhibitor 1; Receptor, IGF Type 2; RNA, Messenger; Smad2 Protein; Smad3 Protein; Smad7 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

LIGHT is a member of the tumor necrosis factor superfamily and is the ligand for LT-betaR, HVEM, and decoy receptor 3. LIGHT has a cytotoxic effect, which is further enhanced by the presence of interferon-gamma (IFN-gamma). Although LIGHT/IFN-gamma can activate caspase activity, neither benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can completely inhibit LIGHT/IFN-gamma-mediated apoptosis. Moreover, overexpression of Bcl-2 further enhances LIGHT/IFN-gamma-mediated apoptosis. It appears that LIGHT and IFN-gamma act synergistically to activate caspase-3, with the resultant cleavage of Bcl-2, removal of the BH4 domain, leading to conversion of Bcl-2 from an antiapoptotic to a proapoptotic form in p53-deficient hepatocellular carcinoma Hep3BT2 cells. Thus, LIGHT seems to be able to override the protective effect of Bcl-2 and induce cell death. Although benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can prevent the cleavage of Bcl-2 by LIGHT/IFN-gamma, they only partially inhibit apoptosis in Hep3BT2 cells that are overexpressing Bcl-2. In contrast, both LIGHT/IFN-gamma-mediated apoptosis and Bcl-2 cleavage are inhibited by free radical scavengers, indicating that free radicals may play an essential role in LIGHT/IFN-gamma-mediated apoptosis at a step upstream of caspase-3 activation. These results suggest that LIGHT signaling may diverge into multiple, separate processes.

Topics: Amino Acid Sequence; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Female; Genes, bcl-2; Humans; Interferon-gamma; Kinetics; Liver Neoplasms; Membrane Proteins; Molecular Sequence Data; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor Ligand Superfamily Member 14; Tumor Necrosis Factor-alpha

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents - transforming growth factor beta1 (TGF-beta1), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) - on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-beta1; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-beta1, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinogens; Carcinoma, Hepatocellular; Cell Culture Techniques; Cycloheximide; Fluorescent Dyes; Hepatocytes; Humans; Indoles; Liver Neoplasms; Okadaic Acid; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured

Transforming growth factor beta-1 (TGF beta 1) has been suggested to play a role in the development, growth or progression of hepatocellular carcinoma (HCC). Genotype and serum titer of HCV also affect the occurrence of HCC in chronic hepatitis C. In this study, we were to evaluate the effects of genotype or serum titer of HCV on the expression of TGF beta 1. We also intended to examine the correlation between the up-regulation of TGF beta 1 and the association with HCC in patients with chronic hepatitis C.. We studied 19 patients with chronic hepatitis C and 18 with HCC associated with HCV infection. HCV genotype was determined by line probe reverse hybridization assay and the amount of HCV-RNA was quantitated by branched DNA signal amplification assay. Serum TGF beta 1 level was measured by enzyme linked immunosorbent assay.. HCV genotypes of patients with HCC were similar to those without it. Serum HCV-RNA titer was higher in genotype 1b than in non-1b (p < 0.05). Serum TGF beta 1 levels were higher in HCC than in chronic hepatitis (p < 0.05). However, there was no significant difference in the serum TGF beta 1 level between genotype 1b and non-1b. Also, it was not correlated with the serum HCV-RNA titer or alanine aminotransferase levels.. TGF beta 1 seems to be overexpressed in HCC compared to that of chronic hepatitis C: it was not affected by serum ALT levels, genotype or serum HCV titer. It is suggested that TGF beta 1 may be associated with the malignant transformation of hepatocyte or the progression of HCV-associated HCC.

Topics: Adult; Aged; Alanine Transaminase; Carcinoma, Hepatocellular; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Liver Neoplasms; Male; Middle Aged; RNA, Viral; Transforming Growth Factor beta

Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been suggested to influence tumor growth. They mediate their functions via specific cell surface receptors (type I ALK5 and type II TGF-beta receptors). The aim of this study was to analyze the roles of the three TGF-betas and their signaling receptors in human hepatocellular carcinoma (HCC).. HCC tissue samples were obtained from 18 patients undergoing partial liver resection. Normal liver tissues from 7 females and 3 males served as controls. The tissues for histological analysis were fixed in Bouin's solution and paraffin embedded. For RNA analysis, freshly obtained tissue samples were snap frozen in liquid nitrogen and stored at -80 degrees C until used. Northern blot analysis was used in normal liver and HCC to examine the expression of TGF-beta1, -beta2, -beta3 and their receptors: type I ALK5 (TbetaR-I ALK5), type II (TbetaR-II), and type III (TbetaR-III). Immunohistochemistry was performed to localize the corresponding proteins.. All three TGF-betas demonstrated a marked mRNA overexpression in HCC in comparison with normal controls, whereas the levels of all three TGF-beta receptors showed no significant changes. Intense TGF-beta1, TGF-beta2, and TGF-beta3 immunostaining was found in hepatocellular carcinoma cells and in the perineoplastic stroma with immunohistochemistry, whereas no or mild immunostaining was present in the normal liver. For TbetaR-I ALK5 and TbetaR-II, the immunostaining in both HCC and normal liver was mild to moderate, with a slightly higher intensity in the normal tissues.. The upregulation of TGF-betas in HCC suggests an important role for these isoforms in hepatic carcinogenesis and tumor progression. Moreover, the localization of the immunoreactivity in both malignant hepatocytes and stromal cells suggests that TGF-betas act via autocrine and paracrine pathways in this neoplasm.

Topics: Activin Receptors, Type I; Adult; Aged; Blotting, Northern; Carcinoma, Hepatocellular; Cell Division; DNA Probes; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Protein Serine-Threonine Kinases; Receptor Cross-Talk; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Transforming Growth Factor beta

We have previously shown in transgenic mice that transforming growth factor (TGF)-alpha dramatically enhances c-myc-induced hepatocarcinogenesis by promoting proliferation and survival of hepatocellular carcinoma (HCC) cells. As transgenic livers display increased levels of mature TGF-beta1 from the early stages of hepatocarcinogenesis, we have now assessed whether impairment of TGF-beta1 signaling contributes to the deregulation of cell cycle progression and apoptosis observed during this process. Focal preneoplastic lesions lacking expression of TGF-beta receptor type II (TbetaRII) were detected in c-myc/TGF-alpha but not in c-myc livers. In c-myc/TGF-alpha mice, 40% (2/5) of adenomas and 90% (27/30) of HCCs showed down-regulation of TbetaRII expression in comparison with 11% (2/18) of adenomas and 47% (14/30) of HCCs in c-myc mice. Down-regulation of the TGF-beta1-inducible p15(INK4B) mRNA and reduced apoptotic rates in TbetaRII-negative HCCs further indicated the disruption of TGF-beta1 signaling. Furthermore, both TbetaRII-negative and -positive c-myc TGF-alpha HCCs, but not c-myc HCCs, were characterized by decreased levels of the cell cycle inhibitor p27. These results suggest 1) an inverse correlation of decreased p27 expression with the particularly strong expression of TGF-alpha in these lesions, consistent with the capacity of TGF-alpha signaling to post-transcriptionally regulate p27, and 2) the presence of alternative, downstream defects of TGF-beta1 signaling in c-myc/TGF-alpha HCCs that may impair the growth-inhibitory response to TGF-beta1. Thus, the accelerated neoplastic development in c-myc/TGF-alpha mice is associated with an early and frequent occurrence of TbetaRII-negative lesions and with reduced levels of p27 in HCC cells, indicating that disruption of TGF-beta1 responsiveness may play a crucial role in the enhancement of c-myc-induced hepatocarcinogenesis by TGF-alpha.

Topics: Animals; Blotting, Northern; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Down-Regulation; Enzyme Inhibitors; Immunohistochemistry; Liver Neoplasms, Experimental; Male; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-myc; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Suppressor Proteins

It has been demonstrated that gamma delta T cells accumulating in early tumor lesions and those purified from spleen cells of tumor-bearing mice attenuate the activity of CTLs and NK cells. We, therefore, investigated whether depletion of gamma delta T cells from early lesions of tumors results in restoration of CTL and NK cell activities and subsequent regression of tumors. A daunomycin-conjugated anti-gamma delta TCR mAb UC7-13D5 (Dau-UC7) was prepared to efficiently deplete gamma delta T cells. An in vitro study revealed that Dau-UC7 specifically lysed gamma delta TCR+ cells and effectively inhibited splenic gamma delta T cells from tumor-bearing mice to produce cytotoxic cell-suppressive factors. Furthermore, intralesional injections of Dau-UC7 at an early stage of tumor development led to augmentation of tumor-specific CTL as well as NK cell activities and to the resultant regression or growth inhibition of the tumors. On analysis of cytokine profile, gamma delta T cells transcribed mRNAs for IL-10 and TGF-beta, but not IL-4 or IFN-gamma, suggesting the T regulatory 1-like phenotype. Finally, a blocking study with mAbs showed that the inhibitory action of gamma delta T cells on CTLs and NK cells was at least partly mediated by IL-10 and TGF-beta. These results clearly demonstrated the novel mechanism by which T regulatory 1-like gamma delta T cells suppress anti-tumor CTL and NK activities by their regulatory cytokines in early tumor formation.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antibody Specificity; Carcinoma, Hepatocellular; Cytokines; Cytotoxicity, Immunologic; Daunorubicin; Interleukin-10; Killer Cells, Natural; Lymphocyte Depletion; Lymphocytes, Tumor-Infiltrating; Male; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Receptors, Antigen, T-Cell, gamma-delta; Suppressor Factors, Immunologic; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Cells, Cultured

Hepatocellular carcinoma (HCC) is a common, potentially lethal tumor in human patients. Because the serum levels of transforming growth factor-beta1 (TGF-beta1) correlate with outcome in patients with HCC and because TGFbeta1 mRNA expression is increased in HCC tissues, it raises the possibility that TGF-beta1 may be of importance in the development, growth, and metastases of HCC. Tamoxifen has been used for the treatment of human HCC. However, clinical trials have produced conflicting results. To further delineate whether tamoxifen may be of benefit in altering the course of HCC, we documented the effects of 4-hydroxytamoxifen and 17beta-estradiol on TGF-beta1 mRNA and protein levels and cell proliferation in a human HCC cell line. PLC/PRF/5 cells were treated with carrier (controls), 4-hydroxytamoxifen, 17beta-estradiol, or TGF-beta1. 4-Hydroxytamoxifen and 17beta-estradiol decreased TGF-beta1 mRNA and protein levels in a time- and dose-dependent manner. TGF-beta1 significantly inhibited PLC/PRF/5 cell proliferation, whereas both 4-hydroxytamoxifen and 17beta-estradiol stimulated PLC/PRF/5 cell proliferation. The stimulatory effects of 4-hydroxytamoxifen on PLC/PRF/5 cell proliferation raise concerns regarding its use in the treatment of HCC in human patients and suggest that 4-hydroxytamoxifen may have no beneficial effects in some patients with HCC.

Topics: Carcinoma, Hepatocellular; Cell Division; Dose-Response Relationship, Drug; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; RNA, Messenger; Tamoxifen; Transforming Growth Factor beta

The multifunctional cytokine interleukin-6 (IL-6) regulates growth and differentiation of many cell types and induces production of acute-phase proteins in hepatocytes. Here we report that IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor-beta (TGF-beta), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF-beta-induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15(INK4B) genes, indicating that IL-6 interferes with only a subset of TGF-beta activities. To further elucidate the mechanism of this anti-apoptotic effect of IL-6, we investigated which signaling pathway transduced by IL-6 is responsible for this effect. IL-6 stimulation of hepatoma cells induced a rapid tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and its kinase activity followed by the activation of Akt. Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the protection of IL-6 against TGF-beta-induced apoptosis. A dominant-negative Akt also abrogated this anti-apoptotic effect. Dominant-negative inhibition of STAT3, however, only weakly attenuated the IL-6-induced protection. Finally, inhibition of both STAT3 and PI 3-kinase by treating cells overexpressing the dominant-negative STAT3 with LY294002 completely blocked IL-6-induced survival signal. Thus, concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways mediates the anti-apoptotic effect of IL-6 against TGF-beta, with the former likely playing a major role in this anti-apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; DNA-Binding Proteins; Enzyme Activation; Gene Expression Regulation; Humans; Interleukin-6; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

TGF-beta is a negative regulator of liver growth. Smad family of genes, as mediators of TGF-beta pathway, are candidate tumor suppressor genes in hepatocellular carcinoma (HCC). We studied 35 HCC and non-tumour liver tissues for possible mutations in Smad2 and Smad4 genes. Three tumours displayed somatic mutations; two in Smad4 (Asp332Gly and Cys401Arg) and one in Smad2 (Gln407Arg) genes. All three mutations were A:T --> G:C transitions suspected to result from oxidative stress as observed in mitochondrial DNA. These observation demonstrate that TGF-beta pathway is altered in hepatocellular carcinoma.

Topics: Base Sequence; Carcinoma, Hepatocellular; DNA-Binding Proteins; Gene Frequency; Genes, Tumor Suppressor; Humans; Liver Neoplasms; Molecular Sequence Data; Mutation; Signal Transduction; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Liver carcinogenesis is associated with striking changes in the integrin repertoire of hepatocytes, including the overexpression of the laminin and collagen receptors alpha1beta1 and the de novo induction of the laminin receptor alpha6beta1. Our aim was to analyze the role of pro-inflammatory cytokines, interferons and fibrogenic cytokines TGF-beta and FGF2 in the regulation of the expression of beta1 integrins by neoplastic hepatocytes. The 2 human hepatocellular cell lines HepG2 and Hep3B were used as models. Integrin expression was assessed by qualitative methods (immunocytochemistry, Western blotting) and semi-quantitative techniques (FACS, cellular ELISA), before and after stimulation by TNFalpha, IL1-beta, TGF-beta, FGF2, interferon gamma and interferon alpha-2b. HepG2 and Hep3B constitutively expressed alpha1, alpha2, alpha6 and beta1 chains. A 24 to 48-hr stimulation with pro-inflammatory cytokines, TGF-beta and FGF2 induced a significant increase in the concentrations of all integrin chains. The maximum induction was registered for beta1 chain, which presented increases amounting up to 3, 4 and 7 times the control values in the presence of, respectively, TNF alpha/IL1-beta, TGF-beta and FGF2. Interferons had no direct effect on integrin expression and partially antagonized the effects of TNF alpha and TGF-beta. The increased concentrations of integrin chains were associated with an increased membrane expression of the corresponding dimers and with an increased adhesion of stimulated hepatocytes to laminin, which was antagonized by neutralizing anti-beta1 and anti-alpha6 antibodies. Finally, anti-alpha6 antibody inhibited the migration of HepG2 and Hep3B cells in reconstituted basement membrane. Our results suggest that the stimulation of alpha6beta1 integrin expression in hepatocarcinoma cells is essential for cell adhesion and migration.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Membrane; Cell Movement; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Flow Cytometry; Humans; Immunoblotting; Immunohistochemistry; Integrin alpha6beta1; Integrins; Interferon alpha-2; Interferon-alpha; Interferon-gamma; Interleukin-1; Liver Neoplasms; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Transforming growth factor-beta1 (TGF-beta1) has been shown to induce apoptosis in normal or transformed hepatocytes. To elucidate the biochemical pathways leading to apoptosis induced by TGF-beta1 in human hepatoma cells (HuH-7), we examined the expression of Bcl-2-related proteins and X-chromosome-linked inhibitor of apoptosis (XIAP), and activation of the caspase cascade following TGF-beta1 treatment. Bcl-xL expression began to decline at 12 hours after TGF-beta1 treatment and progressively decreased to very low levels in a time-dependent manner. Bax expression showed a little change throughout the experiment. On the other hand, activation of caspase-8 was clearly observed at 36 hours after TGF-beta1 treatment, followed by activation of caspase-9, and caspase-3 was activated at 48 hours after treatment at which time apoptosis of HuH-7 cells was observed. TGF-beta1 significantly decreased XIAP expression in HuH-7 cells. Addition of an inhibitor of caspase-8 or caspase-3 (IETD-FMK or DEVD-CHO) markedly inhibited TGF-beta1-induced apoptosis of HuH-7 cells. Fas/Fas ligand (FasL) interactions in HuH-7 cells were not involved in the apoptotic process. Furthermore, epidermal growth factor (EGF) also completely inhibited TGF-beta1-induced apoptosis of HuH-7 cells by inhibiting activation of the caspase cascade. Our results suggested that activation of caspase-3 initiated through caspase-8 activation is involved in the apoptotic process induced by TGF-beta1 in HuH-7 cells. Our results also showed that down-regulation of the expression of Bcl-xL and XIAP by TGF-beta1 may facilitate activation of caspase-3 in these cells.

Topics: Apoptosis; bcl-X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Division; Cysteine Proteinase Inhibitors; Enzyme Activation; Epidermal Growth Factor; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Kinetics; Liver Neoplasms; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Cell growth and division are controlled through the actions of cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CKIs). Treatment of cell lines with Trichostatin A leads to induction of one of these CKIs, p21, and growth arrest. Induction of p21 can also occur through the actions of TGFbeta1. Latent TGFbeta1 can be activated by the M6P/IGF2R. In the present study we have examined the effect of TSA on members of the IGF axis, the CKIs p21 and p27, and also TGFbeta1 in Hep3B cells. The only member of the IGF axis to be affected by treatments was IGF2. Expression of another gene from the same chromosomal location, H19, was also affected. TGFbeta1 expression was greatly enhanced by TSA. In addition, both CKIs, p21 and p27, were upregulated by TSA. Effects of adding IGF-II or TGFbeta1 to TSA-treated cells on p21 induction were examined. The results show that the induction of p21 by TSA can be modulated by additions of IGF-II whereas addition of TGFbeta1 affects its own expression but not p21. In conclusion, the results indicate that the induction of p21 and cell growth arrest caused by Trichostatin A may involve multiple signaling pathways.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Hydroxamic Acids; Insulin-Like Growth Factor II; Liver Neoplasms; Protein Binding; Receptor, IGF Type 2; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

To investigate the gene expression of transforming growth factor-beta1 (TGF-beta(1)) and its type II receptor (TGF-beta R II) in hepatocellular carcinoma.. The expression of TGF-beta(1) and TGF-beta R II at mRNA level and protein level in 30 cases of HCC was separately detected using reverse transcription-PCR and immunohistochemistry technique.. There was no difference in the expression of TGF-beta(1) at mRNA level between HCC tissue (24/30) and surrounding liver tissue (26/30). At the protein level, however, the expression of TGF-beta(1) decreased in HCC tissue compared with that in the surrounding liver tissue (P < 0.01). The expression of TGF-beta R II mRNA decreased significantly in HCC tissue (11/30) compared with that in the surrounding liver tissue (23/30) (P < 0.01). The less HCC expressed TGF-beta R II mRNA, the poorer the tumoral hepatocytes differentiated (P < 0.01) and the more likely the portal vein metastasis and cancer embolus appeared (P < 0.05).. In the negative growth regulation of tumoral hepatocytes by TGF-beta(1), two defects take place: the expression of TGF-beta(1) at the protein level and the expression of TGF-beta R II at the mRNA level.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Female; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta1 (TGF-beta1) arrests growth and/or stimulates apoptosis of a variety of cells. The biochemical pathways involved in the apoptotic processes, however, remain poorly defined. TGF-beta1 induces DNA fragmentation together with morphological changes, which are characteristic of apoptosis in the FaO rat hepatoma cell line. Histones were remarkably enriched in lysates of these cells during TGF beta1-induced apoptosis. We identified U1-70 kd as a death substrate which is cleaved following TGF-beta1 treatment. The tetrapeptide caspase inhibitor carbobenzoxy-valyl-alanly-aspartyl-(beta-O-methyl)-fluoromethyl ketone (ZVAD-FMK) prevented TGF beta1-induced apoptotic DNA fragmentation and cleavage of the U1-70 kd protein, showing that caspase(s) are involved in TGF beta1-mediated apoptosis. To identify specific caspases involved in apoptosis induced by TGF-beta1 in FaO cells, proteolytic activation of several of these caspases and their substrates were studied as a function of time following TGF beta1-treatment. TGF beta1-treatment induced the progressive proteolytic processing of caspase-2 (ICH-1L/Nedd-2), whereas caspase-1 itself did not show any cleavage from the precursor. Pretreatment with ZVAD-FMK abrogated the maturation of caspase-2 and blocked the apoptotic progress. These results suggest that caspase-2, but not caspase-1, may play a crucial role in TGF beta1-induced apoptosis in these cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 1; Caspase 2; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Immunoblotting; Liver Neoplasms; Protease Inhibitors; Proteins; Rats; Ribonucleoprotein, U1 Small Nuclear; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Several novel differentiated cell lines have been derived from a human hepatocarcinoma named HBG. Analysis of their functional properties evidenced a gradual differentiation process as they became confluent and a remarkable stability of the whole quiescent population for at least 6 weeks. However, when replated at low density after several weeks of quiescence, the differentiated cells were able to rapidly reverse to active proliferation, accompanied by transient dedifferentiation. Demonstration that the differentiated hepatic cells were growth-arrested in G1 phase was provided by the increased number of cells with 2C DNA content and decreased expression of S-phase markers. Characteristic features of oncogenes and cell cycle genes were defined during the differentiation process: (a) a biphasic expression of c-myc, with the latter wave covering the quiescence period; (b) opposite kinetics of c-Ki-ras and of N-ras expression with a pattern of changes paralleling that of c-myc; and (c) a decrease of cyclin D1 protein expression and of the cyclin D1-associated kinase activity. The mechanisms by which quiescent differentiated cells might reinitiate active proliferation were analyzed by studying several genes involved in cell growth and death regulation. We found: (a) a point mutation and loss of the specific activity of the tumor suppressor gene p53 without alteration of the apoptotic response to transforming growth factor beta1; (b) a gradual decrease of retinoblastoma protein, which was constantly present, mainly in a hyperphosphorylated form; and (c) an increase of cyclin-dependent kinase inhibitor p27 expression in confluent differentiating cells, as expected, whereas, surprisingly, a disappearance of the p21 protein was observed in parallel. These data may reflect specific mechanisms of cell cycle regulation in liver parenchymal cells through which these cells can proceed to control their reversible differentiation program.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; CDC2 Protein Kinase; Cell Cycle; Cell Differentiation; Cell Division; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; G1 Phase; Gene Expression Regulation; Genes, cdc; Genes, myc; Genes, p53; Genes, Retinoblastoma; Humans; Liver; Mice; Mice, Nude; Mutation; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptosis in normal hepatocytes and hepatoma cells both in vivo and in vitro. However, the mechanism by which TGF-beta induces apoptosis is not clear. The antiapoptotic activity of antioxidants including N-acetyl-L-cysteine (Ac-Cys), ascorbic acid and a novel free radical scavenger, carboxyfullerene (C60) on TGF-beta-treated human hepatoma Hep3B cells was examined. Only the water-soluble hexacarboxylic acid derivative of C60 was found to prevent TGF-beta-induced apoptosis. Antiapoptotic activity of C60 correlated its ability to eliminate TGF-beta-generated reactive oxygen species (ROSs). However, C60 did not interfere with TGF-beta-activated PAI-1 promoter activity in the Hep3B cells. These results indicate that the signaling pathway of TGF-beta-induced apoptosis may be related to the generation of ROSs and may be uncoupled from the TGF-beta-activated gene promoter activity. Furthermore, the regioisomer of C60 with a C3 symmetry was more potent in protecting cells from apoptosis than that with a D3 symmetry, and the C3 isomer had stronger interactions with lipid bilayers than the D3 isomer. The spectroscopic analysis revealed that the C3 isomer had stronger interactions with artificial lipid bilayers than the D3 isomer. Therefore, our study indicates that C60 may interact with membrane to eliminate TGF-beta-induced ROSs and to prevent apoptosis occur in human hepatoma cells.

Topics: Acetylcysteine; Apoptosis; Ascorbic Acid; Carbon; Carboxylic Acids; Carcinoma, Hepatocellular; Cell Survival; Flow Cytometry; Fluorescent Dyes; Free Radical Scavengers; Fullerenes; Humans; Liposomes; Molecular Structure; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Reactive Oxygen Species; Signal Transduction; Stereoisomerism; Transforming Growth Factor beta; Tumor Cells, Cultured

Surgical excision of liver tumors represents the only curative treatment for primary and metastatic liver malignancies. It has been suspected that hepatectomy may stimulate growth of microscopic tumors. To determine whether local or systemic factors after hepatectomy are responsible for enhancement of tumor growth, the effects of hepatectomy on the experimental growth of liver or pulmonary tumors were examined.. One hour after injection of 10(6) Morris hepatoma cells into either the portal or femoral vein, which produces isolated liver and lung tumors, respectively, animals were randomized to undergo 0%, 30%, or 70% partial hepatectomy (PH).. Animals that underwent portal injection of tumor had significantly increased liver tumor burden after PH (sham, 25 +/- 7 vs PH, 94 +/- 17; p < 0.01), whereas animals that underwent femoral injection had no change in lung tumor burden after PH. PH was associated with significantly increased levels of transforming growth factor-alpha, transforming growth factor-beta, and basic fibroblast growth factor in the liver but not in the lung.. Changes in liver cytokine-growth factor activation may contribute to enhanced tumor growth in the liver after hepatectomy.

Topics: Animals; Carcinoma, Hepatocellular; Fibroblast Growth Factor 2; Hepatectomy; Injections, Intravenous; Liver; Liver Neoplasms, Experimental; Lung Neoplasms; Male; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Inbred BUF; Transforming Growth Factor alpha; Transforming Growth Factor beta

The growth of hepatoma cells can be inhibited by treatment with TGFbeta1 or with exogenous reducing agents. To gain information on the molecular mechanisms underlying growth arrest, we visualized and compared gene expression profiles of proliferating versus non proliferating HepG2 cells by computer-assisted gene fishing, an improved technique of RNA fingerprinting that allows the selective amplification of coding regions within transcripts. While many transcripts are selectively regulated by either treatment, a set of bands appear to be coordinately regulated by 2ME and TGFbeta1, suggesting their possible involvement in the mechanisms of growth arrest. Display tags corresponding to 18 differentially expressed genes were cloned and, in most cases, identified as known genes or, more frequently, as their homospecific/cross-specific homologues. A novel member of the kinesin superfamily was identified amongst the genes induced by both 2ME and TGFbeta1. This gene, KIF3C, is upregulated in several cell lines undergoing growth arrest. Taken together, our findings show that computer-assisted gene fishing is a powerful tool for the identification and cloning of genes involved in the control of cell proliferation and indicate that extracellular reducing agents can regulate cell growth through modulation of gene expression.

Topics: Carcinoma, Hepatocellular; Cell Division; Cloning, Molecular; Gene Expression Regulation; Growth Inhibitors; HL-60 Cells; Humans; Kinesins; Mercaptoethanol; Reducing Agents; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptosis on normal hepatocytes and hepatoma cells both in vitro and in vivo. However, how the TGF-beta induces apoptosis is still not clear. We examined the expression of anti-apoptosis proteins and sensitivity to TGF-beta in three well differentiated human hepatoma cell lines. Two TGF-beta sensitive cell lines Hep3B and HuH7 totally lacked Bcl-2. In contrast, the TGF-beta resistant HepG2 cells expressed a substantial amount of Bcl-2. All three cell lines expressed equal amounts of Bcl-X(L), Bcl-X(S) and Bax. Overexpression of Bcl-2 in Hep3B and HuH7 cells protected them from TGF-beta-induced apoptosis. TGF-beta treatment increased intracellular peroxide production and suppressed the expression of glutathione-S-transferase in the Hep3B cells, and these effects were partially suppressed by the overexpression of Bcl-2. These results suggest that Bcl-2 may protect cell from TGF-beta-F-induced apoptosis by interfering TGF-beta generated signals leading to induce reactive oxygen species production.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Carcinoma, Hepatocellular; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glutathione Transferase; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Recombinant Proteins; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Little is known about the influence of amino acid imbalance by supplementation of branched-chain amino acids (BCAAs) on human hepatocellular carcinoma (HCC). We investigated the effect of various BCAA concentrations in culture medium on the growth and metabolism of two human HCC cell lines: Hep G2 and KYN-1. DNA and protein syntheses were studied by radiolabeled thymidine and leucine uptake, Amino acid concentrations in cell and medium were measured with an amino acid analyzer. Expression and excretion of transforming growth factor (TGF)-alpha and TGF-beta1 were studied by immunostaining and enzyme linked immunosorbent assay methods. The cell growth was suppressed in media with higher and lower BCAA concentrations than Dulbecco's modified Eagle's medium, and this was grossly correlated with the incorporation of [3H]thymidine into DNA and incorporation of [3H]leucine into intracellular protein. Pretreatment with 10,000 nmol/ml of BCAA did not suppress the cell growth in subsequent culture in the standard Dulbecco's modified Eagle's medium, indicating that the effect of BCAAs was not cytocidal but cytostatic and reversible. BCAA and aromatic amino acid concentrations in cells increased in parallel as the BCAA level in medium was increased, despite the fixed aromatic amino acid level. Intracellular expression and extracellular excretion of TGF-alpha were higher at BCAA concentrations of 100 and 1,000 nmol/ml than at 10 or 1,000 nmol/ml. The present finding that the in vitro growth of HCC can be suppressed by enriched BCAA levels in medium may indicate that amino acid-imbalanced therapy with enriched BCAAs is useful in the treatment of HCC.

Topics: Amino Acids; Amino Acids, Branched-Chain; Carcinoma, Hepatocellular; Cell Division; Culture Media; Culture Media, Conditioned; DNA; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Protein Biosynthesis; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

In the present retrospective study, liver cell dysplasia (LCD) occurring in cirrhotic livers associated or not associated with hepatocellular carcinoma (HCC) was immunohistochemically analyzed for the expression of hepatocyte growth factor receptor (c-met protein), Rb (retinoblastoma gene) protein, E-cadherin, and transforming growth factor-beta-1 (TGF-beta-1). Cytoplasmic c-met protein staining was observed in about half of the HCC's, and its prevalence was about twice as high in high grade vs. low grade tumors, but it was not correlated with proliferative activity as based on PCNA labelling. In LCD, reactivity for c-met protein was restricted to the small cell type. Nuclear staining for Rb protein was found in HCC's, and was not related to type, grade or proliferative activity, whereas no immunoreactivity was observed in normal, hyperplastic or dysplastic hepatocytes. Expression of E-cadherin prevailed in HCC's of lower grade, and particularly in those with a trabecular or acinar growth pattern. E-cadherin staining was detectable in normal and large dysplastic hepatocytes, but not in small dysplastic liver cells. TGF-beta-1 reactivity was observed in more than half the HCC's, but not in normal or dysplastic hepatocytes. These findings underline the phenotypic difference between large cell and small cell liver dysplasia, and support the hypothesis that small cell dysplasia is a precursor lesion in a hepatocarcinogenic pathway.

Topics: Cadherins; Carcinoma, Hepatocellular; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Precancerous Conditions; Proto-Oncogene Proteins c-met; Retinoblastoma Protein; Retrospective Studies; Transforming Growth Factor beta

The retinoblastoma (RB) gene is one of the most extensively studied tumour-suppressor genes. Deletion or inactivation of both RB alleles is an essential, rate-limiting step in the formation of retinoblastoma and osteosarcoma that arise in families that carry mutant RB (ref. 2). RB inactivation is also found in other human tumours. Whereas loss of RB function is associated with the loss of cellular proliferative control, introduction of a wild-type RB can suppress cell growth and tumorigenicity. Thus, identification of factors that interfere with and/or control the function of the RB protein is critical for understanding both cell-cycle control and oncogenesis. Here we describe a new gene, Bog (for B5T over-expressed gene), which was identified and shown to be overexpressed in several transformed rat liver epithelial (RLE) cell lines resistant to the growth-inhibitory effect of TGF-beta1, as well as in primary human liver tumours. The Bog protein shares homology with other retinoblastoma-binding proteins and contains the Rb-binding motif LXCXE. Using the yeast two-hybrid system and co-immunoprecipitation, we demonstrated that Bog binds to Rb. In vivo, Bog/Rb complexes do not contain E2F-1, and Bog can displace E2F-1 from E2F-1/Rb complexes in vitro. Overexpression of Bog in normal RLE cells conferred resistance to the growth-inhibitory effect of TGF-beta1. Furthermore, normal RLE cells are rapidly transformed when Bog is continuously overexpressed and form hepatoblastoma-like tumours when transplanted into nude mice. These data suggest that Bog may be important in the transformation process, in part due to its capacity to confer resistance to the growth-inhibitory effects of TGF-beta1 through interaction with Rb and the subsequent displacement of E2F-1.

Topics: Amino Acid Sequence; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Epithelial Cells; Humans; Hydrolases; Intracellular Signaling Peptides and Proteins; Liver; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Proteins; Organ Specificity; Rats; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; RNA, Messenger; RNA, Neoplasm; Sequence Homology, Amino Acid; Serine Proteases; Transcription Factor DP1; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Werner Syndrome Helicase; Xenopus Proteins

Transforming growth factor betas (TGF-betas) are the potent growth inhibitors for various cell types. Certain transformed cells, however, show poor response to TGF-beta-induced growth inhibition, which contributes to their uncontrolled proliferation. Recently, we have reported that TGF-beta1 induces degradation of activated Src tyrosine kinase in rat fibroblasts. To elucidate the alteration in TGF-beta signaling pathway in tumor cells that cannot respond to the cytokine, we compared the effects of TGF-beta1 on Src kinase in two human hepatoma cell lines, TGF-beta1-insensitive Mahlavu cells and TGF-beta1-sensitive HepG2 cells. TGF-beta1 decreased Src kinase activity in HepG2 cells, but increased cellular Src levels and Src kinase activity in Mahlavu cells. Co-incubation of Mahlavu cells with TGF-beta1 and 12-O-tetradecanoyl phorbol 13-acetate (TPA) decreased Src protein levels and Src kinase activity, inducing TGF-beta1 sensitivity. TGF-beta1 induced tyrosine dephosphorylation of Ras guanosine triphosphatase-activating protein (Ras-GAP) and Ras inactivation in HepG2 cells, but induced Ras-GAP phosphorylation and Ras activation in Mahlavu cells. The Src kinase inhibitor abolished the increase of Src kinase activity in TGF-beta1-treated Mahlavu cells, and induced TGF-beta1 sensitivity. These findings suggest that regulation of Src kinase by TGF-beta1 is altered in Mahlavu cells. The altered regulation of Src may contribute to TGF-beta1 insensitivity in this cell line, at least in part through activation of Ras.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; GTPase-Activating Proteins; Humans; Liver Neoplasms; Mice; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Rats; Receptors, Transforming Growth Factor beta; src-Family Kinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Tyrosine

To detect the expression of three TGF beta isoforms and its clinicopathologic significance in hepatocellular carcinoma(HCC).. Surgical specimens of 52 patients with small HCC resection during 1992-1995 were used to determine the immunoreactivity of TGF beta protein by ABC immunohistochemistry. These specimens were divided into recurrence group(G1) and recurrence group(G2) according to whether HCC recurred two years after resection. The relations with recurrence of HCC were analyzed.. The positive rates in G1 and G2 were 85% (17/20) versus 47% (15/32) for TGF beta 1, 90% (18/20) vs. 78% (25/32) for TGF beta 2, and 45% (9/20) vs. 63% (20/32) for TGF beta 3. Significant difference was found in TGF beta 1 and TGF beta 2, but not in TGF beta 3.. The high immunoreactivity to TGF beta 1 or TGF beta 2 is positively related to the recurrence of HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Female; Hepatitis B; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Transforming Growth Factor beta

The ability of TGF-beta 1 (transforming growth factor-beta 1) to suppress growth factor induced proliferation of many cell types in vitro is well documented; however, TGF-beta 1 increases within a similar time frame as the hepatocyte mitogens HGF (hepatocyte growth factor), EGF (epidermal growth factor), and TGF-alpha (transforming growth factor-alpha) prior to hepatocyte proliferation during liver regeneration. This has raised the issue that TGF-beta 1 may have effects on hepatocytes additional to mito-inhibition and that these effects may be relevant to the regenerative process. To this end, we examined the effect of TGF-beta 1 on both the mitogenesis and the motility of growth factor stimulated primary rat hepatocytes and the hepatoblastoma cell line HepG2 in vitro. TGF-beta 1 significantly enhanced the chemotactic motility of EGF or TGF-alpha, and not HGF, stimulated hepatocytes on a collagen I substratum. TGF-beta 1 was not chemotactic when added alone and decreased the DNA synthesis of all hepatocyte cultures to near control levels. HepG2 cells were chemotactic toward HGF, EGF, and TGF-beta 1 alone and displayed an additive chemotactic response when TGF-beta 1 was added to either HGF or EGF. Additionally, HepG2 cells were refractory to the growth stimulatory effects of HGF or EGF and the growth inhibitory effects of TGF-beta 1. Hepatocytes plated onto other collagen-containing substrates (collagen IV, Matrigel, or ECL, an entactin-collagen IV-laminin matrix), but not on fibronectin or laminin alone, also displayed enhanced EGF stimulated motility by TGF-beta 1. The data indicate that an additional, novel role for TGF-beta 1 during liver tissue remodeling following PHx may include the synergistic enhancement EGF stimulated hepatocyte motility responses, and this enhancement is observed only on collagen-containing extracellular matrices.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Movement; Collagen; DNA; Drug Synergism; Epidermal Growth Factor; Extracellular Matrix; Hepatocyte Growth Factor; Liver Regeneration; Neoplasm Metastasis; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression.

Topics: Carcinoma, Hepatocellular; Cell Division; DNA, Complementary; Down-Regulation; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Liver Neoplasms; Polyamines; Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; Spermidine Synthase; Transforming Growth Factor beta; Tumor Cells, Cultured

Our aim was to assess the relationship between transforming growth factor beta 1 (TGF-beta 1) and alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC).. Urinary TGF-beta 1 and serum AFP were determined in 123 HCC patients, 50 patients with chronic liver disease (CLD), and 50 healthy controls.. Both TGF-beta 1 and AFP levels were higher in HCC patients than in CLD patients or controls (each, P = 0.0001). There was a negative correlation between TGF-beta 1 and logAFP (r = -0.196, P = 0.029). Multivariate analysis indicated that TGF-beta 1 and AFP were associated with an increased risk of HCC development. By receiver-operating characteristic curve analysis, determination of AFP and TGF-beta 1 in parallel significantly increased the sensitivity and diagnostic accuracy in detecting HCC.. Increased urinary TGF-beta 1 level can be used as a complementary marker to AFP for detection of HCC with low AFP production.

Topics: Adult; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Case-Control Studies; Female; Humans; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Predictive Value of Tests; ROC Curve; Sensitivity and Specificity; Transforming Growth Factor beta

To evaluate the diagnostic application of urinary transforming growth factor-beta1 (TGF-beta1) and serum alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC), TGF-beta1 and AFP were determined in 94 patients with cirrhotic HCC and in 94 sex- and age-matched patients with cirrhosis alone. TGF-beta1 and AFP levels in HCC were higher than in cirrhosis alone (P = 0.0001). There is an inverse correlation between TGF-beta1 and log AFP (r = -0.292, P = 0.004). Multivariate analysis indicated that TGF-beta1 and AFP were closely associated, in a dose-related fashion, with the development of HCC. Receiver-operating characteristic (ROC) curves were used to determine the optimal cut-off values of TGF-beta1 (50 microg g(-1) creatinine) and AFP (100 ng ml(-1)). Both TGF-beta1 and AFP showed a high specificity (99%) and positive likelihood ratio. The sensitivity was 53.1% for TGF-beta1 and 55.3% for AFP. The determination of both markers in parallel significantly increased the diagnostic accuracy (90.1%) and sensitivity (84%), with a high specificity (98%) and positive likelihood ratio (40.0). In conclusion, TGF-beta1 and AFP are independent tumour markers of HCC and may be used as complementary tumour markers to discriminate HCC from cirrhosis.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Evaluation Studies as Topic; Female; Humans; Liver Neoplasms; Male; Middle Aged; ROC Curve; Transforming Growth Factor beta

Primary cultures of rat hepatocytes, rat (FAO) and human (HepG2) hepatoma cells were studied by immunocytochemistry for expression of transforming growth factor (TGF)-beta, for the release of TGF-beta into the medium, and generation of hepatocellular apoptosis by the respective cell-conditioned media.. Using the alkaline-phosphatase anti-alkaline-phosphatase technique, intense TGF-beta immunostaining was shown in all cell types. The cytokine is released almost entirely in the latent form into the culture medium; only the FAO-cells had a substantial fraction of bioactive TGF-beta in the native (unacidified) culture fluid. Exposure of hepatocytes with the respective cell-conditioned media in the activated, but not in the native form (except for FAO-cell media), induced severe detrimental effects as evidenced by: (i) gross morphological alterations, (ii) functional impairment (reduction of WST-1 test, detachment of cells, lactate dehydrogenase increase in the medium), and (iii) generation of apoptosis. The latter phenomenon was confirmed by an increase of internucleosomal DNA fragments, positive TUNEL reaction, and intense binding of the fluorochrome Hoechst 33342 to fragmented nuclei. All these effects, which were mimicked by addition of recombinant human TGF-beta 1, were almost entirely antagonized by pre-incubation of the conditioned media with latency associated peptide. In contrast to hepatocytes, both types of hepatoma cells were completely resistant to the multiple actions of TGF-beta and activated conditioned media.. It is concluded that hepatocytes might have the ability to induce autocrine, TGF-beta-mediated apoptosis, whereas hepatoma cells, because of their TGF-beta resistance, might generate TGF-beta-mediated peritumorous apoptosis of hepatocytes in a paracrine way, which could facilitate their expansion in situ. Both mechanisms, however, are critically dependent on extracellular TGF-beta activation.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Drug Resistance; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Male; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transforming Growth Factor beta

To assess the clinical relevance of transforming growth factor-beta 1 (TGF-beta 1) in the urine of patients with hepatocellular carcinoma (HCC), TGF-beta 1 was measured, by radioimmunoassay, in 140 patients with HCC, 50 cirrhotic patients, 30 patients with chronic active hepatitis, and 50 healthy controls. The results indicate that there were significantly increased urinary TGF-beta 1 levels in patients with HCC. Raised TGF-beta 1 levels were associated, in a dose-related fashion, with increased risk for development of HCC (odds ratio, 1.05, 95% confidence interval, 1.03-1.07). HCC patients with raised TGF-beta 1 levels had shorter survival than those with normal TGF-beta 1 levels (p = 0.038). TGF-beta 1 levels decreased after successful anticancer therapy (p < 0.0001). There was an inverse correlation between TGF-beta 1 and serum alpha-fetoprotein (AFP) (r = -0.199, p < 0.04). Receiver operating characteristics (ROC) curve analysis indicated that parallel determination of TGF-beta 1 and AFP significantly increased the sensitivity and diagnostic accuracy, with a high specificity. In conclusion, raised urinary TGF-beta 1 was associated with HCC development. It is a predictor of poor prognosis, and a tumor marker for diagnosis and therapeutic follow-up of HCC.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Disease Progression; Female; Humans; Liver Neoplasms; Male; Middle Aged; Prognosis; Reference Values; ROC Curve; Survival Analysis; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a potent inducer of programmed cell death in liver as well as some hepatoma cell lines. To explore the mechanism by which TGF-beta induces apoptosis, we investigated the role of caspase family proteases in the apoptotic death of a human hepatoma cell line, Hep3B. We showed that TGF-beta-induced apoptosis was blocked by expression of the cowpox virus protein CrmA, a serpin-like pseudosubstrate for some of the caspase family proteases. CrmA expression, however, did not affect TGF-beta-induced regulation of promoter activities of the cyclin A and plasminogen activator inhibitor type I genes. These results indicate that CrmA inhibits a step specific for the apoptotic effect of TGF-beta. In addition to CrmA, a tripeptide caspase-protease inhibitor, z-Val-Ala-Asp-fluoromethylketone could also suppress TGF-beta-induced apoptosis in a dose-dependent manner. In TGF-beta-treated Hep3B cells, we observed a specific degradation of the catalytic subunit of DNA-dependent protein kinase, which was previously shown to be a substrate of caspase-3 but not several other members of the caspase family. This degradation was not seen in Hep3B cells transfected with CrmA nor in Hep3B cells pretreated with the tripeptide caspase inhibitor. Our study indicates a requirement of caspase family proteases in TGF-beta-induced apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cowpox virus; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Activated Protein Kinase; DNA-Binding Proteins; Humans; Liver Neoplasms; Nuclear Proteins; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Receptors, Transforming Growth Factor beta; Serpins; Transforming Growth Factor beta; Tumor Cells, Cultured; Viral Proteins

Transforming growth factor-beta (TGF-beta) is a pluripotent regulatory molecule, found in at least five different isoforms. It is produced in many different organs. In the liver, TGF-beta is expressed in non-parenchymal cells, but not in hepatocytes. This growth factor is known to induce fibrosis in the course of a variety of pathologic processes. Recently, TGF-beta has also been identified in hepatocellular carcinoma (HCC) cells, and the suggestion has been made that this growth factor may play a role in hepatocarcinogenesis. In this study, we report the findings of immunohistochemical stains for TGF-beta, performed on paraffin sections of 14 human HCCs of the usual type and 11 examples of the fibrolamellar variant (FLC). TGF-beta was detected in tumor cells of 3 HCCs (21%) and 9 FLCs (82%). Compared with the HCCs, the FLCs displayed a more diffuse and intense staining pattern for TGF-beta. Our findings suggest that lamellar fibrosis, which is a histologic hallmark of FLC, may be due to the action of TGF-beta produced by tumor cells.

Topics: Carcinoma, Hepatocellular; Fibrosis; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Transforming Growth Factor beta

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant and suspected human carcinogen, is believed to act by altering expression of a number of genes involved in cell growth control. In a previous study, we demonstrated the transcriptional down regulation of transforming growth factor-beta2 (TGF-beta2) by TCDD. To identify the region of the TGF-beta2 promoter necessary for the observed down-regulation by TCDD, we studied the effect of TCDD on a series of TGF-beta2 gene promoter deletions ranging from 1391 to 64 base pairs upstream of the transcription start site. We demonstrate that the effect of TCDD on TGF-beta2 promoter activity is localized to the TATA box sequence. The effect of TCDD on TGF-beta2 transcription is dose-dependent, exhibiting saturation kinetics maximal by 10 nM. Time course experiments show that the maximum decrease (30-50%) in promoter activity by a 10 nM dose of TCDD is complete by 24 hr. DNAase I footprinting and gel shift experiments indicate a single shifted protein complex in this region that we conclude is the transcription initiation complex. TCDD does not appear to significantly alter this complex suggesting that gross alterations in the proteins associated with this sequence do not occur. Treatment of the cells with various protein kinase inhibitors had no significant effect on the TCDD-induced decrease in promoter activity with the exception of genistein, a tyrosine kinase inhibitor. Genistein reverses the effect of TCDD on TGF-beta2 promoter activity back to control levels. Thus, TCDD can modulate gene transcription by acting at the transcription initiation complex via a tyrosine kinase-dependent pathway.

Topics: 2-Aminopurine; Binding, Competitive; Carcinoma, Hepatocellular; DNA Footprinting; Dose-Response Relationship, Drug; Electrophoresis; Humans; Liver Neoplasms; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Protein-Tyrosine Kinases; TATA Box; Time Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

To define the cis-acting elements involved in the regulation of the murine vitronectin (Vn) gene in inflammation, the 5'-flanking region was isolated, fused to the luciferase reporter gene, and the basal and interleukin 6 (IL-6)-stimulated transcriptional activity was tested in transfection experiments using Hep3B cells. Treatment with IL-6 induced this construct by more than 20-fold, whereas the corresponding 5'-flanking region of the human Vn gene was not stimulated. Transfection studies using murine Vn constructs with serial 5'-deletions revealed that two sequences were important in the IL-6 response, and specific mutations in both sequences abolished the response. A 2-base pair mutation converted the human sequence to that of a murine IL-6 responsive element and partially conveyed IL-6 inducibility. In contrast, transforming growth factor beta stimulated the human construct and the endogenous Vn gene in human Hep3B cells in a dose-dependent manner, whereas the murine construct was not responsive. The transforming growth factor beta responsive region was localized to a 30-base pair fragment with little homology to the murine sequence. These studies reveal that the structural basis for the differential regulation of the human and murine Vn genes resides in the differences in promoter sequence.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Line; DNA Primers; Gene Expression Regulation; Genomic Library; Humans; Interleukin-6; Kinetics; Liver; Liver Neoplasms; Luciferases; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Sequence Homology, Nucleic Acid; Species Specificity; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Vitronectin

Transforming growth factor-beta1 (TGF-beta1) can induce rapid growth arrest and apoptosis in hepatic cells. Its growth suppressive effects appear to be linked to decreased phosphorylation of the protein product of the retinoblastoma gene, pRb. To characterize the role of pRb in apoptosis, we examined endogenous retinoblastoma gene (Rb) expression following treatment with TGF-beta1, okadaic acid, or antisense Rb S-oligonucleotides in cultured primary rat hepatocytes and human hepatoma HuH-7 cells. We also investigated the effects on apoptosis of Rb overexpression following transfection with vectors containing wild-type Rb in HuH-7 cells. Our results indicated that transfection with Rb antisense S-oligonucleotides blocked the expression of pRb in cultured primary hepatocytes and induced apoptosis. Treatment of HuH-7 cells with TGF-beta1 inhibited expression and phosphorylation of pRb, and also induced apoptosis. Furthermore, 93% of viable preapoptotic cells were arrested in the G1 phase of the cell cycle. Incubation with the phosphatase inhibitor okadaic acid maintained pRb in its phosphorylated state, and resulted in significant apoptosis. Overexpression of wild-type Rb inhibited TGF-beta1 induced apoptosis in HuH-7 cells. In contrast, overexpression of transcription factor E2F-1, a known target for the activity of pRb, caused significant apoptosis. However, coexpression of Rb suppressed E2F-1 induced apoptosis in HuH-7 cells. Our results suggest that inhibition of pRb expression is associated with hepatocyte apoptosis. Furthermore, E2F-1 appears to be a target in the pathway through which pRb modulates the apoptotic threshold in hepatic cells. Finally, the data suggest that these cells exit the cell cycle during the G1 phase before progressing into apoptosis and pRb may be a negative regulator of this process.

Topics: Animals; Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Proteins; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; G1 Phase; Humans; Liver; Liver Neoplasms; Male; Molecular Sequence Data; Oligonucleotides, Antisense; Phosphorylation; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; Transcription Factor DP1; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Topics: Amino Acid Sequence; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line; Chlorocebus aethiops; Genes, Dominant; Humans; Liver Neoplasms; Lymphoma, T-Cell, Cutaneous; Molecular Sequence Data; Point Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Sequence Homology, Amino Acid; Signal Transduction; Skin; Skin Neoplasms; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; Cell Division; Cell Line; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP2B1; Dihydrolipoamide Dehydrogenase; Glucuronosyltransferase; Glutathione Transferase; Humans; Isoenzymes; Kinetics; Liver Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

In order to elucidate collagen metabolism in hepatocellular carcinoma (HCC) tissue, we compared levels of different potential markers of collagen metabolism and plasma transforming growth factor-beta 1 in patients with HCC and in patients with liver cirrhosis. Serum levels of prolyl hydroxylase and the tissue inhibitor of metalloproteinase-1 in patients with HCC were significantly higher than those in patients with liver cirrhosis and increased with the size of the HCC tumour, whereas the serum levels of procollagen type III propeptide and type IV collagen 7S domain were similar in the two groups. In HCC, the increased plasma transforming growth factor-beta 1 levels were closely correlated with serum levels of prolyl hydroxylase and the tissue inhibitor of metalloproteinase-1. These findings suggest that, in HCC tissue, the intracellular biosynthesis of collagen is enhanced, whereas the secretion of procollagen is disturbed and the degradation of collagen is suppressed by the excess production of the tissue inhibitor of metalloproteinase-1. The results also suggest that plasma transforming growth factor-beta 1 plays an important role in the altered metabolism of collagen in HCC.

Topics: Aged; Biomarkers; Carcinoma, Hepatocellular; Collagen; Female; Glycoproteins; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Peptide Fragments; Procollagen; Procollagen-Proline Dioxygenase; Protease Inhibitors; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

Insulin-like growth factors initiate tyrosyl phosphorylation of the insulin receptor substrate I (IRS-I) protein and activate multiple signaling pathways essential for liver growth. This gene has been found to be up-regulated in human hepatocellular carcinomas (HCCs), and overexpression of IRS-1 in NIH 3T3 cells leads to malignant transformation with activation of the mitogen-activated protein kinase cascade. To explore another possible role of IRS-I in hepatocarcinogenesis, we examined the capability of transforming growth factor beta1 (TGF-beta1), a known negative regulator of hepatocyte growth, to induce programmed cell death in the context of IRS-I overexpression. Hep3B HCC cells were stably transfected with a retroviral vector containing the IRS-I gene. The overexpressed IRS-I protein was highly tyrosyl phosphorylated following insulin/insulin- like growth factor I stimulation and led to constitutive activation of downstream signal transduction molecules such as phosphatidylinositol-3 kinase and mitogen-activated protein kinase. Although parental Hep3B cells were sensitive to apoptosis, the Hep3B-IRS-I-transfected cells acquired resistance to TGF-beta1-induced programmed cell death. Our investigations suggest that IRS-I-mediated signals may act as survival factors and protect against TGF-beta1-induced apoptosis in HCC; this phenomenon may contribute to hepatic oncogenesis.

Topics: Apoptosis; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Hepatocellular; Enzyme Activation; Humans; Insulin Receptor Substrate Proteins; Liver Neoplasms; Molecular Sequence Data; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Tyrosine

We have demonstrated previously that SV40 T antigen and serum regulate the length of G1 in exponentially growing NIH-3T3 cells in part by inhibiting density dependent negative cell cycle regulation. In these studies it was suggested that T antigen positively regulated G1 in a density independent manner as well. In this report we show that, 24 h after treatment, TGF-beta 1 perturbs the cell cycle of exponentially growing fibroblasts in a manner similar to T antigen. However, prior to 24 h, TGF-beta 1 produced a negative response, elongating the G1 phase of the cell cycle that was followed by a positive response, both of which were density independent. This biphasic response was measured between 0 and 12 h post-treatment and was relative to responses from serum. This switch from an early inhibitory effect to a late stimulatory effect was associated with changes in Rb phosphorylation, the timing and magnitude of which indicated that Rb may be directly regulating TG1 rather than reporting changes in the population. This is further substantiated by abrogation of the inhibitory effect by expression of wild-type SV40 T antigen and retention of the effect in cells that express an Rb-binding mutant of T antigen (K1). The biphasic regulatory effects of TGF-beta 1 were also displayed in WI-38 and IMR-90 human fibroblasts. This suggests that this biphasic effect is a property of fibroblasts.

Topics: 3T3 Cells; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Count; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Transformed; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; G1 Phase; Humans; Kinetics; Mice; Microtubule-Associated Proteins; Oncogene Proteins; Periodicity; Retinoblastoma Protein; S Phase; Time Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins

Hepatocellular carcinoma possesses androgen receptor but its true role is not known. This study aimed to investigate the effect of an anti-androgen cyproterone acetate on the growth of androgen receptor-positive hepatocellular carcinoma.. Androgen receptor-positive human hepatocellular carcinoma cells (KYN-1/SM-10) were subcutaneously transplanted into male nude mice. When the tumor size was about 10 mm, animals were subcutaneously administered cyproterone acetate (0.1 mg/day and 0.8 mg/day) or solvent alone for 21 days. Animals were serially sacrificed for measurements of testicular weight, tumor size, and cytosolic and nuclear androgen receptor levels in tumor. Proliferating cell nuclear antigen, transforming growth factor-alpha, and transforming growth factor-beta 1 in tumor were investigated immunohistochemically, using monoclonal antibodies. Apoptotic activity was also studied by the in situ DNA nick end labeling method.. Cyproterone acetate depressed testicular weight, suppressed tumor growth, and decreased both cytosolic-androgen receptor and nuclear-androgen receptor levels dose-dependently. Numbers of proliferating cell nuclear antigen-positive cells were decreased transiently with the low dose but continuously with the high dose of cyproterone acetate. Transforming growth factor-alpha expression was not influenced by cyproterone acetate, but the high dose of cyproterone acetate induced higher expression of transforming growth factor-beta 1, associated with increased numbers of apoptotic tumor cells, peaking on day 3.. The inhibition of growth of androgen receptor-positive hepatocellular carcinoma with cyproterone acetate in male nude mice could be due to G1-phase cell cycle arrest, and to some extent apoptosis induced by increased synthesis of transforming growth factor-beta 1 in tumor, caused by the direct action of cyproterone acetate through androgen receptors, as well as decreased testosterone levels in blood due to cyproterone acetate-induced testicular atrophy.

Topics: Androgen Antagonists; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Division; Cyproterone Acetate; DNA, Neoplasm; Humans; Immunohistochemistry; Injections, Subcutaneous; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Organ Size; Proliferating Cell Nuclear Antigen; Receptors, Androgen; Testis; Transforming Growth Factor beta; Tumor Cells, Cultured

Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.

Topics: Angiogenesis Inducing Agents; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Chelating Agents; Cobalt; Deferoxamine; Endothelial Growth Factors; Erythropoietin; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Liver Neoplasms; Lymphokines; Molecular Sequence Data; Neovascularization, Pathologic; Placenta Growth Factor; Platelet-Derived Growth Factor; Pregnancy Proteins; RNA, Messenger; RNA, Small Nuclear; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF-alpha, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF-beta family proteins. Affinity crosslinking gels using 125I-labeled TGF-beta showed a significant decrease in the TGF-beta cell-surface type II receptor in Hep40 cells, compared to the TGF-beta-sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c-myc, c-jun, and prothrombin genes, in contrast to the actions of TGF-beta 1 protein, which caused a decrease in the level of c-myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF-beta with a unique TGF-beta receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF-beta as compared to vitamin K suggest that these two growth inhibitors act through differing pathways.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Prothrombin; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Rats; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Vitamin K

A characteristic defect occurs in rat and human hepatocellular carcinoma (HCC) resulting in a loss of function of the vitamin K-dependent enzyme gamma-glutamyl-carboxylase in the tumor but not in the underlying liver. This causes the secretion of elevated levels of the immature or des-gamma-carboxylated form of prothrombin, which is used as a marker of HCC. We investigated whether, using the defined conditions of growing HCC cell lines in tissue culture, addition of the naturally occurring vitamins K1 or K2 or the synthetic vitamin K3 could influence the secretion of immature prothrombin. We found that vitamins K1, K2 and K3 all suppressed the secretion of immature prothrombin into the tissue culture medium. Vitamins K2 and K3 were also found to inhibit growth of the HCC cell line, in an apparently nontoxic and reversible manner. The influence of the vitamins K on the expression of some genes related to vitamin K action was examined and compared with that of another growth inhibitor, TGF beta 1 protein. The vitamins K were found to increase the expression of prothrombin and carboxylase messenger RNA and c-myc messenger RNA, but had no effects on the expression of TGF beta 1 messenger RNA. By contrast, TGF beta 1 increased TGF beta 1 messenger RNA levels, but had no effects on the other genes, suggesting a different pathway. The previously studied vitamin K3-mediated inhibition of growth was antagonized by the addition of catalase to the culture medium, but the inhibitory effects of vitamin K2 were not antagonized.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biomarkers; Biomarkers, Tumor; Carbon-Carbon Ligases; Carcinoma, Hepatocellular; Cell Division; Gene Expression; Genes, myc; Growth Inhibitors; Humans; Ligases; Liver Neoplasms; Protein Precursors; Prothrombin; Transforming Growth Factor beta; Tumor Cells, Cultured; Vitamin K

A new cell line (Hep 3B-TR), which is resistant to growth-inhibition by transforming growth factor beta 1 (TGF-beta 1) up to 10 ng/ml (400 pM), was isolated from parental Hep 3B human hepatoma cells, which are sensitive to growth-inhibition by TGF-beta 1. In the presence of TGF-beta 1 (1 to 10 ng/ml), the growth of the parental cell line (Hep 3B-TS) was inhibited by more than 95%. Under the same conditions, the growth rate of the resistant clone (Hep 3B-TR) however, was identical in the presence or absence of TGF-beta 1 and was almost the same as that of the Hep 3B-TS cells in the absence of TGF-beta 1. Affinity crosslinking with 5 pM 125I-labeled TGF-beta 1 showed that the TGF-beta 1 receptors type I (TGF-beta RI) and type II (TGF-beta RII) were not present on the cell surface of the Hep 3B-TR cells, whereas they were present on the sensitive HEP 3B-TS cells. Hep 3B-TS cells had detectable TGF-beta RII mRNA, which was not found in Hep 3B-TR cells. RNA analysis showed different effects on the expression of TGF-beta 1, c-fos, c-myc, and protein disulfide isomerase (PDI) genes in the two cell lines in response to TGF-beta 1 protein. Addition of TGF-beta 1 (1 ng/ml) strongly increased the expression of TGF-beta 1 mRNA in Hep 3B-TS cells, but not in Hep 3B-TR cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carcinoma, Hepatocellular; Cell Division; Cross-Linking Reagents; Drug Resistance; Gene Expression; Genes, fos; Genes, myc; Humans; Isomerases; Liver Neoplasms; Protein Disulfide-Isomerases; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-Beta 1 (TGF-beta 1) is an important mediator of control of liver cell proliferation and replication. The aim of the current study was to compare TGF-beta 1 gene expression, protein synthesis, and cell membrane receptors in normal liver, cirrhotic nodules, and neoplastic human livers. Five surgical resections for metastasis in an otherwise normal liver and 25 resections for hepatocellular carcinoma with cirrhosis were included in this study. Messenger RNA (mRNA) and TGF-beta 1 protein were detected on serial tissue sections of normal, cirrhotic, and tumoral livers using in situ hybridization and immunohistochemistry. TGF-beta 1 type II receptors were detected on tissue sections using immunohistochemistry. In normal livers, TGF-beta 1 mRNA and protein were not significantly expressed. In cirrhotic nodules, a few sinusoidal cells and mesenchymal cells of fibrous septa displayed TGF-beta 1 mRNA. By immunohistochemistry, protein was detected in the extracellular matrix along the fibrous septa. Hepatocytes from normal and cirrhotic livers did not express TGF-beta 1. In contrast, the cytoplasm of hepatocytes in neoplastic nodules showed intense staining for TGF-beta 1 mRNA and protein. Although TGF-beta 1 receptor II was expressed on the plasma membrane of normal liver cells, tumoral hepatocytes no longer displayed membrane labeling but rather diffuse intracytoplasmic staining with perinuclear accumulation. This study suggests that the escape of tumoral hepatocytes from control of cell proliferation by TGF-beta 1, despite its overexpression by these cells, might be related to a defect in TGF-beta 1 receptor II processing on the liver cell membrane.

Topics: Carcinoma, Hepatocellular; Female; Humans; Immunohistochemistry; In Situ Hybridization; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Receptors, Transforming Growth Factor beta; Reference Values; Transforming Growth Factor beta

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in tumor progression by promoting angiogenesis or suppressing immune system. We reported previously that transforming growth factor-beta 1 (TGF-beta 1) is overproduced by human hepatocellular carcinoma (HCC) tissues and that plasma TGF-beta 1 levels are elevated in patients with HCC. In the present study, we investigated the relationship between plasma TGF-beta 1 levels and tumor vascularity as assessed by conventional celiac angiography in 17 patients with HCC. The plasma TGF-beta 1 level did not correlate with tumor size or underlying liver disease. However, we found that plasma TGF-beta 1 levels correlated positively with the tumor vascularity. These results suggest that excessive TGF-beta 1 production may contribute to tumor angiogenesis in HCC.

Topics: Aged; Angiography; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor beta

The pathway of growth inhibitory cellular signal transduction by transforming growth factor beta 1 is largely unknown. Although several cellular proteins have been shown to be involved in the pathway, others remain to be identified. To search for other involved proteins, differentially expressed genes were examined in two human hepatoma cell lines that were respectively sensitive and resistant to growth inhibition by TGF-beta 1. Two such genes (SB31 and SB16) were characterized and found to be 100% homologous to leucine-rich alpha 2-glycoprotein and the ribosomal protein S25, respectively. SB31 was coordinately expressed with the TGF-beta I type II receptor, implicating a possible interaction. Expression of both genes in different cell lines can be broadly correlated with the sensitivity of the cell lines to growth inhibition by TGF-beta 1. SB16 expression is strongly suppressed in rat liver regeneration after partial hepatectomy, suggesting that it may have a role in liver growth.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; DNA, Neoplasm; Drug Resistance; Gene Expression; Genomic Library; Glycoproteins; Humans; Liver Neoplasms; Liver Regeneration; Rats; Receptors, Transforming Growth Factor beta; Ribosomal Proteins; Sensitivity and Specificity; Signal Transduction; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immunoblot analysis of nuclei isolated from regenerating rat liver after 70% partial hepatectomy (PH). Levels were almost undetectable in quiescent 0-h livers but increased 15- to 60-fold 3 to 24 h post-PH, 105-fold at 30 h, and 20- to 50-fold at 60 to 72 h post-PH. Expression returned to near baseline levels at 18, 42, and 48 h post-PH. A similar pattern of Rb protein expression in the regenerating liver was observed by indirect immunofluorescence microscopy, with peak nuclear expression at 30 h post-PH. Rb-related proteins with apparent molecular masses of 300, 156, and 74 kDa were detected in regenerating liver using mAbs to the Rb protein. Their expression increased 6- to 8-fold during regeneration, and only p156 returned to baseline levels at 60 h post-PH. Rb and its related proteins were detected in cultured primary hepatocytes, and although total protein levels did not change appreciably, there was a dramatic shift from cytosol into nuclei through 96 h. The half-life of the Rb protein was determined to be 1.9 h in regenerating liver and 2.2 h in cultured primary hepatocytes. Rb protein abundance in synchronized HuH-7 human hepatoma cells was cell cycle dependent and exhibited peak nuclear expression during S phase. Rb protein was detected primarily in its hyperphosphorylated state during liver regeneration and through the cell cycle of the HuH-7 cells. In vivo administration of transforming growth factor beta 1, an inhibitor of DNA synthesis in regenerating liver, resulted in reduced expression of Rb as well as its protein partners, cell cycle-dependent kinase 4 and cyclin E. The results suggest that in the regenerating rat liver and in synchronized HuH-7 cells, expression of Rb protein is modulated in a cell cycle-dependent fashion, remains primarily in a hyperphosphorylated state, and exhibits a relatively short half-life. The inhibition of Rb protein expression by transforming growth factor beta 1 may be linked to its simultaneous suppression of cell cycle-dependent kinase 4 and cyclin E protein levels.

Topics: Animals; Blotting, Northern; Carcinoma, Hepatocellular; Cell Cycle; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; DNA; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver; Liver Regeneration; Male; Phosphorylation; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Retinoblastoma; Retinoblastoma Protein; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta 1 (TGF beta 1) is a multifunctional cytokine. The role of TGF beta 1 in hepatocarcinogenesis is still a matter of discussion. To assess the expression of TGF beta 1 in human liver tumors, the following study was conducted.. Formalin fixed, paraffin embedded sections of 50 hepatocellular carcinomas (HCC), 30 cholangiocellular carcinomas (CCC), 15 hepatocellular adenomas (HCA), 15 focal nodular hyperplasias (FNH), and 20 normal livers (NL) were immunostained with a monoclonal anti-TGF beta 1 antibody (clone TB-21). TGF beta 1 mRNA was measured in snap frozen tissue of 2 HCC, 3 HCA, 2 FNH, and 3 NL of the immunohistochemistry group using slot blot hybridizations. Total RNA was extracted, and slot blots were hybridized with 32P endlabeled TGF beta 1 oligonucleotides. TGF beta 1 mRNA was measured in cpm with a scintillation counter.. TGF beta 1 protein was strongly expressed in 14/15 FNH and 13/15 HCA, whereas it was scarcely detectable in 46/50 HCC and 25/30 CCC. All NL were moderately positive. The 2 HCC cases contained far less TGF beta 1 mRNA than the HCA, FNH, and NL examined here.. TGF beta 1 expression is markedly lower in malignant liver tumors than in benign tumors or NL. Therefore, TGF beta 1 down-regulation may be an important step in hepatocarcinogenesis. Additionally, TGF beta 1 immunostaining may be useful in distinguishing well-differentiated HCC from adenomas.

Topics: Adenoma; Carcinoma, Hepatocellular; Cholangiocarcinoma; Humans; Hyperplasia; Immunohistochemistry; In Situ Hybridization; Liver; Liver Neoplasms; Precancerous Conditions; RNA, Messenger; Transforming Growth Factor beta

Many kinds of human malignant tissue, including hepatocellular carcinoma (HCC), were reported to overexpress transforming growth factor-beta 1 (TGF-beta 1) gene. However, little work has been done on the circulating TGF-beta 1 in patients with malignant tumors.. Plasma TGF-beta 1 levels in patients with HCC (n = 26) were compared with those in patients with chronic hepatitis (CH) (n = 12) and cirrhosis (n = 11) and in normal subjects (n = 20) using an enzyme-linked immunosorbent assay system after acid/ethanol extraction.. The patients with HCC had significantly higher plasma TGF-beta 1 levels (19.3 +/- 19.5 ng/ml; mean +/- standard deviation [SD]) than those in normal subjects (1.4 +/- 0.8 ng/ml) and in patients with CH (3.0 +/- 3.1 ng/ml) and cirrhosis (3.7 +/- 2.1 ng/ml) (P < 0.01). Plasma TGF-beta 1 concentrations in the patients with cirrhosis were also significantly higher than those in the normal subjects (P < 0.05). The extracted plasma TGF-beta 1 from the patients with HCC had biologic activity according to a growth inhibitory assay using mink lung epithelial cells. No significant correlation was found between the plasma TGF-beta 1 levels in the patients with HCC and serum alpha-fetoprotein levels. After successful treatment for HCC, the amount of plasma TGF-beta 1 significantly decreased from 22.6 plus or minus 16.7 ng/ml (mean +/- SD) to 10.2 plus or minus 6.5 ng/ml (P < 0.05).. We demonstrated higher levels of plasma TGF-beta 1 in the patients with HCC than those in patients with chronic hepatitis and cirrhosis. Plasma TGF-beta 1 might be a candidate for a novel tumor marker for hepatocellular carcinoma.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Chronic Disease; Enzyme-Linked Immunosorbent Assay; Female; Hepatitis; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Transforming Growth Factor beta

Solid tumours comprise populations of cells whose behaviour is thought to be influenced by growth factors and the local oxygen environment. We investigated the effect of oxygen on steady state levels of transforming growth factor-beta 1 mRNA in cultured human hepatoma cells. Northern blot analysis demonstrated that hypoxia mediated an early and sustained induction of TGF-beta 1 mRNA above that seen at higher oxygen tensions. The data suggests that modulation of TGF-beta 1 expression by the local oxygen environment may be important in tumour development and progression.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Oxygen; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Human hepatoma cell lines (Hep 3B-TS, PLC/PRF/5, and Hep G2), sensitive to growth inhibition by transforming growth factor beta 1 (TGF-beta 1), express TGF-beta receptors type I, type II, and type III. We report that TGF-beta 1 protein selectively increased steady-state levels of the mRNA for the serine/threonine kinase receptor 1 (SKR1), a member of the TGF-beta superfamily receptor genes in these cells, whereas TGF-beta 1 had little effect on expression of the TGF-beta receptor type II gene. This increase of SKR1 mRNA in Hep 3B-TS cells could be detected by Northern blot analysis within 3 h of addition of TGF-beta 1 to the cells, and enhanced message levels peaked at 12 h as long as TGF-beta 1 was present in the culture medium. Hep 3B-TR cells which were resistant to TGF-beta 1 due to lack of both TGF-beta receptors type I and type II, expressed SKR1 mRNA, but it was not induced by TGF-beta 1 protein. The increased expression of SKR1 mRNA in the cells was actinomycin D-sensitive and was not dependent on new protein synthesis. The results indicate that TGF-beta 1 selectively induces SKR1 message at a transcriptional level by a positive regulator.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Division; Cycloheximide; Dactinomycin; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The basic approach in targeted gene delivery relies on the formation of a complex between a vector and a molecule that will be selectively internalized by the target cells. In the case of hepatocytes, asialoglycoproteins are convenient targeting molecules because of the high affinity and avidity of the hepatocyte galactose receptor. In this system, poly-L-lysine is cross-linked to an asialoglycoprotein, and the resulting conjugate is complexed with the expression vector (DNA). The electrostatic binding between DNA and poly-L-lysine-asialoglycoprotein ensures delivery of the intravenously injected complex to the liver, where it is subjected to endocytosis by hepatocytes. However, the poly-L-lysine-asialoglycoprotein complexes tend to be unstable, of limited solubility and of fixed carbohydrate content. For these reasons we searched for a simpler alternative. We exploited the known capacity of reducing sugars to be reductively coupled to the epsilon-amino groups in proteins and used lactose to obtain poly-L-lysine with "exposed" galactose. Glycosylation with sodium cyanoborohydride at high pH in borate buffer is a simple, reproducible procedure. The "lactosylated" poly-L-lysine has proved very stable, highly soluble and easily bound to plasmids. In a set of experiments we compared the asialofetuin-poly-L-lysine vector complexes with lactosylated poly-L-lysine vector complexes by transfecting hepatoma cells (HepG2) in culture. For these experiments we used a pRc/cytomegalovirus eukaryotic expression vector containing a mutant TGF-beta 1 complementary DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Asialoglycoproteins; Blotting, Northern; Carcinoma, Hepatocellular; Female; Gene Transfer Techniques; Genetic Vectors; Glycosylation; Lactose; Liver; Liver Neoplasms; Plasmids; Polylysine; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transfection; Transforming Growth Factor beta

Heterodimers of types I and II serine/threonine kinase receptor monomers compose the active receptor complex for ligands of the transforming growth factor beta family. Here we show that the genomic organization of coding sequences for the intracellular domain of a widely expressed type I serine/threonine kinase receptor is similar to that of the activin type II receptor gene. The genomic structure and cDNA clones indicate that poly(A) addition to alternative exons at each of three carboxyl-terminal coding exon-intron junctions may be a common feature of both type I and II receptor genes. The predicted products are monomers truncated at kinase subdomains VII, IX, and X which vary in kinase activity and potential serine, threonine, and tyrosine phosphorylation sites. These results suggest that combinations of variants that affect the signal-transducing intracellular kinase domain of both type I and II receptor monomers within the transforming growth factor beta ligand family may add to the heterogeneity of biological effects of individual ligands in the family.

Topics: Alternative Splicing; Amino Acid Sequence; Base Sequence; Carcinoma, Hepatocellular; Cell Line; Cloning, Molecular; Conserved Sequence; DNA Primers; DNA, Complementary; Gene Expression; Genetic Variation; Humans; Liver Neoplasms; Macromolecular Substances; Molecular Sequence Data; Poly A; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; RNA, Messenger; Sequence Homology, Amino Acid; Transforming Growth Factor beta; Tumor Cells, Cultured

Mutations affecting the p53 gene abrogate its tumor suppressor activity. It is, however, unclear whether such mutations can generate mutant p53 proteins with an intrinsic transforming ability. More importantly, the mechanism(s) by which they exert such activity is unknown. We report here that p53-deficient hepatoma cells (Hep3B) transfected with mutant p53-249ser (codon 249 Arg-->Ser) acquire a new phenotype with an increased in vitro survival and mitotic activity. However, such a phenotypic change is not sufficient to cause a major shift in the poor tumorigenic potential of these cells. This is apparently due to transforming growth factor beta 1-mediated apoptotic death of Hep3B cells which is not affected by the expression of p53-249ser.

Topics: Amino Acid Sequence; Animals; Apoptosis; Arginine; Base Sequence; Carcinoma, Hepatocellular; Cell Division; Cell Line; DNA Primers; Genes, p53; Humans; Liver Neoplasms; Mice; Mice, Nude; Mitosis; Mitotic Index; Molecular Sequence Data; Point Mutation; Polymerase Chain Reaction; Serine; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Overexpression of the multifunctional growth factor transforming growth factor-beta 1 (TGF beta 1) has been connected to numerous diseases in human. TGF beta 1 expression is largely governed by three AP-1 binding sites located in two different promoters of this gene. We have examined the ability of retinoid receptors to inhibit the activity of the two promoters (especially the promoter 1) by cotransfection assays in the hepatocellular carcinoma cell line HepG2. When the TGF beta 1 promoter activity is induced by 12-O-tetradecanoyl phorbol13-acetate (an activator of AP-1-controlled gene transcription), this activity can be strongly repressed by retinoic acid receptor-alpha (RAR alpha), RAR beta, or retinoid X receptor-alpha (RXR alpha) as well as other members of the nuclear receptor family. Repression was hormone dependent and a function of receptor concentration. Heterodimerization of RAR alpha or RAR beta with RXR alpha did not modify the inhibition activities of these receptors, indicating that heterodimer formation is not required for antagonizing of AP-1 activity. On further examining the anti-AP-1 activity of RXR alpha we observed that three different AP-1-controlled promoters (TGF beta 1, collagenase, and cFos) can be inhibited. Using gel shift assays, we demonstrated that RXR alpha inhibits Jun and Fos DNA binding and that 9-cis RA enhances this inhibition, suggesting that a mechanism involving direct protein-protein interaction between RXR and AP-1 components mediates the inhibitory effect observed in vivo. Transfection analyses with RXR alpha point mutations revealed that residues L422, C432, and, to a lesser extent, residues L418 and L430, are involved in ligand-induced anti-AP1 activity of RXR alpha in vivo. Thus both types of retinoid receptors can inhibit AP-1-activated promoters, including the TGF beta 1 gene promoter, via a mechanism that involves protein-protein interaction.

Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Carcinoma, Hepatocellular; Collagenases; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Genes, fos; Humans; Liver Neoplasms; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Suramin, a drug shown to inhibit the growth of some tumor cell types in vivo and in vitro, was found to strongly inhibit the proliferation of the human hepatoma cell line, HepG2, grown in either serum-supplemented medium or a serum-free, hormonally defined medium tailored for hepatoma cells. In parallel, suramin induced the expression of the 6.0-kilobase transcript of insulin-like growth factor II (IGF II) but had no significant effect on transforming growth factor beta 1 mRNA levels in these cells. The induction in the abundance of IGF II mRNA was posttranscriptionally regulated. The growth-inhibitory effect of suramin was not mediated through IGF II, since addition of IGF II directly to the medium mildly stimulated the growth of HepG2 cells in a dose-responsive manner. Treatment of cells with suramin for 24 h resulted also in increased albumin mRNA levels in both HepG2 cells and normal rat hepatocytes. Suramin's effect on albumin occurred only in cells in medium supplemented with serum and in freshly plated cells, i.e., cells that had not been in culture long enough to form their own extracellular matrix substratum. We hypothesize that, as for heparin, suramin can bind serum factor(s), adversely affecting the stability of albumin mRNA. Addition of either IGF I or IGF II directly to cells resulted in an increase in albumin mRNA in HepG2 cells after 4 days in culture, implicating a role for these factors in differentiation. Yet they showed no effect unless the cells were grown for 2 days in serum-supplemented medium and then switched to a hormonally defined medium. Thus, both mitogenic and differentiation effects of IGFs were observed, and the qualitative responses of the cells to IGFs were dictated by other variables. Neither suramin nor IGF II had an effect on total sulfation levels in cells or medium conditioned by HepG2 cells and rat hepatocytes, suggesting that they have few, if any, effects on glycosaminoglycan synthesis or the extent of sulfation. Therefore, at present, suramin's potent biological effects on the growth and differentiation of HepG2 cells and rat hepatocytes are clearly complex and mediated through as yet unclear mechanisms.

Topics: Animals; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Gene Expression; Glycosaminoglycans; Growth Substances; Humans; Insulin-Like Growth Factor II; Liver; Liver Neoplasms; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sensitivity and Specificity; Suramin; Transforming Growth Factor beta; Tumor Cells, Cultured

The growth of human hepatoma Hep 3B cells is potently inhibited by TGF-beta 1 (ID50 = 0.2 ng/ml, 8 pM). A mutant cell line was derived that was not inhibited in growth by TGF-beta 1 at 5 ng/ml (200 pM) and that lacked TGF-beta receptor type II (TGF-beta RII) gene. Transfection of the cloned cDNA for human TGF-beta RII to this mutant cell line restored receptor expression as well as the inhibition in growth by TGF-beta 1. In both wild-type and mutant cells stably transfected with TGF-beta RII cDNA, TGF-beta RII coimmunoprecipitated with TGF-beta receptor type I in the presence of ligand. These experiments provide direct evidence for the role of TGF-beta RII in the inhibitory effect of TGF-beta on growth and suggest that TGF-beta RII acts by means of a heteromeric surface complex with TGF-beta receptor type I.

Topics: Affinity Labels; Antibodies; Blotting, Southern; Carcinoma, Hepatocellular; Cell Division; Cell Membrane; Drug Resistance; Humans; Iodine Radioisotopes; Kinetics; Liver Neoplasms; Plasmids; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Molecular cloning of complementary DNAs (cDNA) whose expression products bind activin and transforming growth factor beta (TGF-beta 1 and -beta 2) suggests that transmembrane serine/threonine kinases constitute a new class of signaling molecules. A human liver cell cDNA which codes for a new serine/threonine kinase receptor (SKR1) was identified using degenerate oligonucleotide primers complementary to coding sequence for mouse activin and Caenorhabditis elegans daf-1 serine/threonine receptor kinase subdomains VI and VIII in the polymerase chain reaction. The deduced 509-amino acid product consisted of a cysteine-rich extracellular domain and a cytoplasmic serine/threonine kinase domain which are 10-20 and 40% homologous to the respective domains in the activin and transforming growth factor beta receptor kinases. Cells overexpressing SKR1 exhibited no increase in binding of activin, inhibin, TGF-beta 1, TGF-beta 2, or bone morphogenic factor type 2B. Except for its absence in bone and spleen, SKR1 exhibits a tissue expression pattern similar to the TGF-beta receptor II gene. Similarly, SKR1 is expressed in normal parenchymal cells, endothelial cells, fibroblasts, and tumor-derived epithelial cells. The expression pattern and lack of binding to prototypic members of the TGF-beta 1-5 branch of the TGF-beta superfamily suggests that SKR1 is potentially a receptor for a new member of the TGF-beta branch of the ligand superfamily.

Topics: Activins; Amino Acid Sequence; Animals; Base Sequence; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Cell Line; Cloning, Molecular; DNA, Neoplasm; Gene Expression; Growth Substances; Humans; Inhibins; Liver; Liver Neoplasms; Molecular Sequence Data; Moths; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proteins; RNA, Neoplasm; Sequence Homology, Amino Acid; Substrate Specificity; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The HuH-7 human hepatoma cell line was stimulated by IL-1 and IL-6 to increase the synthesis of acute-phase proteins, e.g. serum amyloid A (SAA), alpha 1 antichymotrypsin (ACT), alpha 1-protease inhibitor, alpha 1 acid-glycoprotein and haptoglobin, with the exception of the pentraxins (serum amyloid P and C-reactive protein). Haptoglobin and ACT were stimulated by IL-1 which has not been observed in some other hepatoma cell lines. The concentration of IL-1 required for stimulation of SAA was higher than that required for haptoglobin stimulation. IL-1 receptor antagonist was capable of inhibiting these responses and acted at a lower concentration to inhibit SAA than required to inhibit ACT or haptoglobin induction. Transforming growth factor beta (TGF beta) was also able to inhibit the response to IL-1 but had no effect on acute-phase protein responses to IL-6.

Topics: Acute-Phase Reaction; Animals; Carcinoma, Hepatocellular; Cell Line; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Liver Neoplasms; Serum Amyloid A Protein; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Hepatocytes respond to inflammatory stimuli by changing the synthesis and N-glycosylation of acute phase plasma proteins (APP). So far, interleukin (IL) 6, transforming growth factor beta (TGF beta), tumor necrosis factor alpha (TNF) and IL-1 have been found to control N-glycosylation patterns of APP. Cytokines either increased (type I) or decreased (type II) the ratio of bi-relative to more branched N-glycans on APP. In this study, we describe the effect of leukemia inhibitory factor (LIF), interferon gamma (INF gamma) and dexamethasone (dex) on production of alpha 1-protease inhibitor (PI) and alpha 1-antichymotrypsin (ACT) and on glycosylation of PI in the human hepatoma cell line HepG2. Cytokines and dex were used separately and in various combinations including also IL-6 and TGF beta. Production of the antiproteases was quantitated by immunoelectrophoresis of the proteins accumulated in the culture medium. Glycosylation pattern of PI was assessed by crossed immunoaffinity electrophoresis (CIAE) with Concanavalin A (Con A) as a ligand. The production of ACT and PI was increased by LIF, decreased by INF gamma and unaffected by dex. LIF and INF gamma each like IL-6, decreased PI-Con A reactivity while dex like TGF beta enhanced PI-Con A reactivity. Combination of dex with LIF yielded additive effects while combination of dex with either INF gamma, L-6 or TGF beta acted synergistically on PI-Con A reactivity. Combinations of multiple cytokines and dex produced additive, inhibitory or synergistic effects. The type of glycosylation profile of PI secreted by HepG2 cells depended on the composition and amounts of interacting cytokines and dex.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Carcinoma, Hepatocellular; Dexamethasone; Glycosylation; Growth Inhibitors; Humans; Interferon-gamma; Interleukin-6; Leukemia Inhibitory Factor; Liver Neoplasms; Lymphokines; Transforming Growth Factor beta; Tumor Cells, Cultured

The effects of insulin on cell growth control by transforming growth factor beta 1 (TGF-beta 1) in human hepatoma cell lines were studied. TGF-beta 1 inhibited cell growth and DNA synthesis of Hep3B cells but not that of HA22T/VGH cells. The cell cycle-dependent p34cdc2 kinase activity was inhibited by TGF-beta 1 in a dose-dependent manner in Hep3B cells. In contrast, the p34cdc2 kinase activity of HA22T/VGH cells was not regulated by TGF-beta 1. When insulin (10(-7) M) was added simultaneously with TGF-beta 1, we found that the inhibitory effects of TGF-beta 1 on cell growth and DNA synthesis in Hep3B cells was completely blocked and the p34cdc2 kinase activity of Hep3B cells was recovered after insulin administration. Thus, cell growth inhibition by TGF-beta 1 in Hep3B hepatoma cells can be antagonized by insulin and their interaction may play an important role in the tumor progression stage of hepatocarcinogenesis.

Topics: Carcinoma, Hepatocellular; CDC2 Protein Kinase; Cell Cycle; Cell Division; DNA, Neoplasm; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Humans; Insulin; Liver Neoplasms; Phosphorylation; Transforming Growth Factor beta; Tumor Cells, Cultured

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. Its activity in plasma increases in diverse thrombotic states. The large synthetic capacity of the liver make it a source of potentially large amounts of PAI-1. Because thrombin activity increases in association with thrombotic disorders and because specific binding sites for thrombin have been identified on hepatocytes, we characterized the effect of thrombin on hepatocyte PAI-1 production. Incubation of Hep G2 cells with human alpha-thrombin resulted in a dose- and time-dependent increase in the concentration of PAI-1 in conditioned media. This effect was inhibited completely by hirudin and by antithrombin III. Steady-state levels of both the 3.2-kb and 2.2-kb forms of PAI-1 mRNA increased after stimulation of the cells with thrombin, indicating that thrombin influences PAI-1 expression in Hep G2 cells at the pretranslational level. Incubation of Hep G2 cells with alpha-thrombin and either platelet lysates or purified transforming growth factor-beta (TGF-beta), both previously shown to augment hepatocyte PAI-1 expression, resulted in a synergistic increase in the concentration of PAI-1 in conditioned media. PAI-1 mRNA appeared to be synergistically increased as well. Thus, thrombin increases expression of both PAI-1 protein and mRNA in Hep G2 cells and exerts synergistic effects with TGF-beta. These results underscore the potential importance of inhibition of thrombin under conditions in which thrombolysis is induced pharmacologically.

Topics: Antithrombin III; Blood Platelets; Blotting, Northern; Carcinoma, Hepatocellular; Drug Synergism; Hirudins; Humans; Liver; Liver Neoplasms; Plasminogen Inactivators; RNA, Messenger; Thrombin; Transforming Growth Factor beta; Tumor Cells, Cultured

The effect of transforming growth factor beta 1 (TGF-beta 1) on human hepatoma cells (Hep 3B) was studied. Cell death was observed when the serum starved Hep 3B cells were exposed to a very low concentration of TGF-beta 1. The half-maximal cytocidal concentration of TGF-beta 1 was around 20 pM. Cell death began approximately 24 h following treatment, with more than 80% of the cells dying after 48 h. In contrast, the control cells, which were cultured in serum-free condition, still gradually proliferated. Furthermore, the cytocidal effect of TGF-beta 1 on Hep 3B cells was not altered by either cycloheximide or actinomycin D. It was discovered, using diphenylamine assay, that TGF-beta 1 induced DNA fragmentation in Hep 3B cells. Using gel electrophoresis, the fragmented DNA could be displayed, and showed a characteristic stepladder pattern. Thus, it appeared that TGF-beta 1 induced a particular pathway in Hep 3B cells in which de novo protein synthesis was not actively involved, but endogenous nuclease was activated which cleaves cellular DNA and induces cell death.

Topics: Carcinoma, Hepatocellular; Cell Death; DNA Damage; Humans; In Vitro Techniques; Liver Neoplasms; Nucleosomes; Transforming Growth Factor beta; Tumor Cells, Cultured

The blood level of erythropoietin (Epo) is often anomalously low in anemic patients with inflammatory or malignant diseases. Therefore, we studied effects of pure recombinant immunomodulatory peptides on Epo formation in cultures of the human hepatoma cell line, HepG2. Interleukin (IL)-1 beta, IL-1 alpha, and tumor necrosis factor alpha lowered Epo production with half-maximal inhibition at 2, 5, and 20 U/ml, respectively. IL-6, transforming growth factor beta 2 and interferon gamma did not inhibit. Furthermore, IL-1 beta (10 U/ml) proved to block Epo formation in isolated serum-free perfused rat kidneys. Proposedly, monokines play a role in the pathogenesis of Epo deficiency in various diseases.

Topics: Animals; Carcinoma, Hepatocellular; Erythropoietin; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-1; Kidney; Liver Neoplasms; Monokines; Rats; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

In vitro studies of the mouse erythroleukemia cell system have identified at least 300 agents capable of inducing differentiation by mechanisms that remain to be elucidated. We have recently begun to examine recombinant cytokines as possible agents in inducing differentiation of tumor cells, specifically, malignant cells resistant to cytotoxic drugs. One such cytokine, transforming growth factor-beta (TGF-B1), is a multifunctional peptide that exists in at least five different isoforms in vertebrate species. Recently, there has been a great deal of interest in the role of TGF-B1 as an important multifunctional growth regulator that induces cells of mesenchymal origin to divide while inhibiting the growth of nontransformed epithelial cells. In this study, we combined the effects of the differentiation agent hexamethylene bisacetamide and the inhibiting effects of TGF-B1 on a multidrug-resistant human liver hepatocellular carcinoma and demonstrated the synergistic interaction of these two agents; this synergy resulted in a cell death rate of 80%. These data support the concept of programmed cell death and suggest that drug-resistant tumor cells may be susceptible to the combination of cytokines and differentiating agents.

Topics: Acetamides; Carcinoma, Hepatocellular; Cell Death; Cell Differentiation; Cell Division; Doxorubicin; Drug Resistance; Drug Synergism; Drug Therapy, Combination; Humans; Liver Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

We have investigated the effect(s) of transforming growth factor (TGF)-beta 1 and interleukin (IL)-6 on the expression of fibrinogen and blood coagulation factors VII, IX, X mRNAs in a hepatoma cell line (Hep 3B). The results indicate that TGF-beta induces a decrease of the basal level of fibrinogen and factor VII mRNAs, but does not affect factor X expression. Furthermore, TGF-beta efficiently antagonizes the IL-6 induction of fibrinogen mRNA at late (12-48 h) but not early (6 h) times: this effect is apparently mediated by posttranscriptional mechanism(s). These findings, together with previously reported data on the inhibitory effect of TGF-beta on acute phase genes (e.g., ApoA1 and albumin), suggest a role for TGF-beta in the regulation of liver genes expression. The early stimulatory and late inhibitory effect exerted by IL-6 and TGF-beta respectively on fibrinogen mRNA level may play a role in the regulatory mechanism(s) of clot formation in a variety of conditions.

Topics: Carcinoma, Hepatocellular; Factor IX; Factor VII; Factor X; Fibrinogen; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

We measured the plasma transforming growth factor-beta (TGF-beta) concentration in 14 patients with human hepatocellular carcinoma (HCC) and 9 age-matched normal subjects using growth inhibition assay of mink lung epithelial cells. The calculated plasma TGF-beta concentration in the patients with HCC was 28.6 +/- 27.9 ng/ml (mean +/- SE), showing significant elevation compared with that in 9 normal subjects (5.3 +/- 3.3 ng/ml, P less than 0.01). In three cases, we could measure plasma TGF-beta levels before and after their treatment for HCC. The plasma TGF-beta levels decreased from 59.0 to 18.2 ng/ml after hepatic resection in one case, and from 24.0 to 10.7 ng/ml and from 12.4 to 3.4 ng/ml after transhepatic arterial embolization in the other two cases. These data indicate that plasma TGF-beta level is elevated in patients with HCC, probably due to release from HCC tissues.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Epithelial Cells; Female; Humans; Liver Neoplasms; Lung; Male; Middle Aged; Mink; Transforming Growth Factor beta

Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase-1 (TIMP-1). We show at mRNA and protein levels that TIMP-1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up-regulated by interleukin-6 (IL-6), transforming growth factor beta 1 and phorbol 12-myristate 13-acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL-6-stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP-1 mRNA in liver cells.

Topics: Carcinoma, Hepatocellular; Glycoproteins; Humans; Interleukin-6; Lipopolysaccharides; Liver Neoplasms; Metalloendopeptidases; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

As oligomers inhibited hepatoma growth in vitro. These cells have oligomer-specific binding sites. The oligomers were taken up by liver and have potential for hepatic growth modulation in intact animals.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line, Transformed; Electrophoresis, Polyacrylamide Gel; Humans; Liver; Liver Neoplasms; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-myc; Rats; Transforming Growth Factor beta

Topics: Base Sequence; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Molecular Sequence Data; Oligonucleotides, Antisense; Transforming Growth Factor beta; Tumor Cells, Cultured

Malignant cells in culture express elevated levels of transforming growth factor beta 1 (TGF-beta 1) mRNA and secrete an abundant amount of TGF-beta protein, but little is known about the production of TGF-beta in human malignant tissues in vivo. We estimated the levels of TGF-beta 1 mRNA expression by Northern hybridization and measured TGF-beta protein using a radioreceptor assay in tumor tissues surgically obtained from six patients with hepatocellular carcinoma (HCC). TGF-beta 1 mRNA was expressed at much higher levels in HCC tissues from all the cases compared with normal human liver, suggesting an association of the activated TGF-beta 1 gene transcription with hepatocarcinogenesis. The content of TGF-beta was 207 +/- 121 ng/g wet tissue in the HCC tissue, and it showed correlation with the level of TGF-beta 1 mRNA in the tissue (r = 0.69; P less than 0.05). An immunohistochemical study demonstrated that TGF-beta 1 staining could be observed in HCC cells. These observations suggest that human HCC strongly expresses TGF-beta 1 mRNA in vivo, leading to a high content of TGF-beta protein.

Topics: Aged; Blotting, Northern; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Male; Middle Aged; Radioligand Assay; RNA, Messenger; Transforming Growth Factor beta

Cellular processing of 125I-labeled transforming growth factor-beta 1 was investigated in the human hepatoma cell lines Hep G2 and Hep 3B. Binding of 125I-transforming growth factor-beta 1 to cell surface receptors was specific, saturable and calcium-independent. Both cell lines exhibited a single class of high-affinity (Kd = 2.2 x 10(-10) mol/L) binding sites (4.5 x 10(3) for the Hep G2 cell; 1.5 x 10(3) for the Hep 3B cell) for both human and porcine transforming growth factor-beta 1. Binding was temperature dependent, time dependent and pH dependent. Cell-bound 125I-transforming growth factor-beta 1 was removed by brief exposure to acidic medium (pH less than 4) but was converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Spontaneous dissociation of bound ligand over a 6 hr period at 4 degrees C was less than 10%. Disuccinimidyl suberate was used to covalently label 125I-transforming growth factor-beta 1 to cell-surface binding sites. Labeling of the ligand/receptor complexes was inhibited by unlabeled transforming growth factor-beta 1 but was unaffected by other growth factors. The radiolabeled complexes showed approximate molecular weights of 280,000, 85,000 and 65,000 when run on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell-bound 125I-transforming growth factor-beta 1 was internalized and degraded at 37 degrees C, and the products were released into the medium as trichloroacetic acid-nonprecipitable radioactivity. The lysosomotropic base chloroquine and the carboxylic ionphore monensin inhibited degradation and release of 125I-labeled products from the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding, Competitive; Carcinoma, Hepatocellular; Chloroquine; Cycloheximide; Humans; Hydrogen-Ion Concentration; Liver Neoplasms; Monensin; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with 35S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-beta (TGF-beta) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-alpha) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blood Platelets; Carcinoma, Hepatocellular; Epidermal Growth Factor; Humans; Liver Neoplasms; Plasminogen Inactivators; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta 1 (TGF-beta 1) is known to inhibit hepatocyte growth in vitro and in vivo. In this study, we analyzed the effect of TGF-beta 1 on alpha-fetoprotein (AFP) and albumin gene expression in HuH-7 human hepatoma cells. TGF-beta 1 inhibited cell growth in a dose dependent manner. The cellular secretion rate of AFP but not albumin was suppressed significantly by TGF-beta 1. TGF-beta 1 caused a significant reduction in the level of AFP mRNA. In contrast, the levels of albumin mRNA or beta-actin mRNA were not changed by TGF-beta 1. In transient transfection experiments, TGF-beta 1 resulted in selective repression of AFP promoter activity. These results suggest that TGF-beta 1 is one of the key factors involved in the differential regulation of the AFP gene and the albumin gene.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Division; Cell Line; Chloramphenicol O-Acetyltransferase; Humans; Liver Neoplasms; Recombinant Fusion Proteins; RNA, Messenger; Serum Albumin; Transfection; Transforming Growth Factor beta

Alpha-2-macroglobulin (alpha 2-M), a serum protease inhibitor that also binds cytokines, neutralized the inhibitory effect exerted by transforming growth factor-beta (TGF-beta) on IL-6-induced C-reactive protein (CRP) production by the human hepatoma cell line PLC/PRF/5. alpha 2-M was found to bind noncovalently with TGF-beta to form a complex that, upon acidification, released TGF-beta inhibitory activity as detected by IL-6-induced CRP production. Although alpha 2-M also binds IL-6, it did not alter IL-6-induced CRP production by the hepatoma cells. The interaction between alpha 2-M and TGF-beta may influence the production of acute-phase proteins by liver hepatocytes.

Topics: alpha-Macroglobulins; C-Reactive Protein; Carcinoma, Hepatocellular; Gene Expression Regulation; Humans; Interleukin-6; Liver Neoplasms; Protein Binding; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Liver cells are considered the principal source of plasma vitronectin. The human hepatoma cell line HepG2 produces vitronectin into its culture medium. In the current work we have analyzed the regulation of vitronectin by transforming growth factor-beta 1 (TGF beta 1) in this hepatoma cell line by Northern hybridization, polypeptide and immunoprecipitation analyses and compared the response to another TGF beta-regulated gene, plasminogen activator inhibitor (PAI-1). Rabbit antibodies raised against human plasma-derived vitronectin were used in immunodetection. Polypeptide and immunoprecipitation analyses of the medium and cells, as well as immunoblotting analysis of the cells and their extracellular matrices, indicated enhanced TGF beta 1-induced production and extracellular deposition of vitronectin. Accordingly, TGF beta 1 enhanced the expression of vitronectin mRNA at picomolar concentrations (2-20 ng/ml) as shown by Northern hybridization analysis. Comparison of the temporal TGF beta induction profiles of vitronectin and PAI-1 mRNAs showed that vitronectin was induced more slowly but the vitronectin mRNAs persisted longer. In addition, platelet-derived and epidermal growth factors had an effect on vitronectin expression, but it was of lower magnitude. TGF beta 1 enhanced the expression of PAI-1 but, unlike previous reports, epidermal growth factor did not have any notable effect on PAI-1 in these cells. The results indicate that TGF beta 1 is an efficient regulator of the production of vitronectin by HepG2 cells and that PAI-1 and vitronectin are not coordinately regulated. In addition, with affinity purified antibodies to vitronectin receptor, we observed strong enhancement of the alpha subunit of the receptor in response to TGF beta 1. These effects of TGF beta are probably involved in various processes of the liver where matrix induction and controlled pericellular proteolysis is needed, as in tissue repair.

Topics: Blood Proteins; Carcinoma, Hepatocellular; Cell Line; Cycloheximide; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Humans; Kinetics; Liver Neoplasms; Plasminogen Inactivators; Poly A; Receptors, Immunologic; Receptors, Vitronectin; RNA, Messenger; Transforming Growth Factor beta; Vitronectin

Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells. Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-1 and PLC/PRF/5. Heparin's inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF beta). HepG2 cells expressed high basal levels of mRNAs encoding IGF II and TGF beta that were inducible, through transcriptional and posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II and TGF beta, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects of insulin, glucagon, and growth hormone in combination with heparin; TGF beta was regulated primarily by the synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations regulated TGF beta posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-1 either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGF beta, whose mRNA synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and the biological responses to them.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Clone Cells; Growth Substances; Heparin; Heparitin Sulfate; Hormones; Humans; In Vitro Techniques; Insulin-Like Growth Factor II; Liver Neoplasms; Neoplasm Metastasis; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated the expression of transforming growth factor beta 1 (TGF-beta 1) mRNA in tumor tissues surgically removed from ten patients with hepatocellular carcinoma (HCC). All HCC tissues expressed TGF-beta 1 mRNA at different levels, indicating the presence of activated transcription of TGF-beta 1 gene in human HCC tissues in vivo. The level of TGF-beta 1 mRNA expression showed no relationship to main tumor size of plasma alpha-fetoprotein level. Some HCC tissues presenting a relatively low grade of histological differentiation showed the highest levels of TGF-beta 1 mRNA expression.

Topics: Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Male; Middle Aged; RNA, Messenger; Transforming Growth Factor beta

Phorbol ester treatment of HepG2, a human tumorigenic cell line, caused rapid morphological changes characterized by a flattening and spreading of the cells that coincided with a rapid inhibition of thymidine incorporation. Within 24 h, cell division was completely inhibited, suggesting the cells had entered a quiescent state. Continued incubation in the presence of phorbol esters resulted in the resumption of thymidine incorporation and cell division, but this coincided with only a partial down-regulation of PKC activity. Seventy-two hours of treatment was required to obtain down-regulation of greater than 80% of the PKC activity, but reversal of the inhibitory effects occurred between 24 and 48 h after the addition of phorbol esters, when a large proportion of the PKC activity was still present. Northern blot analysis of a number of transcripts showed that the steady-state levels of c-myc and transforming growth factor beta 1 (TGF-beta 1) messages increased only after 3 h of phorbol ester treatment and returned to normal levels after 24 h. C-fos, albumin, and alphafetoprotein messages were not affected, suggesting the differentiation state of the cells was not altered. Therefore, phorbol ester activation of PKC causes an inhibition of HepG2 cell growth initially, but this is unlike the promotion of differentiation seen in other systems. Partial down-regulation of PKC activity causes a reversal of the growth inhibition and the cells return to a normal growth rate. This effect is also clearly different from systems in which phorbol esters have been shown to have a mitogenic effect on cells.

Topics: Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Down-Regulation; Gene Expression Regulation; Humans; Liver; Liver Neoplasms; Phenotype; Phorbol Esters; Poly A; Protein Kinase C; Proto-Oncogene Proteins c-myc; Receptor, Insulin; Receptors, Cell Surface; Receptors, Somatomedin; RNA, Messenger; Thymidine; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) modified production of the major human acute phase reactant, C-reactive protein (CRP), induced by the inflammatory cytokines, IL-1 beta or IL-6. CRP mRNA accumulation in the hepatoma PLC/PRF/5 cell line was slightly more rapid, but of smaller magnitude in response to IL-1 beta (fourfold increase) than to IL-6 (10-fold increase); however, the amount of CRP protein accumulating in the culture medium was similar for both cytokines. TGF-beta at concentrations greater than or equal to 0.1 pg/ml inhibited the induced IL-1 or IL-6 CRP production; whereas concentrations less than 0.1 pg/ml slightly enhanced CRP synthesis. Addition of TGF-beta to the cultures up to 16 h after the PLC/PRF/5 cells were already exposed to IL-1 or IL-6 resulted in the cessation of CRP production. CRP mRNA accumulated in hepatoma cells treated with both TGF-beta and IL-6, although CRP protein synthesis was inhibited. A similar pattern of inhibition of CRP production by TGF-beta occurred when Hep 3B.2 cells were treated with a mixture of IL-1 and IL-6. Enhanced production of CRP was observed only when TGF-beta was added to the cells before the cytokine. This enhanced CRP response was sensitive to cycloheximide. TGF-beta added along with IL-6 inhibited the metabolic labeling of CRP with [35S]methionine; however, enhanced incorporation of [35S]methionine into CRP was observed when the cells were exposed to TGF-beta before IL-6 addition. Therefore, TGF-beta is potentially a potent regulator of CRP synthesis by hepatocytes at the post-transcriptional level.

Topics: Antibodies, Monoclonal; Blotting, Northern; C-Reactive Protein; Carcinoma, Hepatocellular; Humans; Interleukin-1; Interleukin-6; Molecular Weight; Recombinant Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

We have previously shown that changes in acute-phase protein glycosylation result from alterations occurring within hepatocytes as a result of regulation by cytokines, that the glycosylation patterns of proteins secreted by Hep 3B and Hep G2 cells respond differently to the crude mixtures of cytokines found in conditioned medium from LPS-stimulated monocytes, and that interleukin-6 (IL-6) causes increased concanavalin A (Con A) binding of alpha 1 protease inhibitor in Hep 3B cells and decreased Con A binding of this protein in Hep G2 cells. In the present study we found that transforming growth factor beta 1 (TGF-beta), like IL-6, led to secretion of forms of alpha 1-protease inhibitor with increased Con A binding in Hep 3B cells, and that IL-6 and TGF-beta in combination were additive. In contrast, in Hep G2 cells, TGF-beta had an effect opposite to that produced by IL-6, leading to secretion of forms of alpha 1-protease inhibitor with increased Con A binding. When employed in combination with IL-6. TGF-beta abolished the effect of that cytokine. These studies indicate that TGF-beta influences glycosylation of alpha 1-protease inhibitor in two human hepatoma cell lines in a manner that can be differentiated from that of IL-6. The identification of TGF-beta as a second defined cytokine capable of influencing glycoprotein glycosylation and the demonstration that the effect of one cytokine can be modulated by another cytokine support the view that changes in glycosylation of plasma proteins are mediated by combinations of cytokines.

Topics: alpha 1-Antitrypsin; Carcinoma, Hepatocellular; Concanavalin A; Drug Interactions; Glycosylation; Humans; Interleukin-6; Liver Neoplasms; Neoplasm Proteins; Protein Binding; Protein Processing, Post-Translational; Transforming Growth Factor beta; Tumor Cells, Cultured