cyclin-d1 and Carcinoma--Hepatocellular

Gain-of-function (GOF), the most malicious oncogenic activity of a cancer-promoting protein, is well illustrated to three hotspot p53 mutations at R248, R175, and R273 with distinct molecular mechanisms. Yet, less is known about another hotspot p53 mutant, R249S (p53-R249S). p53-R249S is the sole hotspot mutation in hepatocellular carcinoma (HCC) that is highly associated with chronic hepatitis B virus (HBV) infection and dietary exposure to aflatoxin B1 (AFB1). Its GOF is suggested by the facts that this mutant is associated with earlier onset of HCC and poorer prognosis of cancer patients and that its overexpression drives HCC proliferation and tumorigenesis. By contrast, simply knocking in this mutant in normal mice did not show apparent GOF activity. Hence, the GOF activity for p53-R249S and its underlying mechanisms have been elusive until recent findings offered some new insights. This review will discuss these findings as well as their clinical significance and implications for the development of a strategy to target multiple molecules as a therapy for p53-R249S-harboring HCC.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cyclin D1; Gain of Function Mutation; Humans; Liver Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Proto-Oncogene Proteins c-myc; Tumor Suppressor Protein p53

Some studies investigated the association between CCND1 rs9344 polymorphism and hepatocellular carcinoma (HCC) risk. However, the results were inconclusive. Thus, we did a meta-analysis to determine this relationship.. Relevant studies were systematically searched using the PubMed, CNKI, and EMBASE databases. The strength of the association was calculated with the odds ratio (OR) and respective 95% confidence intervals (Cis).. We investigated the association between CCND1 rs9344 polymorphism and HCC risk in the dominant models. The result of this meta-analysis showed that CCND1 rs9344 polymorphism did not significantly associated with HCC risk (OR = 1.09; 95% CI 0.88-1.34). In the stratified analysis by ethnicity, we found that this polymorphism was significantly associated with HCC risk in Caucasians (OR = 1.55; 95% CI, 1.05-2.29). However, we did not find any significant association between this polymorphism and HCC risk in Asians (OR = 0.91; 95% CI, 0.71-1.18).. This meta-analysis suggested that CCND1 rs9344 polymorphism might be associated with the risk of HCC among Caucasians.

Topics: Alleles; Asian People; Carcinoma, Hepatocellular; Case-Control Studies; Cyclin D1; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Liver Neoplasms; Odds Ratio; Polymorphism, Single Nucleotide; Publication Bias; Risk Assessment; Risk Factors; White People

Cyclin D1 (CCND1) is a key protein involved in cell-cycle regulation, and the CCND1 G870A polymorphism is associated with many types of malignancy. Studies examining the associations between this G870A polymorphism and susceptibility to leukemia and hepatocellular carcinoma (HCC) have shown inconsistent results. Therefore, we conducted a meta-analysis to clarify these associations. A search of the PubMed database yielded 7 relevant articles: 3 pertaining to leukemia and 4 to HCC. The odds ratios (ORs) from individual studies were pooled using a fixed or random-effect model. A significant association was observed between the CCND1 G870A variant and leukemia under the allele contrast model [P = 0.003, OR = 1.49, 95% confidence interval (CI) = 1.15-1.95], the homozygote contrast model (P = 0.003, OR = 2.30, 95%CI = 1.34-3.96), and the recessive model (P = 0.002, OR = 2.03, 95%CI = 1.29-3.21). A significant association was observed between this variant and HCC under the recessive model (P = 0.0006, OR = 1.62, 95%CI = 1.23-2.14), the dominant model (P = 0.002, OR = 1.59, 95%CI = 1.19-2.14), the homozygote contrast model (P < 0.0001, OR = 2.06, 95%CI = 1.45-2.94), and the allele contrast model (P < 0.0001, OR = 1.43, 95%CI = 1.20-1.69). Our findings suggest that heritable CCND1 status may influence the risk of developing leukemia and HCC, and that more attention should be given to carriers of these susceptibility genes.

Topics: Alleles; Carcinoma, Hepatocellular; Cyclin D1; Gene Expression; Genetic Predisposition to Disease; Homozygote; Humans; Inheritance Patterns; Leukemia; Liver Neoplasms; Models, Genetic; Odds Ratio; Polymorphism, Single Nucleotide

The molecular mechanism of the cell-cycle machinery in hepatocellular carcinoma (HCC) has not yet been fully elucidated. Among the various types of cell-cycle regulators, p16 and p27 are now considered to be potent tumor suppressors. p16 is a G1-specific cell-cycle inhibitor that prevents the association of cyclin-dependent kinase (CDK) 4 and CDK6 with cyclin D(1). Many studies have reported that p16 is inactivated not only in aggressive types of HCC but also in preneoplastic liver cirrhosis. In many cases of HCC, p16 is mainly inactivated by extensive CpG methylation, suggesting that epigenetic changes in the p16 gene may be important events during hepatocarcinogenesis. p27, an inhibitor of CDK2, is presently regarded as a potent adverse prognostic factor in many aggressive cancers. It should be noted that some cases of HCC show increased cell proliferation despite the expression of considerable amounts of p27. In these cases, p27 is inactivated by sequestration into cyclin D(1)-CDK4-containing complexes. Although the reason for the compositional changes in the p27-containing complexes is unclear, our experimental results indicate that loss of p16 following DNA methylation is closely related to the functional inactivation of p27 in HCC. We suggest that assessment of the p16 status may be useful for a precise prognostic prediction for individuals with HCCs expressing high levels of p27.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; DNA Methylation; Humans; Liver Neoplasms

Many steps in the Wnt signalling pathway may be altered during the process of carcinogenesis. This Review focuses on the changes observed in gastrointestinal cancers. A literature search was undertaken and the currently available data summarised. Understanding the alterations to this signalling pathway may help to reveal future targets for therapeutic agents. In addition, since in some tumours, levels of components of the Wnt pathway have been found to correlate with clinical stage, their potential use as prognostic indicators is highlighted.

Topics: Axin Protein; beta Catenin; Carcinoma, Hepatocellular; Cyclin D1; Cytoskeletal Proteins; DNA-Binding Proteins; Frizzled Receptors; Gastrointestinal Neoplasms; Genes, APC; Genes, myc; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Matrix Metalloproteinase 7; Oligopeptides; Pancreatic Neoplasms; Prognosis; Proteins; Repressor Proteins; Signal Transduction; Trans-Activators; Wnt Proteins

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Genes, Retinoblastoma; Humans; Liver Neoplasms; Mutation; Retinoblastoma Protein

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cyclin D; Cyclin D1; Cyclins; Genes, bcl-1; Humans; Liver Neoplasms

Hepatocellular carcinoma (HCC) is a common malignant tumor with limited treatment options. Conventional antitumor therapy combined with traditional Chinese medicine (TCM) to limit tumor progression has gradually become the focus of complementary and alternative therapies for HCC treatment. The Fuzheng Jiedu Xiaoji formulation (FZJDXJ) alleviates the clinical symptoms of patients and inhibits tumor progression, but its curative effect still requires extensive clinical research and mechanistic analysis.. To explore the effectiveness of FZJDXJ in HCC patients and investigate its biological function and mechanism underlying anticancer therapy.. This randomized controlled clinical trial enrolled 291 HCC patients receiving transcatheter arterial chemoembolization (TACE) therapy; patients received either FZJDXJ combined with standard treatment, or standard treatment alone, for 48 weeks. Statistical analyses were performed according to survival time at the end of the trial. The main constituents of the FZJDXJ extracts were identified and evaluated using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and molecular docking. The antitumor effects of FZJDXJ and its specific biological mechanism of action were studied.. After 48 weeks of treatment, one-year overall survival (OS) and progression-free survival (PFS) were significantly different between the two groups. Co-administration of FZJDXJ and TACE prolonged the OS of HCC patients, especially in BCLC A or B stage. FZJDXJ and TACE treatment effectively extended the PFS of patients, especially in the BCLC B stage. HPLC-MS/MS identified 1619 active constituents of FZJDXJ, including formononetin, chlorogenic acid (CGA), caffeic acid, luteolin, gallic acid, diosgenin, ergosterol endoperoxide, and lupeol, which may function through the AKT/CyclinD1/p21/p27 pathways. Through molecular docking, CGA and gallic acid could effectively combine with Thr308, an important phosphorylation site of AKT1. FZJDXJ inhibited tumor growth in nude mice. In vitro, FZJDXJ-mediated serum inhibited the proliferation, migration, and invasion of liver cancer cells, and promoted cell apoptosis.. Clinically, FZJDXJ combined with TACE therapy significantly prolonged OS and PFS and reduced the mortality rate of HCC patients. Mechanistically, FZJDXJ effectively inhibited the proliferation and migration of liver cancer cells through the modulation of the AKT/CyclinD1/p21/p27 pathways, and may be a promising TCM drug for anti-HCC therapy.

Topics: Animals; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Molecular Docking Simulation; Neoplasms, Experimental; Phytotherapy; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; Retrospective Studies; Tandem Mass Spectrometry

To assessthe potential role of tissue inhibitor of metalloproteinase 3 as a staging marker of hepatocellular carcinoma.. The experimental study was conducted at Faculty of Pharmacy, Kafrelsheikh University, Egypt, from December 2020 to March 2022 after approval from the Faculty of Pharmacy, Kafrelsheikh University, Egypt, and comprised male albino rats. The subjects were divided into 4 groups. The control group was administrated a single intraperitoneal injection of normal saline, while the other groups were generated post-induction of hepatocellular carcinoma. The induction was done by injecting rats intraperitoneally with a single dose of 200mg/kg diethyl nitrosamine, followed by the administration of 0.05% phenobarbital sodium in drinking water daily. Rats were euthanised at 8, 16 and 24 weeks after the injection to obtain three groups related to the 3 stages of hepatocellular carcinoma. Serum was used for measuring the alpha protein level. Liver homogenates were used for the assessment of the hepatic tissue inhibitor of metalloproteinase 3 expression, B-cell lymphoma 2-associated X protein expression, and the hepatic content of matrix metalloproteinase -9 and cyclin D1. Data was analysed using Graph Prism 8.. Of the 24 rats with weight range 120-130g, 6(25%) were in each of the 4 groups. The relative protein and messenger ribonucleic acid tissue inhibitor of metalloproteinase 3 expressions were significantly decreased in the intervention groups compared to the control group, with more decline as the hepatocellular carcinoma stage increased. The matrix metalloproteinase -9 and cyclin D1 concentrations and the relative hepatic protein Ki67 expression were significantly raised in the intervention groups compared to the control group (p<0.05). The relative expression of hepatic B-cell lymphoma 2-associated X protein was markedly decreased in the intervention groups compared to the control group (p<0.05).. Tissue inhibitor of metalloproteinase 3 might be a promising diagnostic and staging biomarker in hepatocellular carcinoma.

Topics: Animals; Biomarkers; Carcinoma, Hepatocellular; Cyclin D1; Liver Neoplasms; Male; Proto-Oncogene Proteins c-bcl-2; Rats; Tissue Inhibitor of Metalloproteinase-3

The present study was aimed to investigate the APC expression, its promoter methylation status, the expression of β-Catenin, c-Myc and Cyclin D1 and further explore their prognostic value in Hepatocellular carcinoma (HCC).. Serum samples from 90 HCC patients and 27 healthy donors were collected in this study. The methylation-specific PCR (MSP) was performed to evaluate promoter methylation status of APC gene. RT-qPCR was used to detect the mRNA expression of APC, β-Catenin, c-Myc and Cyclin D1, meanwhile the protein expression were analyzed by Western blot.. The positive rate of APC gene methylation in HCC patients (46.67%) was higher than healthy donors (11.11%). APC gene exhibited marked hypermethylation in the patients of TNM III-IV stage when compared to the patients of TNM I-II stage , the methylation status of APC gene was correlated with tumor size and lymph node metastasis whereas the APC gene methylation showed no relationship with the patient's sex and age. APC methylation may be associated with APC expression level, APC expression in HCC cells is silenced by aberrant promoter hypermethylation. In HCC patients with methylated APC, the mRNA and protein expression of β-Catenin, c-Myc and Cyclin D1 were higher than the unmethylated patient subgroup and healthy donors.. The downregulation of APC in HCC samples was associated with promoter hypermethylation. APC methylation could be used as a novel diagnostic biomarker in HCC, which was associated with regulation of Wnt/β-Catenin signal pathway.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cyclin D1; DNA Methylation; Humans; Liver Neoplasms; Prognosis; Promoter Regions, Genetic; RNA, Messenger

The current study investigates the anti-inflammatory and hepatoprotective effects of selenium (Se) formulated as nanoparticles (SeNPs) and in combination with quercetin (QCT) against thioacetamide (TAA)-induced hepatocellular carcinoma (HCC) in rats. ​​​​​​Seventy-two male Sprague-Dawley rats were divided into six groups (n = 12). Three control groups; normal, SeNPs; group received SeNPs only and HCC; group received TAA. In addition, three preventive groups; SeNPs + TAA, QCT + TAA, and QCT + SeNPs + TAA. Induction of HCC was detected histopathologically and by the raise of the serum level of alpha-fetoprotein (AFP). Oxidative stress was evaluated by the hepatic levels of reduced glutathione (GSH), glutathione peroxidase (GPx), and malondialdehyde (MDA) spectrophotometrically. The oncogenic pathway of p53/β-catenin/cyclin D1 was assessed by immunohistochemistry. The inflammatory markers; interleukin-33 (IL-33), IL-6, and IL-1β were assessed by enzyme-linked immune sorbent assay. SeNPs prevented the elevation of serum AFP and hepatic IL-33, IL-1β, and IL-6 in comparison to HCC or QCT + TAA groups. SeNPs + TAA exhibited a lower positive hepatic staining of p53, β-catenin, and cyclin D1 in comparison to HCC or QCT + TAA groups. Moreover, SeNPs improved the overall oxidative balance indicated by low hepatic MDA and enhanced GSH and GPx when compared to HCC or QCT + TAA groups. ​​SeNPs alone and in combination with QCT were found to suppress the progression of HCC in rats via the enhancement of the oxidative stress and then inflammatory status and the prevention of the deregulation of the oncogenic axis pathway of p53/β-catenin/cyclin D.

Topics: alpha-Fetoproteins; Animals; beta Catenin; Carcinoma, Hepatocellular; Cyclin D1; Inflammation; Interleukin-33; Interleukin-6; Liver; Liver Neoplasms; Male; Nanoparticles; Oxidative Stress; Quercetin; Rats; Rats, Sprague-Dawley; Selenium; Thioacetamide; Tumor Suppressor Protein p53

RNA helicases are dysregulated in tumors. Here, we identified DHX37 as one of the top RNA helicase genes with upregulated expression in hepatocellular carcinoma (HCC). DHX37 promoted proliferation of liver cancer cells in vitro and in vivo. Epigenomic profiling of DHX37-knockdown and control HCC cells revealed that DHX37 is associated with superenhancer activity. Mechanistically, DHX37 interacted with pleiotropic regulator 1 (PLRG1) to transcriptionally activate cyclin D1 (CCND1) expression via co-occupation of its promoter and superenhancer elements. DHX37 and PLRG1 promoted liver cancer cell proliferation and contributed to the poor prognosis of patients with HCC. Importantly, CCND1 inhibitors were effective as antiproliferative agents for liver cancer. These results together demonstrate a cooperative mechanistic interaction between DHX37 and PLRG1 that regulates CCND1 expression and promotes liver cancer progression, advancing our understanding of the epigenetic and transcriptional dysregulations mediated by RNA helicases and superenhancers in HCC.. This work characterizes a novel mechanism of superenhancer-driven cyclin D1 upregulation by DHX37 and PLRG1, implicating this pathway as a potential therapeutic target in hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Nuclear Proteins; RNA Helicases

Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model.. Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays.. Exosomes released from cancer cells contain. We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.

Topics: Carboxypeptidase H; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Exosomes; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Liver Neoplasms; MicroRNAs; Phenotype; Pilot Projects; RNA, Messenger; RNA, Small Interfering

The present study was targeted at investigating the effects of hsa_circRNA_0008092 (circ_0008092) on hepatocellular carcinoma (HCC) cell proliferation, migration, invasion and apoptosis, and its related mechanism.. The gene expression profiles of GSE166678 were downloaded from the Gene Expression Omnibus database, and differentially expressed circRNAs in human HCC were screened out. Besides, circ_0008092, microRNA-502-5p (miR-502-5p) and cyclin D1 (CCND1) expressions in HCC tissues and cell lines were detected by quantitative real-time polymerase chain reaction (qRTPCR). Cell countering kit-8 (CCK-8), Transwell and flow cytometry assays were used to detect the proliferation, migration, invasion and apoptosis of HCC cells. Bioinformatics was utilized to predict the targeted relationships between miR-502-5p and circ_0008092, as well as miR-502-5p and CCND1 mRNA 3'-untranslated region (3'UTR). Western blot assay was applied to detect CCND1 protein expression in HCC cells.. Circ_0008092 was highly expressed in HCC tissues and cells, which was associated with a shorter survival time in patients with HCC. Circ_0008092 overexpression promoted proliferation, migration and invasion, and inhibited apoptosis of HCC cells; circ_0008092 knockdown worked oppositely. Circ_0008092 directly targeted miR-502-5p and negatively modulated miR-502-5p expression. CCND1 Conclusion: Our data suggest that circ_0008092 promotes HCC progression by regulating the miR- 502-5p/CCND1 axis.

Topics: 3' Untranslated Regions; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; RNA, Circular; Sincalide

The stromal antigen 3 (STAG3) gene encodes an adhesion complex subunit that can regulate sister chromatid cohesion during cell division. Chromosome instability caused by STAG3 gene mutation may potentially promote tumor progression, but the effect of STAG3 on hepatocellular carcinoma (HCC) and the related molecular mechanism are not reported in the literature. The mechanism of the occurrence and development of HCC is not adequately understood. Therefore, the biological role of STAG3 in HCC remains to be studied, and whether STAG3 might be a sensitive therapeutic target in HCC remains to be determined.. The expression and clinical significance of STAG3 in HCC tissues and cell lines were determined by RT-qPCR and immunohistochemistry analyses. The biological functions of STAG3 in HCC were determined through in vitro and in vivo cell function tests. The molecular mechanism of STAG3 in HCC cells was then investigated by western blot assay.. The mRNA expression of STAG3 was lower in most HCC cells than in normal cells. Subsequently, an immunohistochemical analysis of STAG3 was performed with 126 samples, and lower STAG3 expression was associated with worse overall survival in HCC patients. Moreover, cytofunctional tests revealed that the lentivirus-mediated overexpression of STAG3 in HCC cells inhibited cell proliferation, migration, and invasion; promoted apoptosis; induced G1/S phase arrest in vitro; and inhibited tumor growth in vivo. Furthermore, studies of the molecular mechanism suggested that the overexpression of STAG3 increased Smad3 expression and decreased CDK4, CDK6, cyclin D1, CXCR4 and RhoA expression.. STAG3 exhibits anticancer effects against HCC, and these effects involve the Smad3-CDK4/CDK6-cyclin D1 and CXCR4/RhoA pathways. STAG3 is a tumor-suppressor gene that may serve as a potential target for molecular therapy, which provides a new idea for the treatment of HCC.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Receptors, CXCR4; rhoA GTP-Binding Protein; Smad3 Protein; Up-Regulation

Hepatocellular carcinoma (HCC) is a highly aggressive and solid malignancy with a poor prognosis. Cell division cycle associated 2 (CDCA2) is highly expressed in HCC and is considered to be closely related to the prognosis of patients with HCC. In this research, we aimed to investigate the function and potential mechanism of CDCA2 in HCC cells.. Gain- and loss-of-function experiments were conducted to determine the biological function of CDCA2 in HCC cells. Quantitative reverse transcription-polymerase chain reaction and western blot were utilized to examine the Messenger RNA (mRNA) and protein levels of CDCA2 in HCC cells. The malignant behaviors of HCC cells were analyzed by several biological experiments including cell viability, cell colony formation, and transwell assays. Western blot was also implemented to examine the expression of : AKT, protein kinase B and mTOR, mammalian target of rapamycin (AKT-mTOR) pathway related proteins and Cyclin D1.. A significant increase of CDCA2 was observed in HCC cell lines. Upregulation of CDCA2 resulted in the enhancement of the growth, migration, and invasion of HCC cells. Inversely, depletion of CDCA2 displayed the opposite results. Furthermore, the protein levels of p-AKT, p-mTOR, and Cyclin D1 were elevated with CDCA2 upregulation and reduced with CDCA2 depletion in HCC cells.. Our observations revealed that CDCA2 promoted the malignant development of HCC cells, and AKT-mTOR pathway might involve in the underlying mechanism.

Topics: Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Nuclear Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Tacrolimus is a calcineurin inhibitor widely used for immunological disorders. However, there is significant controversy regarding its effect on the liver. The present study was conducted to evaluate the anticancer effects of tacrolimus on an induced murine hepatocellular carcinoma (HCC) model and its possible hepatotoxicity at standard therapeutic doses.. Fifty-four male mice were divided into five groups: a control healthy group, control HCC group, tacrolimus-treated group, doxorubicin (DOXO)-treated group, and combined tacrolimus- and DOXO-treated group. The activity of liver enzymes, including alkaline phosphatase, gamma- glutamyl transferase, lactate dehydrogenase, alanine transaminase, and aspartate transaminase, was determined. Serum vascular endothelial growth factor (VEGF) was measured using an enzyme- linked immunosorbent assay. A quantitative real time- polymerase chain reaction (qRTPCR) was conducted to measure the expression of proliferating cell nuclear antigen (PCNA), Bax, and p53 mRNA. Immunohistochemical staining for cyclin D1 and VEGF was performed.. Mice that received combined treatment with tacrolimus and DOXO exhibited the best improvement in all parameters when compared with the groups that received DOXO or tacrolimus alone (p < 0.001).. The combination of DOXO and tacrolimus was more effective in the management of HCC compared with either agent alone. This improvement was detected by the reduction of liver enzymes and the improvement of the histopathological profile. The involved mechanisms included significant apoptosis induction demonstrated by upregulation of bax along with a reduction in angiogenesis demonstrated by downregulation of VEGF. This was accompanied by inhibition of cell cycle progression mediated by upregulated p53 and downregulated PCNA and cyclin D1.

Topics: Animals; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cyclin D1; Doxorubicin; Liver Neoplasms; Male; Mice; Proliferating Cell Nuclear Antigen; Tacrolimus; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

Aldehyde dehydrogenases (ALDHs) are considered as markers for normal and cancer stem cells (CSC) and are involved in cell metabolism, proliferation, differentiation, stemness, and retinoic acid (RA) biosynthesis. The aim of the present study was to identify the ALDH isoforms that are associated with the CSC phenotype in non-small cell lung and hepatocellular carcinomas.. We utilized lung (A549) and hepatocellular (HepG2) cancer cells and generated tumor spheres to isolate the CSC sub-population.. The CSC enrichment was confirmed by the up-regulation of various CSC-related genes. Comparative qPCR analysis indicated the up-regulation of several ALDH isoforms in A549 and HepG2 spheres. Interestingly, cyclin D1 and Akt, down-stream targets of the RA signaling pathway, were also shown to be significantly up-regulated in both sphere populations.. Specific ALDH isoforms appear to be important mediators for the acquisition of an CSC phenotype and thus, are potential promising targets for CSC-based therapeutic approaches in lung and hepatocellular carcinomas.

Topics: A549 Cells; Aldehyde Dehydrogenase; Carcinoma, Hepatocellular; Cyclin D1; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Isoenzymes; Liver Neoplasms; Lung Neoplasms; Neoplastic Stem Cells; Phenotype; Proto-Oncogene Proteins c-akt; Signal Transduction; Spheroids, Cellular

Hepatocellular carcinoma (HCC) is a common malignant tumor with insidious onset and rapid progression. Its treatment is often difficult owing to tumor resistance. In this study, we aimed to understand the different biological characteristics, gene expression profiles, and drug resistance mechanisms of HCC cells cultured under different conditions. A conventional adherence method and a liquid overlay technique were used to prepare two- and three-dimensional cultures of Bel-7402 and 5-fluorouracil (5-Fu)-resistant Bel-7402 (Bel-7402/5-Fu) cells. Morphological characteristics were assessed via microscopy, and cell cycle distribution and apoptotic rate were obtained using flow cytometry. Cell sensitivity to different concentrations of drugs was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Gene expression profiles and signal transduction pathways of Bel-7402 and Bel-7402/5-Fu cells under different culture conditions were determined using gene chips. Cells in three-dimensional culture were suspended and they grew into dense multicellular spheroid (MCS) structures, aggregating with each other. In contrast to cells in the two-dimensional culture, cell cycle arrest was observed in MCSs. The sensitivity of Bel-7402 cells in the two-dimensional culture to drugs at high concentrations was significantly higher than that of cells in the three-dimensional culture (p < .05). The apoptotic rate of Bel-7402 and Bel-7402/5-Fu cells was also higher in the two-dimensional culture (p < .05). Signal transduction pathway analysis showed that after Bel-7402 cells acquired resistance to 5-Fu, CCND1, MCM2, and MCM3 gene expression was upregulated in the G1 to S cell cycle control signal transduction pathway, CDKN1C and CCNG2 gene expression was downregulated, and MCM2 and MCM3 gene expression was upregulated in the DNA replication signal transduction pathway. Therefore, the liquid overlay technique is a simple, low-cost procedure to successfully construct three-dimensional culture models of HCC. This study provides new information and methods for exploring the molecular mechanisms of liver cancer resistance, clinical treatment, development of molecular information, and interventional prevention.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Cisplatin; Cyclin D1; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Minichromosome Maintenance Complex Component 2; Minichromosome Maintenance Complex Component 3; Mitomycin; Paclitaxel; Transcriptome

Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is a group of isoforms produced by alternative splicing and is overexpressed in human malignancies including hepatocellular carcinoma (HCC). However, the prognostic value and biological functions of its major protein isoforms, named CABYR-a/b (combined CABYR-a and CABYR-b), in HCC remain to be established.. CABYR-a/b expression was detected in HCC tissues and cell lines by quantitative real-time polymerase chain reaction and Western blot analysis. The correlation of CABYR-a/b expression with clinical characteristics and its prognosis impact were determined by statistical analysis. Finally, the biological functions and molecular mechanism of CABYR-a/b were also investigated using molecular biology approaches.. The present research found that CABYR-a/b was markedly elevated in HCC specimens and cell lines. Upregulated CABYR-a/b level had positive association with tumor size and differentiation in patients. Moreover, cases with elevated CABYR-a/b level had poorer overall survival (OS) and disease-free survival (DFS) than those with reduced CABYR-a/b level. Multivariate analysis and prognostic nomograms demonstrated that CABYR-a/b overexpression was an independent predictive indicator for OS and DFS. The calibration curve for the odds of OS and DFS demonstrated that the prediction by nomograms was in excellent accordance with actual situation. CABYR-a/b downregulation suppressed cell proliferation and induced G1-phase arrest via decreasing cyclin D1 and cyclin dependent kinase 4, while promoted apoptosis by reducing B-cell lymphoma 2 (Bcl-2) and increasing Bcl-2-associated death promoter.. Our research indicates that CABYR-a/b exerts an oncogenic effect on HCC development and may become a new prognostic indicator for patients with HCC.

Topics: Aged; Alternative Splicing; Apoptosis; Biomarkers, Tumor; Calcium; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Middle Aged; Phosphorylation; Prognosis; Protein Binding; Protein Isoforms; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Treatment Outcome; Tyrosine

The overall survival rate of patients with hepatocellular carcinoma (HCC) has remained unchanged over the last several decades. Therefore, novel drugs and therapies are required for HCC treatment. Isoliquiritigenin (ISL), a natural flavonoid predominantly isolated from the traditional Chinese medicine Glycyrrhizae Radix (Licorice), has a high anticancer potential and broad application value in various cancers. Here, we aimed to investigate the anticancer role of ISL in the HCC cell line Hep3B. Functional analysis revealed that ISL inhibited the proliferation of Hep3B cells by causing G1/S cell cycle arrest in vitro. Meanwhile, the inhibitory effect of ISL on proliferation was also observed in vivo. Further analysis revealed that ISL could suppress the migration and metastasis of Hep3B cells in vitro and in vivo. Mechanistic analysis revealed that ISL inhibited cyclin D1 and up-regulated the proteins P21, P27 that negatively regulate the cell cycle. Furthermore, ISL induced apoptosis while inhibiting cell cycle transition. In addition, phosphatidylinositol 3'-kinase/protein kinase B (PI3K/AKT) signal pathway was suppressed by ISL treatment, and the epithelial marker E-cadherin was up-regulated when the mesenchymal markers Vimentin and N-cadherin were down-regulated. In brief, our findings suggest that ISL could be a promising agent for preventing HCC tumorigenesis and metastasis.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chalcones; Cyclin D1; Down-Regulation; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Wogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far.. The aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression.. Herein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC.. Our results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased.. In summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Down-Regulation; Enzyme Activation; Flavanones; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Molecular Docking Simulation; Phosphorylation; Xenograft Model Antitumor Assays

Upregulated ubiquitin-conjugating enzyme E2M (UBE2M) is associated with poor prognosis in malignancies; However, the phenotype and mechanism of action of UBE2M in hepatocellular carcinoma (HCC) remain elusive. Here, we report that UBE2M is overexpressed and correlated with poor prognosis in HCC patients. The UBE2M level is an independent prognostic factor for HCC patients. UBE2M knockdown inhibits HCC cell proliferation, migration, and invasion, whereas its overexpression has an opposite effect. Mechanistically, upregulated UBE2M exerts oncogenic effects by translocation of accumulated β-catenin from the cytoplasm to the nucleus, thus activating downstream β-catenin/cyclin D1 signaling. In summary, our study demonstrates a notable role of UBE2M in promoting the growth of HCC, providing a novel strategy for HCC prevention and treatment.

Topics: Adult; Aged; beta Catenin; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Female; Humans; Liver Neoplasms; Male; Middle Aged; Prognosis; Signal Transduction; Ubiquitin-Conjugating Enzymes

Inhibitors of the CDK family of proteins have been approved for the treatment of a variety of tumours; however, the development of new drugs administered in combination with CDK inhibitors is expected to improve the therapeutic effect. We identified the function of suppressor of cytokine signalling 1 (SOCS1) in hepatocellular carcinoma (HCC) cell models and the xenograft mouse model. When SOCS1 expression was artificially upregulated, HCC cell lines were arrested at the G1-S transition in the cell cycle. Interestingly, during this process, total CyclinD1 protein increased, but the effective proportion decreased. We found that the deficiency of CyclinD1 in the nucleus is probably due to the decrease in the stability of nuclear CyclinD1 caused by the ubiquitin-based degradation of P21, thus inhibiting the progression of the cell cycle to S phase. After P21 expression was increased, the levels of the component that inactivates CyclinD1 decreased as expected. It showed that P21 has a partial promoting effect on cancer. SOCS1 is a good indicator of prognosis, tumour size and long-term survival after resection. SOCS1 is expected to become a drug target in combined with CDK family inhibitors.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Hepatocytes; Heterografts; Liver Neoplasms; Mice; Suppressor of Cytokine Signaling 1 Protein

Cyclin D1 is a key regulatory factor of the G1 to S transition during cell cycle progression. Aberrant cyclin D gene amplification and abnormal protein expression have been linked to hepatocellular carcinoma (HCC) tumorigenesis. Intrabodies, effective anticancer therapies that specifically inhibit target protein function within all intracellular compartments, may block cyclin D1 function. Here, a single-chain variable fragment (scFv) antibody against cyclin D1 (ADκ) selected from a human semi-synthetic phage display scFv library is expressed in Escherichia coli as soluble ADκ. Purified ADκ specifically binds to recombinant and endogenous cyclin D1 with high affinity. To enable blocking of intracellular cyclin D1 activity, an endoplasmic reticulum (ER) retention signal sequence is added to the ADκ sequence to encode anti-cyclin D1 intrabody ER-ADκ. Transfection of HepG2 cells with expression vector encoding ER-ADκ elicited intracellular ER-ADκ expression leading to cyclin D1 binding, significant G1 phase arrest, and apoptosis that are mechanistically tied to decreased intracellular phosphorylated retinoblastoma protein (Rb) levels. Meanwhile, ER-ADκ dramatically inhibited subcutaneous human HCC xenografts growth in nude mice in vivo after injection of tumors with expression vector encoding ER-ADκ. These results demonstrate the potential of intrabody-based cyclin D1 targeting therapy as a promising treatment for HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Proliferation; Cyclin D1; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; Single-Chain Antibodies

Ultrasonic microbubbles in combination with microRNA (miRNAs/miRs) exhibited promising effects on cancer treatments. The aim was to investigate the role of miR‑378 in hepatoma cells and the efficiency of it in combination with ultrasonic irradiation and SonoVue® microbubbles method for cell transfection. HuH‑7, Hep3B and SK‑Hep1 cells were transfected with an miR‑378 mimic using only Lipofectamine® 3000 or combined with SonoVue microbubbles and ultrasonic irradiation at 0.5 W/cm2 for 30 sec. mRNAs and protein levels of Cyclin D1, Bcl‑2, Bax, Akt, p53 and Survivin were detected by reverse transcription‑quantitative PCR and western blotting, respectively. Cell survival rate, proliferation, cell cycle and apoptosis were determined by Cell Counting Kit‑8, cell double cytochemical staining and flow cytometry, respectively. It was found that using a combination of ultrasonic irradiation and the SonoVue microbubbles method increased the effectiveness of miR‑378 transfection into hepatocellular carcinoma (HCC) cells, and increased the inhibition of cell survival and proliferation. Moreover, miR‑378 increased the rate of apoptosis and upregulated the expression of Bax and p53, and suppressed the cell cycle and downregulated the expression of Cyclin D1, Bcl‑2, Akt, β‑catenin and Survivin much more effectively in the HCC cell line by applying the combined method. Thus, miR‑378 was shown to be a suppressive factor to reduce proliferation and increase apoptosis in HCC cells. Additionally, the combination of ultrasonic irradiation and SonoVue microbubbles method was more efficient in the transfection of miRNA.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Humans; Liver Neoplasms; Microbubbles; MicroRNAs; Phospholipids; Proto-Oncogene Proteins c-bcl-2; Sulfur Hexafluoride; Survivin; Transfection; Ultrasonic Therapy; Ultrasonics

Quercetin, an abundant flavonoid found in various fruits and vegetables, displays multiple biological activities, including anticancer effects. Therefore, quercetin is receiving increasing attention as a potential adjuvant anticancer treatment. Gemcitabine (GEM) resistance is a major issue for clinicians and patients with advanced cancers, making it crucial to determine ways to bolster its effects. In this study, we explored the anticancer effects and mechanistic actions of quercetin in GEM-resistant cancer cells. Pancreatic cancer (BxPC-3, PANC-1) and hepatocellular carcinoma (HepG2, Huh-7) cell lines were studied. Proliferation assays showed that quercetin had cytotoxic effects on GEM-resistant cell lines (HepG2 and PANC-1), and flow cytometric analysis indicated a significant pro-apoptotic effect on these cell lines. GEM treatment, in combination with quercetin, resulted in increased anticancer effects compared with GEM alone. Quercetin led to S phase arrest in GEM-resistant cell lines, and western blot analysis revealed tumour protein p53 upregulation and cyclin D1 downregulation. This study provides mechanistic insight into the anticancer effects of quercetin and suggests that quercetin adjuvant treatment may benefit patients who are resistant to GEM therapy.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Deoxycytidine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Gemcitabine; Hep G2 Cells; Humans; Liver Neoplasms; Pancreatic Neoplasms; Quercetin; Tumor Suppressor Protein p53

Studies show that patients with hepatocellular carcinoma (HCC) have poor prognosis, particularly when patients are diagnosed at late stages of the disease development. The flap endonuclease 1 (FEN1) gene is overexpressed in multiple malignant tumors and may promote tumor aggressiveness. However, its expression profile and functional roles in HCC are still unclear. Here, we evaluated the molecular mechanisms of FEN1 in HCC.. The expression of FEN1 in HCC was evaluated using HCC mRNA expression data from TCGA and GEO databases. The expression of FEN1 was also confirmed by immunohistochemistry (IHC) using a tissue microarray (TMA) cohort with a total of 396 HCC patients. Kaplan-Meier analysis and univariate and multivariate Cox regression analyses were used to determine the correlation between FEN1 expression and survival rate of HCC patients. The molecular mechanism and biological functions of FEN1 in HCC were predicted using functional and pathway enrichment analysis. FEN1 was overexpressed in multiple HCC cohorts at both mRNA and protein levels. The receiver operating characteristic (ROC) curve showed that FEN1 can serve as a diagnostic predictor of HCC. Meanwhile, patients with high FEN1 expression levels showed lower overall survival (OS) and relapse-free survival (RFS) rates than those with low FEN1 expression. More importantly, we found that FEN1 elevation was an independent prognostic factor for OS and RFS in HCC patients based on univariate and multivariate analyses, indicating that FEN1 might be a potential prognostic marker in HCC. Furthermore, knocking down FEN1 resulted in suppressed cell proliferation and migration. Our study indicated that high FEN1 expression might function as a biomarker for diagnosis and prognosis. In addition, the study confirms that FEN1 is an oncogene in HCC progression.

Topics: Adult; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Proliferation; Cohort Studies; Cyclin D1; Delayed Diagnosis; Disease Progression; Female; Flap Endonucleases; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Prognosis; Proportional Hazards Models; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; ROC Curve; Survivin

Chemoresistance is one of the major barriers in chemotherapy-based hepatocellular carcinoma (HCC) intervention. 5-Fluorouracil (5-Fu) is a widely used as an anticancer drug. Liver cancer stem cells (LCSCs) are considered the origin of tumor recurrence and resistance. CCND1 (Cyclin D1) plays an important role in tumorigenesis and metastasis in multiple cancers including HCC. Herein, this study was designed to explore the role of CCND1 in regulating LCSCs differentiation and 5-Fu resistance in HCC cells.. The CCND1 mRNA level was examined by qRT-PCR. The protein levels of γ-H2AX (a DNA damage marker) and RAD51 (a DNA repair protein) were examined by Western blot. CD133 was used as a LCSC marker and CD133+ cell percentage in HCC cells was detected by flow cytometry.. CCND1 silencing decreased CD133+ cell percentage in HepG2 and SMMC-7721 cells. Furthermore, CCND1 silencing significantly increased protein level of γ-H2AX and decreased that of RAD51 under 5-Fu exposure. Moreover, CCND1 silencing enhanced the sensitivity of HepG2 and SMMC-7721 cells to 5-Fu, which was effectively abrogated by RAD51 upregulation.. Collectively, CCND1 silencing suppresses LCSCs differentiation and overcomes 5-Fu resistance in HCC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Culture Techniques; Cell Differentiation; Cyclin D1; Drug Resistance, Neoplasm; Fluorouracil; Gene Silencing; Hep G2 Cells; Humans; Liver Neoplasms; Neoplastic Stem Cells; Rad51 Recombinase; Up-Regulation

The hypomethylation of the Cyclin D1 (CCND1) promoter induced by excess oxidative stress likely promotes the development of hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC). We aimed to evaluate methylation status of the CCND1 promoter as a new plasma marker for the detection of HBV-HCC.We consecutively recruited 191 participants, including 105 patients with HBV-HCC, 54 patients with chronic hepatitis B (CHB), and 32 healthy controls (HCs). Using methylation-specific polymerase chain reaction, we identified the methylation status of the CCND1 promoter in plasma samples. We analyzed the expression levels of the CCND1 mRNA in peripheral blood mononuclear cells by using quantitative real-time PCR. We assessed the plasma levels of superoxide dismutase, 8-hydroxydeoxyguanosine and malondialdehyde by using enzyme-linked immunosorbent assays.Patients with HBV-HCC (23.81%) presented a reduced methylation frequency compared with patients with CHB (64.81%) or HCs (78.13%) (P < .001). When receiver operating characteristic curves were plotted for patients with HBV-HCC versus CHB, the methylation status of the CCND1 promoter yielded diagnostic parameter values for the area under the curve of 0.705, sensitivity of 76.19%, and specificity of 64.81%, thus outperforming serum alpha-fetoprotein (AFP), which had an area under the curve of 0.531, sensitivity of 36.19%, and specificity of 90.74%. Methylation of the CCND1 promoter represents a prospective diagnostic marker for patients with AFP-negative HBV-HCC and AFP-positive CHB. The expression levels of CCND1 mRNA was increased in patients with HBV-HCC compared with patients with CHB (Z = -4.946, P < .001) and HCs (Z = -6.819, P < .001). Both the extent of oxidative injury and antioxidant capacity indicated by the superoxide dismutase, 8-hydroxydeoxyguanosine and malondialdehyde levels were increased in patients with HBV-HCC. Clinical follow up of patients with HBV-HCC revealed a worse overall survival (P = .012, log-rank test) and a decreased progression-free survival (HR = 0.109, 95%CI: 0.031-0.384) for the unmethylated CCND1 group than methylated CCND1 group.Our study confirms that oxidative stress appears to correlate with plasma levels of CCND1 promoter methylation, and the methylation status of the CCND1 promoter represents a prospective biomarker with better diagnostic performance than serum AFP levels.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cyclin D1; DNA Methylation; Early Detection of Cancer; Enzyme-Linked Immunosorbent Assay; Female; Hepatitis B, Chronic; Humans; Liver Neoplasms; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Promoter Regions, Genetic; Prospective Studies; Real-Time Polymerase Chain Reaction; ROC Curve; Sensitivity and Specificity; Superoxide Dismutase

This study investigated the role of lncRNA CTBP1-AS2 in hepatocellular carcinoma (HCC). The authors found that CTBP1-AS2 was upregulated in HCC by analyzing TCGA dataset. The downregulation of CTBP1-AS2 in HCC was confirmed by measuring the expression level of CTBP1-AS2 in both HCC and nontumor tissues from HCC patients. MiR-623 is predicted to target CTBP1-AS2, while it failed to downregulate its expression. Interestingly, CTBP1-AS2 overexpression led to the upregulation of cyclin D1, a target of miR-623. CCK-8 analysis showed that CTBP1-AS2 and cyclin D1 overexpression promoted the proliferation of HCC cells. MiR-623 overexpression played an opposite role and reduced the effects of CTBP1-AS2 and cyclin D1 overexpression. Therefore, CTBP1-AS2 promotes cell proliferation in HCC by regulating the miR-623/cyclin D1 axis.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Humans; Liver Neoplasms; MicroRNAs; RNA, Long Noncoding; Signal Transduction; Transfection; Up-Regulation

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Gene Knockdown Techniques; Hepatocyte Nuclear Factor 4; Hepatocytes; Liver; Liver Regeneration; Male; Mice, Inbred BALB C; Mice, Knockout

Tumor necrosis factor-related apoptotic induction ligand can induce cell apoptosis in various tumor cells. However, many cancer cells are resistant to tumor necrosis factor-related apoptotic induction ligand. Therefore, overcoming the tumor necrosis factor-related apoptotic induction ligand resistance makes it possible for tumor necrosis factor-related apoptotic induction ligand-based anti-cancer therapies. In this study, we took mesenchymal epithelial transition factor as the research target to study its role in tumor necrosis factor-related apoptotic induction ligand-resistant hepatocellular carcinoma. Mesenchymal epithelial transition factor gene has been proved to be an effective predictor of recurrence after hepatocellular carcinoma resection. The expression of mesenchymal epithelial transition factor and cyclin B1 were measured in tumor necrosis factor-related apoptotic induction ligand-resistant and non-resistant hepatocellular carcinoma tissues. Cyclin B1-knockdown and cyclin B1-overexpression hepatocellular carcinoma cells were treated with tumor necrosis factor-related apoptotic induction ligand; mesenchymal epithelial transition factor knockout, mesenchymal epithelial transition factor re-introduction and cyclin B1 restored in hepatocellular carcinoma cells treated with tumor necrosis factor-related apoptotic induction ligand were established. And MTT, bromodeoxyuridine, flow cytometry and western blotting were performed to evaluate the effect of mesenchymal epithelial transition factor and cyclin B1 on hepatocellular carcinoma cells treated with tumor necrosis factor-related apoptotic induction ligand. In addition, subcutaneous tumor transplantation in nude mice was conducted to access the effect of mesenchymal epithelial transition factor and cyclin B1 on tumor formation in vivo. In conclusion, cyclin B1 enhanced the cell growth and inhibited apoptosis in tumor necrosis factor-related apoptotic induction ligand-resistant hepatocellular carcinoma cells. And mesenchymal epithelial transition factor promoted the cell growth and apoptosis in tumor necrosis factor-related apoptotic induction ligand-resistant hepatocellular carcinoma cells by regulating cyclin B1. Therefore, mesenchymal epithelial transition factor regulates the cyclin B1 to regulate tumor necrosis factor-related apoptotic induction ligand resistance in hepatocellular carcinoma cells. Our results suggest a novel molecular mechanism for regulating tumor necrosis factor-related apopt

Topics: Apoptosis; Carcinoma, Hepatocellular; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Tumor Necrosis Factor-alpha

Thyroid hormones (TH) are multifunctional mediators that fine‑tune several physiological processes, including metabolic rate, digestive function and tissue development via interactions with type II nuclear thyroid hormone receptors (TR). Upon binding of TH, TRs interact specifically with thyroid hormone response elements of target gene promoter regions to regulate their transcription. Earlier studies suggested a correlation between aberrant TR regulation and hepatocellular carcinoma (HCC). THs are involved in a crosstalk between hepatoma and stromal cells, and disruption of TH signaling is associated with tumorigenesis. Previous cDNA microarray analysis of target gene expression following T3 treatment of wild‑type TR‑expressing hepatoma cells led to the identification of forkhead box M1 (FOXM1) as a factor negatively regulated by T3 and associated with poor prognosis in several cancer types. Increased FOXM1 expression during late stages of HCC was associated with poorer overall and recurrence‑free survival in patients with HCC. However, the specific mechanisms underlying FOXM1 activity in liver cancer progression remain to be elucidated. Experiments from the present study showed that TH/TR signaling suppresses FOXM1 mRNA and protein expression. Depletion of FOXM1 induced inhibition of the cell growth rate and a decline in oncogenic cyclin D1, cyclin E and CDK2 expression. Conversely, overexpression of FOXM1 enhanced cell proliferation and expression of oncogenic factors, which was decreased upon FOXM1 depletion. Re‑expression of FOXM1 partially rescued suppression of cell proliferation induced by T3. Collectively, the present findings suggest that TH/TR participates in HCC progression via modulation of FOXM1 expression.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Disease Progression; Forkhead Box Protein M1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Thyroid Hormone Receptors beta; Triiodothyronine

In this study, we comprehensively analysed the role of ubiquitin-specific protease 1(USP1) in hepatocellular carcinoma (HCC) using data from a set of public databases.. We analysed the mRNA expression of USP1 in HCC and subgroups of HCC using Oncomine and UALCAN. Survival analysis of USP1 in HCC was conducted with the Kaplan-Meier Plotter database. The mutations of USP1 in HCC were analysed using cBioPortal and the Catalogue of Somatic Mutations in Cancer database. Differential genes correlated with USP1 and WD repeat domain 48 (WDR48) were obtained using LinkedOmics. USP1 was knocked down with small interfering RNA (siRNA) or pharmacologically inhibited by ML-323 in MHCC97H or SK-Hep-1 cell lines for function analysis.. High USP1 expression predicted unfavourable overall survival in HCC patients. USP1 showed positive correlations with the abundances of macrophages and neutrophils. We identified 98 differential genes that were positively correlated with both USP1 and WDR48. These genes were mainly involved in the cell cycle, aldosterone synthesis and secretion and oestrogen signalling pathways. Interactions between USP1 and WDR 48 were confirmed using co-immunoprecipitation. USP1 knockdown or ML-323 treatment reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1.. Overall, USP1 is a promising target for HCC treatment with good prognostic value. USP1 and WDR48 function together in regulating cancer cell proliferation via the cell cycle.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin E; Disease-Free Survival; Enzyme Inhibitors; Estrogens; Humans; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Liver Neoplasms; Oncogene Proteins; Prognosis; Proliferating Cell Nuclear Antigen; Protein Interaction Maps; RNA Interference; RNA, Small Interfering; Signal Transduction; Ubiquitin-Specific Proteases

Rho guanine nucleotide exchange factor 11 (ARHGEF11) has been proved to promote tumor metastasis in glioblastoma and ovarian carcinoma. However, the role of ARHGEF11 in hepatocellular carcinoma (HCC) progression is largely unknown. Here, we found that ARHGEF11 was upregulated in HCC samples and highly metastatic hepatoma cell lines. Knockdown of ARHGEFF11 inhibited the cell proliferation and invasion in both HCCLM3 and SKHEP1 cell lines. Subsequent mechanistic investigation showed that downregulation of ARHGEF11 significantly attenuated β-catenin nuclear translocation, thereafter repressed the expression of ZEB1 and cyclinD1, finally contributing to inhibition of epithelial-mesenchymal transition (EMT) and cell cycle arrest. Moreover, high levels of ARHGEF11 were found to be associated with shorter disease free and overall survival. A prognostic nomogram model that integrated ARHGEF11, tumor size and BCLC classification showed good performance in predicting clinical outcomes of HCC patients. Overall, this study demonstrated that ARHGEF11 could promote proliferation and metastasis of HCC via activating β-catenin pathway, suggesting that ARHGEF11 might serve as a potential prognostic biomarker for HCC.

Topics: Adolescent; Adult; Aged; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Decision Support Techniques; Disease-Free Survival; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Nomograms; Predictive Value of Tests; Rho Guanine Nucleotide Exchange Factors; Tumor Burden; Wnt Signaling Pathway; Young Adult; Zinc Finger E-box-Binding Homeobox 1

Pyruvate kinase M2 (PKM2) is not only a key rate-limiting enzyme that guides glycolysis, but also acts as a non-metabolic protein in regulating gene transcription. In recent years, a series of studies have confirmed that post-translational modification has become an important mechanism for regulating the function of PKM2, which in turn affects tumorigenesis. In this study, we found that K62 residues were deacetylated, which is related to the prognosis of HCC. Further studies indicate that HDAC8 binds and deacetylates the K62 residue of PKM2. Mechanistically, K62 deacetylation facilitate PKM2 transport into the nucleus and bind β-catenin, thereby promoting CCND1 gene transcription and cell cycle progression. In addition, the deacetylation of K62 affects the enzyme activity of PKM2 and the flux of glucose metabolism. Therefore, these results suggest that HDAC8 / PKM2 signaling may become a new target for the treatment of HCC.

Topics: Acetylation; Animals; beta Catenin; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; G1 Phase; Gene Expression Regulation, Neoplastic; Glucose; Glycolysis; Histone Deacetylases; Humans; Liver Neoplasms; Lysine; Male; Membrane Proteins; Mice; Middle Aged; Models, Biological; Protein Binding; Protein Transport; Repressor Proteins; S Phase; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Up-Regulation

Combination treatment is a promising strategy to improve prognosis of hepatocellular carcinoma (HCC). Sorafenib is a traditional first-line agent approved for the treatment of advanced HCC, though with limited efficacy. Previously, we reported that lonafarnib, an orally bioavailable non-peptide inhibitor targeting farnesyltransferase, synergizes with sorafenib against the growth of HCC cells. In the present study, we aim to clarify the underlying mechanism of this combination strategy. Initially, using in vitro HCC cell model, we confirmed that synergistic treatment of lonafarnib and sorafenib suppressed cell viability and colony formation, and induced cell death. We then found conversion of LC3-I to LC3-II via combination the treatment and observed formation of autophagosomes by electron microscopy. Knockdown of ATG3 inhibited the autophagic flux induced by the combination treatment. Furthermore, we demonstrated that drug-eliciting autophagy selectively promoted the degradation of cyclin D1 in a lysosome-dependent manner and subsequently inhibited DNA synthesis through downregulating the phosphorylation of Rb protein. In conclusion, our results provide a deeper insight into the mechanism for the combination treatment of lonafarnib and sorafenib in HCC therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Autophagy-Related Proteins; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Enzyme Inhibitors; Farnesyltranstransferase; Female; Gene Knockdown Techniques; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; Models, Biological; Piperidines; Protein Kinase Inhibitors; Proteolysis; Pyridines; Sorafenib; Ubiquitin-Conjugating Enzymes; Xenograft Model Antitumor Assays

Polycomb group (PcG) proteins have recently been identified as critical regulators in tumor initiation and development. However, the function of CBX8 in human hepatocellular carcinoma (HCC) remains largely unknown. Our study was designed to explore the biological function and clinical implication of CBX8 in HCC. We investigated the interplay between CBX8 and cell cycle through Gene Set Enrichment Analysis and western blotting. Bioinformatics tools and co-immunoprecipitation were used to explore cell cycle regulation. Finally, we studied the expression and clinical significance of CBX8 in HCC through 3 independent datasets. CBX8 was upregulated in HCC and its expression correlated with cell cycle progression. CyclinD1 was downregulated by CBX8 knockdown but upregulated by CBX8 overexpression. YBX1 interacted with CBX8 and regulated the cell cycle. Moreover, targeting YBX1 with specific siRNA impaired CBX8-mediated regulation of CyclinD1. CBX8 overexpression boosted HCC cell growth, while CBX8 knockdown suppressed cell proliferation. Further, YBX1 interacted with CBX8. YBX1 knockdown compromised the proliferation of CBX8 overexpressing cells. CBX8 promotes HCC cell proliferation through YBX1 mediated cell cycle progression and is related to poor HCC prognoses. Therefore, CBX8 may serve as a potential target for the diagnosis and treatment of HCC.

Topics: Carcinoma, Hepatocellular; Case-Control Studies; Cell Cycle; Cell Proliferation; Cohort Studies; Cyclin D1; Humans; Liver Neoplasms; Polycomb Repressive Complex 1; Y-Box-Binding Protein 1

Topics: Amnion; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Culture Media, Conditioned; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; MicroRNAs; Placenta; Pregnancy; Proto-Oncogene Proteins c-mdm2; Stem Cells; Tumor Suppressor Protein p53

Hepatocellular carcinoma (HCC) is one of the primary causes of cancer‑associated deaths worldwide. Current treatment methods include surgical resection, chemotherapy and radiotherapy; however the curative rate remains low, thus novel treatments are required. The aim of the present study was to investigate the role of targeting protein for Xenopus kinesin‑like protein 2 (TPX2) in the growth of HCC and its underlying molecular mechanism. Immunohistochemistry staining, reverse transcription‑quantitative (RT‑q)PCR and western blotting were used to detect the expression of TPX2 mRNA and protein in liver cancer tissue samples, adjacent normal liver tissue samples, and the HCC cell lines Huh7, Hep3B, PLC/PRF/5 and MHCC97‑H. The recombinant plasmid pMagic4.1‑shRNA‑TPX2 was constructed and transfected into Huh7 and Hep3B HCC cells to silence TPX2 expression. The proliferation, apoptosis, migration and invasion of Huh7 cells and Hep3B cells were evaluated before and after TPX2 silencing. The mRNA and protein expression levels of multiple signaling pathway‑associated genes were detected by RT‑qPCR and western blotting. The expression levels of TPX2 mRNA and protein were significantly higher in HCC tissue samples compared with adjacent normal liver tissue sample. TPX2 mRNA and protein expression levels were detected in the different HCC cell lines. The recombinant plasmid pMagic4.1‑shRNA‑TPX2 was successfully transfected into Huh7 and Hep3B cells, resulting in TPX2 silencing. TPX2 knockdown significantly reduced cell proliferation, cell migration and cell invasion of Huh7 and Hep3B cells, whilst also increasing the rate of apoptosis in these cells. Following TPX2 silencing, the expression levels of PI3K, phospho‑AKT, Bcl‑2, c‑Myc and Cyclin D1 were significantly decreased, whereas the expression levels of P21 and P27 were significantly increased. In conclusion, TPX2 may suppress the growth of HCC by regulating the PI3K/AKT signaling pathway and thus, TPX2 may be a potential target for the treatment of liver cancer.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Neoplasms; Microtubule-Associated Proteins; Neoplasm Proteins; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Sorafenib (SO) is a multi-kinase inhibitor that targets upstream signals in the MAPK pathway. Drug resistance and transient survival benefits are the main obstacles associated with SO treatment in Hepatocellular carcinoma (HCC) patients. Mebendazole (MBZ), an anthelmintic agent, has demonstrated activity against various cancer types. Therefore, we aimed to investigate the possible mechanisms of MBZ other than its anti-tubulin activity. MBZ (100 mg/kg/day, P.O.) was administered to N-nitrosodiethylamine-induced HCC mice as a monotherapeutic agent or in combination with SO. Our results revealed that MBZ decreased AFP levels, improved liver function and histology and increased survival in HCC mice, particularly when administered in combination with SO. MBZ also reduced hepatic inflammation and fibrogenesis as evidenced by reductions in TNF-α and TGF-β1 levels, respectively. Increased hepatic caspases-3 and -9 and decreased BCL-2 levels suggest induced-cell death. In addition, MBZ demonstrated anti-angiogenic, anti-metastatic, and anti-proliferative effects, as indicated by reduced VEGF levels, MMP-2:TIMP-1 ratios, and reduced cyclin D1 levels and Ki67 immunostaining, respectively. Our main finding was that MBZ targeted downstream signal of the MAPK pathway by inhibiting ERK1/2 phosphorylation. Targeting downstream MAPK signalling by MBZ and upstream signalling by SO is a novel approach to minimizing resistance and prolonging survival.

Topics: Alanine Transaminase; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cyclin D1; Diethylnitrosamine; Hep G2 Cells; Humans; Kaplan-Meier Estimate; Ki-67 Antigen; Liver Neoplasms; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Mebendazole; Mice; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Signal Transduction; Sorafenib; Tissue Inhibitor of Metalloproteinase-1; Tumor Burden; Vascular Endothelial Growth Factor A

Hepatocellular carcinoma (HCC) is a common human malignant cancer due to a high metastatic capacity and the recurrence rate is also high. This study is aim to investigate the role of musashi1 as a potential biomarker for therapy of HCC.. The mRNA and protein expression levels of musashi1 were detected in HCC samples and cell lines. The malignant properties of HCC cells, including proliferation, invasion and migration were measured by overexpressing or knocking down expression of musashi1. Additionally, the correlation between musashi1 and clinicopathological indexes and prognosis were analyzed. The expression of CD44 was measured and the correlation between CD44 and musashi1 was analyzed.. In vitro cytological experiments demonstrated that musashi1 was elevated in HCC samples and cell lines and this increased expression affected cancer cell viability, migration and invasive capacity by activating of the Wnt/β-catenin signaling pathway. Analysis of clinicopathological characteristics suggested that up-regulation of musashi1 was related to metastasis potential and a poor prognosis. Besides, there was a positive correlation between CD44 and musashi1 expression. Upregulation of musashi1 in malignant liver tumors may have contributed to the maintenance of stem-cell like characteristics of HCC cells.. Upregulation of musashi1 could enhance malignant development of HCC cells and thus might be a novel marker for HCC therapy.

Topics: Actins; Adenomatous Polyposis Coli Protein; beta Catenin; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Female; Humans; Hyaluronan Receptors; Liver Neoplasms; Male; Middle Aged; Molecular Targeted Therapy; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Nerve Tissue Proteins; Prognosis; RNA-Binding Proteins; RNA, Messenger; Up-Regulation; Wnt Signaling Pathway

The aim of this study was to make a clinical characterisation of patients with hepatocellular carcinoma and to investigate the expression of a set of molecular markers in patients from the Republic of North Macedonia. We analysed 60 patients for clinicopathologic factors, and we investigated tumour tissue and surrounding liver tissue for immunoexpression of E-cadherin, β-catenin, cyclin D1, and p53. Infection with hepatitis virus B and C (p < 0.001), tumour dimension (p < 0.001), vascular invasion (p < 0.002), and tumour differentiation (p < 0.021) significantly influenced the survival of the patients. E-cadherin and β-catenin expression reduction and cyclin D1 and p53 overexpression were significantly higher in the tumour than in the non-tumour tissue (p < 0.001; p < 0.001; p = 0.001; p < 0.001, respectively). No significant correlation was found between clinicopathological characteristics and the analysed molecules nor between the molecules themselves. The immunoexpression of E-cadherin, β-catenin, cyclin D1, and p53 was not related to the tumour aggressiveness and prognosis. However, their significantly higher expression in HCC tissue compared to that in non-tumour tissue indicate their important role in hepatocarcinogenesis. The clinicopathological characteristics of the neoplasm remain highly predictive factors for the survival of the patients.

Topics: Antigens, CD; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cyclin D1; Humans; Immunohistochemistry; Liver Neoplasms; Prognosis; Tumor Suppressor Protein p53

Increasing studies have indicated that long noncoding RNAs (lncRNAs) exert important roles in hepatocellular carcinoma (HCC). Therefore, it is of great significance to identify the dysregulated lncRNAs in HCC. According to the previous reports, it has been suggested that DiGeorge syndrome critical region gene 5 (DGCR5) might participate in HCC and can serve as potential biomarker for HCC. In our current study, we concentrated on the biological function and roles of lncRNA-DGCR5 in HCC. It was indicated that DGCR5 was decreased in HCC tissues and HCC cells including HepG2, Hep3B, MHCC-97L, SNU-449, and SNU-182 cells compared with the normal human liver cell line LO2. Overexpression of DGCR5 was able to restrain HCC growth, migration, and invasion capacity in HepG2 and SNU-449 cells. In addition, whether lncRNA-DGCR5 can regulate Wnt/β-catenin pathway during HCC progression is unclear. In our study, it was found that upregulation of DGCR5 inactivated Wnt signaling pathway through inhibiting β-catenin, cyclin D1 and increasing GSK-3β levels. Subsequently, in vivo tumor xenografts were established using HepG2 cells to investigate the function of DGCR5 in HCC development. Inconsistent with the in vitro findings, increase of DGCR5 dramatically suppressed HCC tumor progression in vivo. Taken these together, it was uncovered in our research that DGCR5 could play tumor suppressive role by targeting Wnt signaling in HCC progression.

Topics: Animals; Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Cell Movement; Cell Survival; Cyclin D1; Disease Progression; Female; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; RNA, Long Noncoding; Transfection; Transplantation, Heterologous; Tumor Burden; Up-Regulation; Wnt Signaling Pathway

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most lethal cancer worldwide. Although gene mutations associated with HCC development have been intensively studied, how epigenetic factors specifically modulate the functional properties of HCC by regulating target gene expression is unclear. Here we demonstrated the overexpression of KDM3B in liver tissue of HCC patients using public RNA-seq data. Ablation of KDM3B by CRISPR/Cas9 retarded the cell cycle and proliferation of hepatocarcinoma HepG2 cells. Approximately 30% of KDM3B knockout cells exhibited mitotic spindle multipolarity as a chromosome instability (CIN) phenotype. RNA-seq analysis of KDM3B knockout revealed significantly down-regulated expression of cell cycle related genes, especially cell proliferation factor CDC123. Furthermore, the expression level of Cyclin D1 was reduced in KDM3B knockout by proteosomal degradation without any change in the expression of CCND1, which encodes Cyclin D1. The results implicate KDM3B as a crucial epigenetic factor in cell cycle regulation that manipulates chromatin dynamics and transcription in HCC, and identifies a potential gene therapy target for effective treatment of HCC.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin D1; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genes, cdc; Hep G2 Cells; Humans; Jumonji Domain-Containing Histone Demethylases; Liver; Liver Neoplasms; Transcription, Genetic

Fam134b (JK-1, RETREG1) was first identified as an oncogene in esophageal squamous cell carcinoma. However, the roles of FAM134B during tumorigenesis of hepatocellular carcinoma (HCC) and in epithelial-to-mesenchymal transition (EMT) were previously unclear. In this study, we investigated the function of FAM134B in HCC and the related tumorigenesis mechanisms, as well as how FAM134B induces EMT. We detected the expression of FAM134B in a normal hepatic cell line, HCC cell lines, fresh specimens, and a HCC tissue microarray. A retrospective study of 122 paired HCC tissue microarrays was used to analyze the correlation between FAM134B and clinical features. Gain- and loss-of-function experiments, rescue experiments, Akt pathway activator/inhibitors, nude mice xenograft models, and nude mice lung metastasis models were used to determine the underlying mechanisms of FAM134B in inducing tumorigenesis and EMT in vitro and in vivo. The expression level of FAM134B was highly elevated in HCC, as compared with that in normal liver tissues and normal hepatic cells. Overexpression of FAM134B was significantly associated with tumor size (P = 0.025), pathological vascular invasion (P = 0.026), differentiation grade (P = 0.023), cancer recurrence (P = 0.044), and portal vein tumor thrombus (P = 0.036) in HCC. Patients with high expression of FAM134B had shorter overall survival and disease-free survival than patients with non-high expression of FAM134B. Furthermore, knockdown of FAM134B with shRNAs inhibited cell growth and motility, as well as tumor formation and metastasis in nude mice, all of which were promoted by overexpression of FAM134B. Our study demonstrated that Fam134b is an oncogene that plays a crucial role in HCC via the Akt signaling pathway with subsequent glycogen synthase kinase-3β phosphorylation, accumulation of β-catenin, and stabilization of Snail, which promotes tumorigenesis, EMT, and tumor metastasis in HCC.

Topics: Aged; Animals; beta Catenin; Cadherins; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Enzyme Activation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Membrane Proteins; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Metastasis; Protein Stability; Proto-Oncogene Proteins c-akt; Signal Transduction; Snail Family Transcription Factors; Up-Regulation

Overexpressed CCND1 (cyclin D1) is associated with hepatocellular carcinoma (HCC) and we used 147 tumor tissue samples from HCC patients and 3 murine models to reveal an inverse correlation between low autophagic activity and high CCND1 expression. These 2 phenomena in combination correlated with poor overall survival in HCC patients. Mechanistic analysis showed that activated autophagy triggered CCND1 ubiquitination followed by SQSTM1 (sequestosome 1)-mediated selective phagophore recruitment, autophagosome formation, fusion with a lysosome, and degradation. Functional studies revealed that autophagy-selective degradation of CCND1 suppresses DNA synthesis, cell proliferation, and colony, and liver tumor formation by arresting the cell cycle at the G

Topics: Animals; Autophagy; Carcinogenesis; Carcinoma, Hepatocellular; Cyclin D1; Humans; Liver Neoplasms; Mice; MicroRNAs

To investigate the interaction between interleukin-17 (IL-17) and interferon-γ (IFN-γ) and how their interaction affects the growth of mouse hepatoma Hepa1-6 cells.. Hepa1-6 cells treated with IL-17 and IFN-γ either alone or in combination were examined for changes in cell proliferation using MTT assay and in cell cycle distribution using flow cytometry. Western blotting was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, P21 and P16 and the phosphorylation of p38MAPK, ERK1/2 and Stat1 in the cells.. Compared with control group, IFN-γ treatment obviously inhibited the growth and proliferation of Hepa1-6 cells, induced cell cycle arrest at G0/G1 phase, reduced the protein expression of PCNA and cyclin D1, and increased the protein expression of P21. IL-17 alone had no effect on the growth of Hepa1-6 cells. In the combined treatment, IL-17 significantly antagonized the effects of IFN-γ. Compared with those treated with IFN-γ alone, the cells with the combined treatment showed significantly decreased G0/G1 cell population, increased the protein expressions of PCNA and cyclin D1, and decreased the protein expression of P21. IL-17 significantly inhibited IFN-γ-induced phosphorylation of p38MAPK and ERK1/2 without affecting the phosphorylation of Stat1.. IL-17 obviously reverses the antitumor effects of IFN-γ to promote the proliferation of mouse hepatoma cells and accelerate the development of hepatocellular carcinoma.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Interferon-gamma; Interleukin-17; Liver Neoplasms; Mice; Neoplasm Proteins; Proliferating Cell Nuclear Antigen

The effects of epigallocatechin-3-gallate (EGCG) and metformin single treatment have been tested against hepatocellular carcinoma (HCC). This study aimed to assess the combination effects of EGCG and metformin on proliferation and apoptosis of HepG2cells and identified new potential molecular targets. The effect of EGCG and metformin against cell proliferation in HepG2 was determined using MTT assay. Reverse transcription polymerase chain reaction was applied to examine the gene expression of cyclin D1, lncRNA-AF085935, caspase-3, survivin and VEGF. The level of protein expression of glypican-3 was assessed by western blot. In HepG2 cells, EGCG and metformin combination treatment exhibited high significant effect against tumor proliferation. It significantly reduced cyclin D1, lncRNA-AF085935, glypican-3 and promoted apoptosis through increasing caspase3 and decreasing survivin compared to control cells. Moreover, EGCG and metformin treated cells showed decreased expression levels of VEGF. Our study provided new insights of the anticarcinogenic effects of EGCG and metformin on HCC through their effects on glypican-3 and lncRNA-AF085935.

Topics: Anticarcinogenic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Catechin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Glypicans; Hep G2 Cells; Humans; Liver Neoplasms; Metformin; RNA, Long Noncoding; Signal Transduction; Survivin; Vascular Endothelial Growth Factor A

Chemokine-like factor (CKLF)-like, MAL and related proteins for vesicle trafficking and membrane link (MARVEL) transmembrane domain-containing family proteins (CMTMs) have significant roles in the immune system, in male reproduction, as well as in tumorigenesis. Previous studies have shown that CMTM family member 7 (CMTM7) was broadly expressed in various normal tissues, but not in lung, gastric, esophageal, pancreas, and cervix cancers. To explore its relationship with liver cancer, we examined the expression of CMTM7 in liver cancers and its correlation with clinical and pathological conditions. We found that CMTM7 expression was markedly reduced in liver cancer tissues, and negatively correlated with TNM staging and tumor metastasis. In vitro studies showed that enforced expression of CMTM7 inhibited the cell growth and migration of liver cancer cells. Further analysis revealed that CMTM7 suppressed AKT signaling and induced cell cycle arrest at the G0/G1 phase in the liver cancer cells, likely as the consequent of decreased levels of cyclin D1, cyclin-dependent kinase 4 (CDK4), and CDK6, and increased p27 expression. Thus, CMTM7 functions as a tumor suppressor in liver cancer through suppressing cell cycle progression.

Topics: Aged; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chemokines; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Cirrhosis; Liver Neoplasms; Lymphatic Metastasis; Male; MARVEL Domain-Containing Proteins; Middle Aged; Neoplasm Staging; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; Signal Transduction

The present study aims to investigate the effects of ginsenoside Rg3 combined with oxaliplatin on the proliferation and apoptosis of hepatocellular carcinoma cells and the related mechanism. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to examine the proliferation rate of hepatocellular carcinoma cell SMMC-7721 with different treatment. Flow cytometry was performed to examine apoptosis rate of hepatocellular carcinoma cells with different treatment. Immunofluorescence and Western blot methods were used to evaluate the expressions of proliferating cell nuclear antigen (PCNA) and cyclin D1 in different groups. We found that ginsenoside Rg3, oxaliplatin or ginsenoside Rg3 + oxaliplatin significantly suppressed the proliferation and promoted the apoptosis of SMMC-7721. Meanwhile, ginsenoside Rg3, oxaliplatin or ginsenoside Rg3 + oxaliplatin also significantly inhibited the expressions of PCNA and cyclin D1. Moreover, compared with ginsenoside Rg3 group and oxaliplatin group, the effect of ginsenoside Rg3 + oxaliplatin was more remarkable. Taken together, cells treated with oxaliplatin+ ginsenoside enhanced the anti-tumor effect and may inhibit the proliferation and promoted apoptosis of hepatocellular carcinoma via regulating the expression of PCNA and cyclin D1.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Synergism; Ginsenosides; Humans; Liver Neoplasms; Oxaliplatin; Proliferating Cell Nuclear Antigen

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumor growth and glutamine metabolism. However, the role of GLS1 in hepatocellular carcinoma (HCC) progression remains to be elucidated. In this study, we reported that GLS1 expression was significantly increased in HCC tissues and correlated with serum AFP, tumor differentiation, lymphatic metastasis, TNM stage, and poorer patient outcome. We further showed that GLS1 promoted colony formation and cell proliferation of HCC cells. Furthermore, our data showed that GLS1 inhibitor compound 968 inhibited the proliferation of HCC cells in a dose-dependent manner. Importantly, we found that GLS1 overexpression increased p-AKT, p-GSK3β and cyclinD1 expression, and had no influence on total AKT and GSK3β protein level, indicating that GLS1 was involved in AKT/GSK3β/CyclinD1 pathway. It is suggested that GLS1 promotes proliferation in HCC cells probably via AKT/GSK3β/CyclinD1 pathway and may be a potential target for anti-hepatocellular carcinoma cancer.

Topics: Animals; Benzophenanthridines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Drug Delivery Systems; Female; Glutaminase; Glycogen Synthase Kinase 3 beta; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Oncogenes; Proto-Oncogene Proteins c-akt; Retrospective Studies; Signal Transduction; Up-Regulation

DEK is an oncogene that has been identified as part of the DEK-CAN fusion gene. DEK plays a role in carcinogenesis through WNT signaling and induces cell proliferation through cyclin-dependent kinase signaling. DEK overexpression has been reported in HCC, but the clinical significance is unclear. This study enrolled 221 cases of HCC. The expression of DEK protein was evaluated by immunohistochemical staining. Cdk4, cyclin D1, Wnt10b, E-cadherin, and β-catenin were also immunohistochemically stained and analyzed for correlation. The association of clinicopathologic factors with DEK expression was analyzed. DEK expression was observed in 44.8% (99/221) of cases. DEK expression showed a statistical association with clinicopathologic factors, including Edmondson-Steiner grade, presence of vascular emboli, and multiplicity (p<0.05). Among the other IHC markers, the expression of cdk4 was correlated with DEK expression (p<0.05). Patients with high DEK expression showed a significantly lower overall survival rate (p=0.006). However, the disease-free survival rate did not differ significantly. In addition, in a Cox regression model analysis, DEK expression was an independent prognostic factor. In summary, high expression of DEK was observed in HCC and was associated with poor prognostic marker expression and poor prognosis.

Topics: Adult; Aged; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Chromosomal Proteins, Non-Histone; Cyclin D1; Cyclin-Dependent Kinase 4; Disease-Free Survival; Female; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Oncogene Proteins; Poly-ADP-Ribose Binding Proteins; Prognosis; Proto-Oncogene Proteins; Survival Rate; Wnt Proteins

Plumbagin exerts effective anti-hepatocellular carcinoma (HCC) benefits, however, the detailed mechanisms behind these effects are not yet completely elucidated. The pharmacological targets and molecular mechanisms of plumbagin against HCC were revealed through conducting network pharmacology approach before experimentative verification.. The web-accessible databases of herbal ingredients' targets (HIT), Swiss-Target-Prediction and Super-Pred were used to predict the therapeutic targets of plumbagin, followed by combined with pathogenic targets of HCC from oncogenomic database of hepatocellular carcinoma (OncoDB.HCC) and Liverome databases to obtain the predominant targets of plumbagin-treating HCC. The database for annotation, visualization and integrated discovery (DAVID) was applied to output the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment by use of all predominant targets for computerized visualization. The validated data of human and cell culture were subjected to a group of medical imaging, biochemical tests and immunostaining, respectively.. As revealed in bioinformatic data, 19 predominant targets of plumbagin-treating HCC were obtained, and 5 top targets of TP53, MAPK1, MAP2K1, RAF1 and CCND1 were the most important biomolecules in anti-HCC effects exerted by plumbagin. Other identifiable 102 GO items were showed, including 66 biological processes, and 12 cellular components, 24 molecular functions. And 67 KEGG pathways were mainly involved in neoplastic signaling. In human data, HCC sections showed increased expressions of hepatocellular TP53, MAPK1, accompanied with positive clinical imaging results for HCC. In plumbagin-treated HepG2 cells, reduced TP53, MAPK1 protein expressions were observed, accompanied with cell arrest and apoptosis.. Collectively, the pharmacological targets and mechanisms of plumbagin-treating HCC were predicted and integrated through the method of network pharmacology, followed by some investigative validations. Interestingly, these 5 predominant biomolecules may be the potential targets for screening and treating HCC.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Computational Biology; Cyclin D1; Female; Hep G2 Cells; Humans; Liver Neoplasms; Male; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Naphthoquinones; Proto-Oncogene Proteins c-raf; Tumor Suppressor Protein p53

Hepatocellular carcinoma (HCC) is a highly heterogeneous, multigene-driven malignant tumor. ZNF384 is an overexpressed gene with a high frequency of alteration in HCC, but research on the function of ZNF384 in HCC is lacking. In this study, the expression level of ZNF384 in HCC was analyzed through immunohistochemical (IHC) staining, Western blot analysis and qRT-PCR. We also generated ZNF384 knockdown and knockout HCC cell lines using short hairpin RNA (shRNA) and CRISPR/Cas9 systems. MTS, colony formation, and 5-ethynyl-20-deoxyuridine (EdU) assays; flow cytometry; and a xenograft mouse model were used to evaluate the effects of ZNF384 on cell proliferation. Western blot analysis, a dual luciferase reporter assay and a ChIP assay were performed to explore the potential mechanism. We found that overexpression of ZNF384 in HCC and elevated expression of ZNF384 in HCC tissues was significantly correlated with tumor recurrence (P = 0.0097). Kaplan-Meier survival analysis revealed that high expression levels of ZNF384 were correlated with poor overall survival (P = 0.0386). Downregulation of ZNF384 expression suppressed HCC cell proliferation by inhibiting the expression of Cyclin D1. These findings suggest that ZNF384 tends to act as an oncogene in the development of HCC. ZNF384 promotes the proliferation of HCC cells by directly upregulating the expression of Cyclin D1 and might serve as a prognostic predictive factor for HCC patients.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Prognosis; Promoter Regions, Genetic; Protein Binding; Trans-Activators; Transplantation, Heterologous; Up-Regulation; Zinc Fingers

Emerging evidence indicates that Long non-coding RNAs (LncRNAs) and microRNAs (miRNAs) play crucial roles in tumor progression, including hepatocellular carcinoma (HCC). However, whether there is a crosstalk between LncRNA pituitary tumor-transforming 3 (PTTG3P) and miR-383 in HCC remains unknown. This study is designed to explore the underlying mechanism by which LncRNA PTTG3P sponges miR-383 during HCC progression.. qPCR and Western blot were used to analyze LncRNA PTTG3P, miR-383 and other target genes' expression. CCK-8 assay was performed to examine cell proliferation. Annexin V-PE/PI and PI staining were used to analyze cell apoptosis and cell cycle distribution by flow cytometry, respectively. Transwell migration and invasion assays were used to examine cell migration and invasion abilities. An in vivo xenograft study was performed to detect tumor growth. Luciferase reporter assay and RNA pull-down assay were carried out to detect the interaction between miR-383 and LncRNA PTTG3P. RIP was carried out to detect whether PTTG3P and miR-383 were enriched in Ago2-immunoprecipitated complex.. In this study, we found that PTTG3P was up-regulated in HCC tissues and cells. Functional experiments demonstrated that knockdown of PTTG3P inhibited cell proliferation, migration and invasion, and promoted cell apoptosis, acting as an oncogene. Mechanistically, PTTG3P upregulated the expression of miR-383 targets Cyclin D1 (CCND1) and poly ADP-ribose polymerase 2 (PARP2) by sponging miR-383, acting as a competing endogenous RNA (ceRNA). The PTTG3P-miR-383-CCND1/PARP2 axis modulated HCC phenotypes. Moreover, PTTG3P also affected the PI3K/Akt signaling pathway.. The data indicate a novel PTTG3P-miR-383-CCND1/PARP2 axis in HCC tumorigenesis, suggesting that PTTG3P may be used as a potential therapeutic target in HCC.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Liver; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Oncogenes; Poly(ADP-ribose) Polymerases; RNA, Long Noncoding; Up-Regulation

Hepatocellular carcinoma (HCC) is characterized by bad prognosis and is the second most common reason for cancer-linked mortality. Treatment with sorafenib (SRF) alone increases patient survival by only a few months. A causal link has been determined between angiotensin II (Ang-II) and HCC. However, the mechanisms underlying the tumorigenic effects of Ang-II remain to be elucidated. N-Nitrosodiethylamine was utilized to examine the effects of telmisartan (TEL) (15 mg/kg), SRF (30 mg/kg), and a combination of these two agents on HCC mice. Downregulation of NF-кBP65 mRNA expression and inhibition of the phosphorylation-induced activation of both ERK1/2 and NF-кB P65 were implicated in the anti-tumor effects of TEL and SRF. Consequent regression of malignant changes and improvements in liver function associated with reduced levels of AFP, TNF-α, and TGF-β1 were also confirmed. Anti-proliferative, anti-metastatic, and anti-angiogenic effects of treatment were indicated by reduced hepatic cyclin D1 mRNA expression, reduced MMP-2 levels, and reduced VEGF levels, respectively. TEL, but not SRF, demonstrated agonistic activity for PPARγ receptors, as evidenced by increased PPARγ DNA binding activity, upregulation of CD36, and HO-1 mRNA expression followed by increased liver antioxidant capacity. Both TEL and SRF inhibited TAK1 phosphorylation-induced activation, indicating that TAK1 might act as a central mediator in the interaction between ERK1/2 and NF-кB. TEL, by modulating the ERK1/2, TAK1, and NF-кB signaling axis in the context of PPARγ agonistic activity, exerted anti-tumor effects and increased tumor sensitivity to SRF. Therefore, TEL is an encouraging agent for further clinical trials regarding the management of HCC.

Topics: Animals; Antihypertensive Agents; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Diethylnitrosamine; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Male; MAP Kinase Kinase Kinases; Matrix Metalloproteinase 2; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; PPAR gamma; Telmisartan; Transcription Factor RelA; Vascular Endothelial Growth Factor A

Dysfunction of degradation machineries causes cancers, including hepatocellular carcinoma (HCC). Overexpression of cyclin D1 in HCC has been reported. We previously reported that autophagy preferentially recruits and degrades the oncogenic microRNA (miR)-224 to prevent HCC. Therefore, in the present study, we attempted to clarify whether cyclin D1 is another oncogenic factor selectively regulated by autophagy in HCC tumorigenesis. Initially, we found an inverse correlation between low autophagic activity and high cyclin D1 expression in tumors of 147 HCC patients and three murine models, and these results taken together revealed a correlation with poor overall survival of HCC patients, indicating the importance of these two events in HCC development. We found that increased autophagic activity leads to cyclin D1 ubiquitination and selective recruitment to the autophagosome (AP) mediated by a specific receptor, sequestosome 1 (SQSTM1), followed by fusion with lysosome and degradation. Autophagy-selective degradation of ubiquitinated cyclin D1 through SQSTM1 was confirmed using cyclin D1/ubiquitin binding site (K. Taken together, our data demonstrate that autophagic degradation machinery and the cell-cycle regulator, cyclin D1, are linked to HCC tumorigenesis. We believe these findings may be of value in the development of alternative therapeutics for HCC patients. (Hepatology 2018;68:141-154).

Topics: Adult; Aged; Aged, 80 and over; Animals; Autophagosomes; Autophagy; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Liver Neoplasms; Male; Mice, SCID; Mice, Transgenic; Microtubule-Associated Proteins; Middle Aged; Rats, Sprague-Dawley; Sequestosome-1 Protein; Taiwan; Ubiquitination

Insulin receptor substrate 4 (IRS-4) is an adaptor protein for which new evidence suggests plays a role in tumour promotion.. We described nuclear IRS-4 in RKO colon cancer cell lines in biopsies of patients with colorectal cancer (CRC) (n = 20) and in matched adjacent normal colorectal (MANC) tissue (n = 20).. Treatment with physiological doses of IGF-1 promoted nuclear influx of IRS-4 from cellular cytosol in RKO cells. When exogenous IRS-4 was overexpressed in RKO cells, there was an increase in cyclin D1, cyclin E, E2F1, pRB Ser 809/811 and pRB Ser 705 levels compared with the empty vector-transfected cells. Some of these changes returned to control values after wortmannin treatment. Subcellular fractionation showed an overexpression of IRS-4 in the cytoplasm, membrane, and nuclei of tumour samples, whereas the levels of the protein were barely detectable in the three compartments of normal samples. Immunohistochemical studies showed positive nuclear IRS-4 staining in over 74% of the tumour cells. IRS-4 was strongly overexpressed in tumoural tissues from CRC patients compared to MANC tissues. The up-regulation of IRS-4 in CRC samples correlated significantly with the increase of several G1 checkpoint proteins including cyclin D1 (r = 0.6662), Rb (r = 0.7779), pRb Serine 809/811 (r = 0.6864), pRb serine 705 (r = 0.6261) and E2F1 (r = 0.8702).. Taken together, our findings suggest that IRS-4 promotes retinoblastoma-cyclin-dependent kinase activation and it may serve as a pharmacological target since its expression is very low in normal tissue, including colonic epithelium.

Topics: Adenocarcinoma; Adenoma; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell Proliferation; Colon; Colorectal Neoplasms; Cyclin D1; Cytoplasm; E2F1 Transcription Factor; Female; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Liver Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Proliferating Cell Nuclear Antigen; Protein Transport; Rectum; Retinoblastoma Protein; Signal Transduction; Up-Regulation

Hepatocellular carcinoma (HCC) is the common primary liver cancer and the third leading cause of cancer related mortality worldwide. It is generally thought that the estrogen-signaling pathway is not related to the development and progression of human HCC. However, accumulating evidences indicate the existence of a rapid estrogen signaling in HCC cells that is able to promote cell growth. However, the receptor that mediates the rapid estrogen signaling in HCC cells has not been established. Previously, our laboratory identified a variant of ER-α, ER-α36, and found that ER-α36 mediates the rapid estrogen signaling such as the activation of the MAPK/ERK signaling in breast carcinoma cells. Our current experiments studied the role of the rapid estrogen signaling mediated by ER-α36 in growth of HCC HepG2 and PLC/PRF/5 cells that highly express ER-α36 and found these cells were strongly responsive to the rapid estrogen signaling. Knockdown of ER-α36 expression in these HCC cells using the shRNA method attenuated their responsiveness to estrogen and destabilized EGFR protein. ER-α36 mediated estrogen-induced phosphorylation of Src and the MAPK/ERK as well as cyclin D1 expression. In addition, there existed an ER-α36/EGFR positive regulatory loop in HCC cells that was important for the maintenance and positive regulation of HCC tumorsphere cells. Our results thus indicated that the rapid estrogen receptor is mediated by ER-α36 in HCC cells through the EGFR/Src/ERK signaling pathway and suggested that the ER-α36/EGFR signaling loop is a potential target to develop novel therapeutic approaches for HCC treatment.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; ErbB Receptors; Estrogens; Gene Knockdown Techniques; Humans; Liver Neoplasms; Nuclear Receptor Coactivators; Signal Transduction

The current study was designed and performed to investigate the effect of mefloquine on the proliferation and tumor formation potential of liver cancer stem cells. CD133 + HepG2 cells were identified using MACS and showed markedly higher tumor formation potential compared to the parental cells. The secondary tumors formed by CD133 + cells were markedly large in size and more in number compared to the parental cells. Mefloquine treatment of CD133 + HepG2 cells inhibited the proliferation selectively in concentration based manner. The rate of proliferation was inhibited to 82 and 12% in parental and CD133 + sphere forming cells, respectively on treatment with 10 μM concentration of mefloquine. The number of secondary tumors formed by primary tumors was decreased significantly on treatment with 10 μM mefloquine concentration. Treatment of the liver cancer stem cells with mefloquine markedly decreased the potential to undergo self-renewal at 10 μM concentration after 48 h. The results from western blot analysis showed significantly higher expression of cancer stem cell molecules β-catenin and cyclin D1 in LCSCs. Treatment of the LCSCs with various concentrations of mefloquine reduced the expression levels of β-catenin and cyclin D1. Administration of the CD133 + cell tumor xenografts in the mice led to the formation of large sized tumors in the control group. However, the tumor growth was inhibited significantly in the mice on treatment with 10 mg/kg doses of mefloquine after day 21. The tumor weight was significantly lower in the animals of mefloquine treatment group compared to the control group. Thus, mefloquine treatment inhibits self-renewal and proliferation potential of cells through targeting β-catenin pathway.

Topics: AC133 Antigen; Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cyclin D1; Disease Models, Animal; Drug Combinations; Hep G2 Cells; Humans; Lithium Chloride; Liver Neoplasms; Male; Mefloquine; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Transplantation, Heterologous

ClC-3 is a type of chloride channel that has multiple functions in tumorigenesis and tumor growth, and can be blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). In the present study, we found that DIDS inhibited the proliferation of Hep3B hepatocellular carcinoma (HCC) cells in a concentration-dependent manner. More in-depth research demonstrated that DIDS downregulated the protein expression levels of cyclin D1 and cyclin E, which are key proteins of the G1 phase. Additionally, we found that ClC-3 siRNA transfection induced G1 arrest in the Hep3B cells, confirming that ClC-3 is involved in the DIDS-induced inhibition of Hep3B cells. Moreover, the level of α-fetoprotein (AFP), a negative prognostic indicator of HCC, was decreased after treatment with DIDS and ClC-3 siRNA. In conclusion, we demonstrated that ClC-3 can arrest the cell cycle at the G1 phase to inhibit cell proliferation, suggesting that ClC-3 has the potential to be a novel target for HCC therapy and potentially improve the prognosis of HCC patients.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chloride Channels; Cyclin D1; Cyclin E; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; RNA, Small Interfering

Taraxasterol has potent anti-inflammatory and anti-tumor activity. However, the effect and potential mechanisms of Taraxasterol on the growth of human liver cancer have not been clarified. Histidine triad nucleotide-binding protein 1 (Hint1) is a tumor suppressor and its downregulated expression is associated with the development of cancer. Here, we report that Taraxasterol treatment significantly suppressed cell proliferation and induced cell cycle arrest at G0/G1 phase and apoptosis in liver cancer cells, but not in non-tumor hepatocytes. Furthermore, Taraxasterol upregulated Hint1 and Bax, but downregulated Bcl2 and cyclin D1 expression, accompanied by promoting the demethylation in the Hint1 promoter region in liver cancer cells. The effects of Taraxasterol were abrogated by Hint1 silencing and partially mitigated by Bax silencing, Bcl2 or cyclin D1 over-expression in HepG2 cells. Moreover, oral administration with Taraxasterol did not affect body weight, urinary protein levels, and the heart, liver, and kidney morphology in BALB/c mice but effectively inhibited the growth of implanted SK-Hep1 tumor in vivo. Collectively, we demonstrate that Taraxasterol inhibits the growth of liver cancer at least partially by enhancing Hint1 expression to regulate Bax, Bcl2, and cyclin D1 expression. Taraxasterol may be a drug candidate for the treatment of human liver cancer.. Taraxasterol inhibits growth and induces apoptosis in human liver cancer cells. Taraxasterol enhances Hint1 expression by promoting demethylation in Hint1 promoter. Taraxasterol increases Hint1 levels to regulate Bax, Bcl2, and cyclinD1 expression. The effects of Taraxasterol are abrogated by Hint1 silencing in liver cancer cells. Taraxasterol inhibits the growth of subcutaneously implanted liver cancers in mice.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Nerve Tissue Proteins; Proto-Oncogene Proteins c-bcl-2; Sterols; Triterpenes; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is a major global health problem. Therapeutic interventions of HCC are still limited because of its complicated molecular pathogenesis. Many reports showed that renin-angiotensin system (RAS) contributes to the development of different types of malignancies. Therefore, the present study aimed to examine the effect of RAS inhibition using perindopril (1 mg/kg), fosinopril (2 mg/kg), or losartan (10 mg/kg) on diethylnitrosamine-induced HCC compared to sorafenib (30 mg/kg). The administration of RAS inhibitors resulted in improved liver function and histologic picture with a reduction in AFP levels. These effects found to be mediated through inactivation of NFкB pathway by the inhibition of NFĸB p65 phosphorylation at the Ser536 residue and inhibition of the phosphorylation-induced degradation of NFĸBia. Consequently, expression levels of cyclin D1 mRNA were significantly lowered. In addition, NFкB-induced TNF-α and TGF-β1 levels were reduced leading to lower levels of MMP-2 and VEGF. We concluded that RAS inhibition either through inhibiting the ACE or the blockade of AT1R has the same therapeutic benefit and that the tissue affinity of the ACEIs has no impact on its anti-tumor activity. These results suggest that ACEIs and ARBs can serve as promising candidates for further clinical trials in the management of HCC.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cyclin D1; Diethylnitrosamine; Fosinopril; Liver Neoplasms, Experimental; Losartan; Male; Matrix Metalloproteinase 2; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Niacinamide; Perindopril; Phenylurea Compounds; Phosphorylation; Renin-Angiotensin System; Signal Transduction; Sorafenib; Time Factors; Transcription Factor RelA; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Long intergenic non-coding RNA 00152 (LINC00152) is aberrantly expressed in various human malignancies and plays an important role in the pathogenesis. Here, we found that LINC00152 is upregulated in hepatocellular carcinoma (HCC) tissues as compared to adjacent non-neoplastic tissues; gain-and-loss-of-function analyses in vitro showed that LINC00152 facilitates HCC cell cycle progression through regulating the expression of CCND1. LINC00152 knockdown inhibits tumorigenesis in vivo. MS2-RIP analysis indicated that LINC00152 binds directly to miR-193a/b-3p, as confirmed by luciferase reporter assays. Furthermore, ectopic expression of LINC00152 partially halted the decrease in CCND1 expression and cell proliferation capacity induced by miR-193a/b-3p overexpression. Thus, LINC00152 acts as a competing endogenous RNA (ceRNA) by sponging miR-193a/b-3p to modulate its target gene, CCND1. Our findings establish a ceRNA mechanism regulating cell proliferation in HCC via the LINC00152/miR-193a/b-3p/CCND1 signalling axis, and identify LINC00152 as a potential therapeutic target for HCC.

Topics: Base Sequence; Carcinogenesis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Liver Neoplasms; MicroRNAs; RNA, Long Noncoding; Signal Transduction; Up-Regulation

MicroRNAs (miRNAs) are solid factors involved in the initiation and progression of hepatocellular carcinoma (HCC). Recently, miR-298 is recognized as a cancer-associated miRNA in breast, gastric and ovarian cancer. However, the functional role of miR-298 and its underlying mechanism are rarely reported in HCC. Herein, we found that the expression of miR-298 was down-regulated in HCC tissues and cell lines. The in vitro experiments showed that miR-298 overexpression inhibited cell proliferation, migration and invasion, and induced G1 arrest and apoptosis of HCC cells. miR-298 knockdown exerted an opposite effect on these cellular behaviors of HCC cells. Moreover, miR-298 restoration suppressed HCC tumor growth and metastasis in vivo. Additionally, catenin delta 1 (CTNND1) was demonstrated to be a direct target of miR-298 in HCC cells. CTNND1 knockdown led to similar effects with miR-298 overexpression on HCC cell proliferation, cell cycle progression, apoptosis and mobility. CTNND1 restoration reversed miR-298-induced inhibitory effects on HCC cells. Mechanistically, both miR-298 overexpression and CTNND1 knockdown repressed Wnt/β-catenin signaling and resulted in reduced expression of β-catenin, WNT11, Cyclin D1 and MMP7 in HCCLM3 cells. While, CTNND1 restoration abolished miR-298-induced inactivation of Wnt/β-catenin signaling. In conclusion, our findings provide the first evidence that miR-298 suppresses HCC progression at least partially by targeting CTNND1-mediated Wnt/β-catenin signaling. MiR-298 may be a target for new therapies in HCC patients.

Topics: Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Catenins; Cell Movement; Cell Proliferation; Cyclin D1; Delta Catenin; Disease Progression; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 7; MicroRNAs; Neoplasm Invasiveness; Time Factors; Wnt Proteins; Wnt Signaling Pathway

Developing reliable biomarkers for hepatocellular carcinoma (HCC) patients who are at a high risk of recurrence after curative hepatic resection is very important for determining subsequent therapeutic strategies. We investigated the role of the cell cycle factor NIMA-related kinase 2 (NEK2) in HCC progression in hepatoma cells and post-surgery patients.. The effects of NEK2 on proliferation, invasion and migration of hepatoma HuH7 and SK-Hep1 cells were evaluated. In a post-surgery HCC cohort (N = 97), the Nek2 induction levels in the tumors were examined with real-time RT-PCR analysis, and the results were analyzed for their correlations with recurrence.. NEK2 promoted G1 to S phase cell cycle progression by causing increases in cyclin D1 and AKT phosphorylation and decreases in the cyclin-dependent kinase inhibitor p27, indicating that NEK2 plays an important role during interphase in addition to its previously identified role in M phase. NEK2 also enhanced the proliferation, migration and invasion of hepatoma cells and regulated the expression of E-cadherin and MMP9. The Nek2 mRNA levels in the tumors were highly correlated with recurrence rates in the post-surgery HCC patients. Combined evaluation of the tumor AJCC stage and the Nek2 level can serve as a reliable method for predicting the relative risk of HCC recurrence in these patients.. NEK2 plays a significant role in cell cycle progression in the inter- and M-phases. NEK2 enhances HCC metastasis and is correlated with recurrence and thus can potentially serve a promising high-risk biomarker for HCC.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antigens, CD; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Hepatectomy; Humans; Liver Neoplasms; Male; Matrix Metalloproteinase 9; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; NIMA-Related Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Risk Factors; Signal Transduction; Time Factors; Treatment Outcome

Propofol is a commonly used anaesthetic with controversial effects on cancer cells. We aimed to explore the functional roles of propofol in hepatocellular carcinoma (HCC) cells as well as the underlying mechanisms.. HepG2 and SMMC-7721 cells were used in this study. Firstly, the effects of propofol on cell viability, migration, invasion, apoptosis, and involved proteins were assessed by Cell Counting Kit-8 assay, Transwell assay, flow cytometry assay and Western blot analysis, respectively. Subsequently, alteration of miR-374a after stimulation of propofol was analyzed by qRT-PCR. miR-374a was overexpressed and the alteration of proteins in the Wnt/β-catenin and PI3K/AKT pathways was detected by Western blot analysis. The downstream factor of miR-374a was finally studied.. Propofol inhibited cell viability, migration and invasion but promoted apoptosis of HepG2 and SMMC-7721 cells. Meanwhile, cyclinD1, matrix metalloproteinase (MMP)-2 and MMP-9 were down-regulated while Bax/Bcl-2, cleaved caspase-3 and cleaved caspase-9 were up-regulated by propofol. Then, miR-374a level was reduced by propofol. Expression of Wnt3a, β-catenin, p-PI3K and p-AKT was decreased by propofol, whereas these decreases were reversed by miR-374a overexpression. Finally, TP53 was proven to be target of miR-374a in HepG2 cells.. Propofol inhibited cell proliferation, migration and invasion while promoted cell apoptosis of HepG2 and SMMC-7721 cells through inhibiting the Wnt/β-catenin and PI3K/ AKT pathways via down-regulation of miR-374a. Besides, miR-374a affected propofol-treated HepG2 cells by targeting TP53.

Topics: 3' Untranslated Regions; Antagomirs; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MicroRNAs; Propofol; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53; Up-Regulation; Wnt Signaling Pathway

There is increasing evidence that liver cancer stem cells (LCSCs) contribute to hepatocellular carcinoma (HCC) initiation and progression. MicroRNA (miRNA) plays a significant functional role by directly regulating respective targets in LCSCs-triggered HCC, however, little is known about the function of the miRNA-302 family in LCSCs.. MiRNAs microarray was used to detect the miRNAs involved in LCSCs maintenance and differentiation. Biological roles and the molecular mechanism of miRNA-302a/d and its target gene E2F7 were detected in HCC in vitro. The expression and correlation of miRNA-302a/d and E2F7 in HCC patients was evaluated by quantitative PCR and Kaplan-Meier survival analysis.. We found that the miRNA-302 family was downregulated during the spheroid formation of HCC cells and patients with lower miRNA-302a/d expression had shorter overall survival (OS) and progression-free survival (PFS). Moreover, E2F7 was confirmed to be directly targeted and inhibited by miRNA-302a/d. Furthermore, concomitant low expression of miRNA-302a/d and high expression of E2F7 correlated with a shorter median OS and PFS in HCC patients. Cellular functional analysis demonstrated that miRNA-302a/d negatively regulates self-renewal capability and cell cycle entry of liver cancer stem cells via suppression of its target gene E2F7 and its downstream AKT/β-catenin/CCND1 signaling pathway.. Our data provide the first evidence that E2F7 is a direct target of miRNA-302a/d and miRNA-302a/d inhibits the stemness of LCSCs and proliferation of HCC cells by targeting the E2F7/AKT/β-catenin/CCND1 signaling pathway.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cyclin D1; E2F7 Transcription Factor; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; Spheroids, Cellular

Hepatocellular carcinoma is the major form of primary liver cancer, which is the second and sixth leading cause of cancer-related death in men and women, respectively. Extensive research indicates that Wnt/β-catenin signaling pathway, which plays a pivotal role in growth, development, and differentiation of hepatocellular carcinoma, is one of the major signaling pathways that is dysregulated in hepatocellular carcinoma. Cyclin D1 is a proto-oncogene and is one of the major regulators of Wnt signaling pathway, and its overexpression has been detected in various types of cancers including hepatocellular carcinoma. Using several validated bioinformatic databases, we predicted that the microRNAs are capable of targeting 3'-untranslated region of Cyclin D1 messenger RNA. According to the results, miR-20a was selected as the highest ranking microRNA targeting Cyclin D1 messenger RNA. Luciferase assay was recruited to confirm bioinformatic prediction results. Cyclin D1 expression was first assessed by quantitative real-time polymerase chain reaction in HepG2 cell line. Afterward, HepG2 cells were transduced by lentiviruses containing miR-20a. Then, the expression of miR-20a and Cyclin D1 was evaluated. The results of luciferase assay demonstrated targeting of 3'-untranslated region of Cyclin D1 messenger RNA by miR-20a. Furthermore, 238-fold decline in Cyclin D1 expression was observed after lentiviral induction of miR-20a in HepG2 cells. The results highlighted a considerable effect of miRNA-20a induction on the down-regulation of Cyclin D1 gene. Our results suggest that miR-20a can be used as a novel candidate for therapeutic purposes and a biomarker for hepatocellular carcinoma diagnosis.

Topics: 3' Untranslated Regions; Carcinoma, Hepatocellular; Computational Biology; Cyclin D1; HEK293 Cells; Hep G2 Cells; Humans; Liver Neoplasms; MicroRNAs; Proto-Oncogene Mas; Signal Transduction

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

The triterpenoid, bauerenol, from Suregada angustifolia (Baill. ex Muell.-Arg.) Airy Shaw (Euphorbiaceae) was screened for anti-cancer property using hepatocellular carcinoma cell line, HepG2. Bauerenol exhibited growth inhibitory and apoptosis inducing potential against HepG2 cancer cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxic assay revealed that bauerenol treatment significantly reduced the growth of HepG2 cells in a time- and dose-dependent manner with 50% growth inhibitory concentration doses of 45 and 25 µg/mL at 24 and 48 h treatments, respectively. Bauerenol-induced cell death reflected apoptotic morphological features, that is, cell membrane blebbing, vacuolization, chromatin condensation, and nuclear fragmentation. In addition, bauerenol treatment diminished the mitochondrial membrane potential, by inducing the efflux of cytochrome c, downregulating the levels of anti-apoptotic Bcl-2 as well as upregulating the levels of pro-apoptotic Bax, and inducing caspase activation and poly (ADP-ribose) polymerase cleavage. Moreover, bauerenol treatment activates p38MAPK and inactivates the anti-apoptotic kinases Akt and ERK1/2 through the induction of reactive oxygen species. Furthermore, bauerenol-mediated S-phase arrest was associated with downregulation of cell cycle-rate-limiting factor (cyclin D1) and upregulation of cyclin-dependent kinase inhibitor p21 and tumor suppressor p53. Interestingly, pre-treatment of cells with reactive oxygen species inhibitor and p38 inhibitor significantly decreases bauerenol-induced cytotoxicity, Bax upregulation, and p38 activation. This study clearly states that bauerenol induces cell cycle arrest and apoptosis through the reactive oxygen species-dependent p38MAPK activation in HepG2 cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cyclin D1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; p38 Mitogen-Activated Protein Kinases; Plant Extracts; Reactive Oxygen Species; Suregada; Triterpenes

Xiao-Chai-Hu Tang (XCHT) is an extract of seven herbs with anticancer properties, but its mechanism of action is unknown. In this study, we evaluated XCHT-treated hepatocellular carcinoma (HCC) for anti-proliferative and pro-apoptotic effects.. Using a hepatic cancer xenograft model, we investigated the. We found that XCHT reduced tumor size and weight, as well as significantly decreased cell viability both. Our data suggest that XCHT enhances expression of pro-apoptotic pathways, resulting in potent anticancer activity.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Drugs, Chinese Herbal; Genes, bcl-2; Humans; Liver Neoplasms

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Prognosis; Proportional Hazards Models; Signal Transduction; Transfection; Tumor Suppressor Proteins

M2 macrophages are a major component of the tumor microenvironment and are important promoters of tumor occurrence and progression. In this study, we detected large numbers of M2 macrophages in hepatocellular carcinoma tissues using immunohistochemistry and immunofluorescence. Moreover, upon oxaliplatin treatment, the M2 macrophages overexpressed interleukin-17, an important inflammatory cytokine, and thus inhibited oxaliplatin-induced apoptosis. By knocking down the interleukin-17 receptor and lysosome-associated membrane protein 2A (a key protein in chaperone-mediated autophagy) in hepatocellular carcinoma cells, we found that interleukin-17 stimulated chaperone-mediated autophagy, which further suppressed apoptosis upon oxaliplatin treatment. Chaperone-mediated autophagy induced tolerance to oxaliplatin treatment by reducing cyclin D1 expression; thus, cyclin D1 overexpression stimulated oxaliplatin-induced apoptosis. In addition, cyclin D1 expression was inhibited by interleukin-17, but increased when the interleukin-17 receptor was knocked down. Thus M2 macrophages in the hepatocellular carcinoma microenvironment generate large amounts of interleukin-17, which suppress oxaliplatin-induced tumor cell apoptosis by activating chaperone-mediated autophagy and in turn reducing cyclin D1 expression. These findings may facilitate the development of novel therapeutic strategies for chemorefractory liver cancer.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Gene Expression; Humans; Immunohistochemistry; Interleukin-17; Liver Neoplasms; Lysosomal-Associated Membrane Protein 2; Macrophages; Organoplatinum Compounds; Oxaliplatin; Receptors, Interleukin-17; RNA Interference; RNA, Small Interfering; Tumor Microenvironment

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide, and therapeutic agents for this malignancy are lacking. MicroRNAs play critical roles in carcinogenesis and present tremendous therapeutic potential. Here, we report that microRNA-206 is a robust tumor suppressor that plays important roles in the development of HCC by regulating cell-cycle progression and the cMet signaling pathway. MicroRNA-206 was underexpressed in livers of two HCC mouse models, human individuals bearing HCC, and human HCC cell lines. Combining bioinformatic prediction and molecular and cellular approaches, we identified cMET (Met proto-oncogene), cyclin D1 (CCND1), and cyclin-dependent kinase 6 (CDK6) as functional targets of microRNA-206. By inhibiting expression of cMET, CCND1, and CDK6, microRNA-206 delayed cell-cycle progression, induced apoptosis, and impaired proliferation of three distinct human HCC cell lines. Systemic administration of microRNA-206 completely prevented HCC development in both cMyc and V-Akt murine thymoma viral oncogene homolog 1/neuroblastoma RAS viral oncogene homolog (AKT/Ras) HCC mice, whereas 100% of control mice died from lethal tumor burdens. Conversely, reintroduction of cMet or Cdk6 into livers of cMyc and AKT/Ras HCC mice recovered growth of HCC inhibited by microRNA-206. These results strongly suggested that cMet and Cdk6 were two functional targets that mediated the inhibitory effect of microRNA-206 on the development of HCC. MicroRNA-206 overexpression demonstrated a profound therapeutic effect on HCC in xenograft and cMyc HCC mice.. In summary, this study defines a potentially critical role of microRNA-206 in preventing the growth of HCC and suggests its use as a potential therapeutic strategy for this malignancy. (Hepatology 2017;66:1952-1967).

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 6; Genes, myc; Humans; Liver; Liver Neoplasms, Experimental; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Proto-Oncogene Mas; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; ras Proteins; Xenograft Model Antitumor Assays

Liver cancer is one of the common gastrointestinal cancers. This study was designed to investigate the effect of the cytokine-induced apoptosis inhibitor 1 (CIAPIN1) on hepatocellular carcinoma cell proliferation and invasion.. To establish a low and high expression of CIAPIN1 in hepatoma cell lines, pGPU6/GFP/Neo and CIAPIN1 siRNA vectors were constructed. The growth curve of liver cancer cells with a low and high expression of CIAPIN1 was measured by MTT assay and colony formation in soft. The effect of overexpression and inhibition of CIAPIN1 on the expressions of cell cycle proteins Cyclin D1, CDK2, CDK4, and Cyclin E were detected by western blot.. As compared with the low expression group, the cells in CIAPIN1 high expression group showed a significant decrease in proliferation (p < 0.05). In addition, the colony-forming ability of cells with high expression of CIAPIN1 was decreased significantly (p < 0.01). Furthermore, the expressions of Cyclin D1 CDK2, CDK4, and Cyclin E in high expression group were significantly increased (p < 0.01).. CIAPIN1 played an important role in the proliferation of liver cancer cells through increasing the expressions of cell cycle related proteins Cyclin D1, CDK2, CDK4, and Cyclin E.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Microscopy, Fluorescence; Oncogene Proteins; Plasmids; RNA Interference; RNA, Small Interfering

The long noncoding RNA, homeobox transcript antisense RNA (Hotair), has been demonstrated to have an important role in regulating various biological processes in various cancers, including hepatocellular carcinoma (HCC). However, the importance of Hotair in HCC proliferation and cell cycle progression remains to be elucidated. In the present study, knockdown of HOTAIR expression by RNA interference inhibited cell proliferation and induced G0/G1 cell cycle arrest in Huh7 hepatocellular carcinoma cells. In addition, the expression levels of CCND1 mRNA and its cyclin D1 protein product were reduced in Huh7 cells following knockdown of HOTAIR. Knockdown of HOTAIR reduced the expression of phosphorylated signal transducer and activator of transcription 3 (STAT3) and HOTAIR knockdown combined with STAT3 inhibition led to an additional decrease in cyclin D1 expression. The present study suggested that Hotair may have a critical role in the proliferation of HCC by regulating cell cycle, STAT3 activity and cyclin D1 expression. Therefore, Hotair may be a novel potential therapeutic target for HCC treatment.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Liver Neoplasms; RNA Interference; RNA, Long Noncoding; STAT3 Transcription Factor

Invasion and metastasis are the primary causes of mortality from hepatocellular carcinoma (HCC). Effective inhibition against participants in the tumourigenesis and metastasis process is critical for treatment of HCC. Wnt3a is involved in the development and metastasis of many malignant tumours. However, the specific mechanisms of Wnt3a-mediated cell proliferation, invasion and metastasis in HCC remain unclear. In this study, we found that Wnt3a and its target gene c‑Myc showed higher expression in tumour tissues than normal liver tissues in HCC patients; 71.8% of the cases studied had high Wnt3a and c‑Myc expression levels (n=32); Wnt3a expression positively correlated with its target genes MMP‑7 and c‑Myc. Intriguingly, the expression of Wnt3a, MMP‑7 and c‑Myc is significantly correlated with Notch3 and Hes1 expression. In vitro experiments showed that Wnt3a was highly expressed in MHcc97H and SK‑Hep‑1 cells. Therefore, Wnt3a expression was silenced with siRNA, and then, MTT, flow cytometry, wound healing and Transwell assays were performed to analyse cell proliferation, cycle, migration and invasion. The results demonstrated that downregulation of Wnt3a expression inhibited cell viability and induced G0/G1 cell cycle arrest via decreased expression of cyclin D1 and c‑Myc and increased expression of p21 and p27. In addition, deletion of Wnt3a significantly inhibited migration and invasion by downregulating MMP‑2/-7/-9 expression via the MAPK (p38, ERK1/2 and JNK) pathway. In conclusion, our data show that Wnt3a is involved in HCC development. Wnt3a may be an effective target for treatment of HCC.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Neoplasm Metastasis; Proto-Oncogene Proteins c-myc; Up-Regulation; Wnt Signaling Pathway; Wnt3A Protein

Sex affects the risk, treatment responses and outcome of many types of cancers. The mechanism of gender disparity in development of hepatocellular carcinoma (HCC) remains obscure. Sex-determining region on Y chromosome (SRY) was overexpressed in approximate 84% male patient HCC. Moreover, we are the first to generate a liver-specific transgenic (TG) murine model with overexpression of the male specific gene SRY. Subject to a single intraperitoneal injection N-nitrosodiethylamine (DEN) at day 14, TG and wildtype (WT) mice of both genders were sacrificed at different time points (6-13.5 months). Overexpression of SRY in male TG and ectopic expression of SRY in female TG livers promoted DEN-induced hepatocarcinogenesis compared to age- and sex-matched WT. This accelerated tumorigenesis in TG of both genders was a consequence of increased injury and inflammation, fibrosis, and compensatory enhancement in hepatocytes proliferation secondary to activation of downstream targets Sox9 and platelet-derived growth factor receptor α (PDGFRα)/phosphoinositide 3-kinase (PI3K)/Akt and c-myc/CyclinD1. In conclusion, activation of SRY and its downstream Sox9 and PDGFRα pathways are commonly involved in male hepatocarcinogenesis, which provides novel insights into gender disparity and sex-specific therapeutic strategies of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cyclin D1; Diethylnitrosamine; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Health Status Disparities; Humans; Liver Neoplasms; Male; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Receptor, Platelet-Derived Growth Factor alpha; Sex Factors; Sex-Determining Region Y Protein; Signal Transduction; SOX9 Transcription Factor; Time Factors; Tumor Microenvironment; Up-Regulation

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with highest incidence in Asia and Africa. MicroRNAs (miRNAs), a class of non-coding single stranded RNA, which not only post transcriptionally regulate gene expression but also respond to signaling molecules to affect cell functions such as Wnt/β-catenin signaling specifically in HCC. The goal of this study is to investigate the crosstalk between Wnt/β-catenin signaling proteins and microRNAs expression in HCC patients.. Fresh tissue samples of 30 primary HCC patients and 10 control subjects were included. Expression level of 13 different miRNAs (miR-10a- miR-106b- miR-99a- miR-148a- miR-125b- miR-30e- miR-183- miR-155- miR-199a- miR-199a3p- miR-24- miR-122 and miR-215) were examined using real-time PCR assay. Five proteins involved in the Wnt/β-catenin pathway (β-catenin, APC, c-myc, survivin and cyclin D1) were analysed by immunohistochemistry technique. The correlation between miRNAs expression levels with protein expressions was assessed.. Up-regulation of miR-155 and miR-183 was reported in HCC patients compared to normal controls and this up-regulation was significantly correlated with liver cirrhosis in the case of miR-155 (p<0.05) referring to their oncogenic activity. Down-regulation was observed for 11 miRNAs in HCC indicating their tumour suppression activity. MiRNA-10a, miR-30e, miR-215, miR-125b and miR-148a were significantly correlated with the expression of important players in Wnt/β-catenin pathway including β-catenin, APC and c-myc (p<0.05). Detailed analysis revealed that miR-215 is associated with the grade of the disease and miR-125b is associated with HCV infection.. Collectively, our data showed potential role of miR-10a, miR-30e, miR-215, miR-125b and miR-148a as important mediators in HCC progression. Furthermore, their association with Wnt/β-catenin cascade proteins could be exploited to develop new therapeutic target strategies in HCC.

Topics: Adenomatous Polyposis Coli Protein; Aged; beta Catenin; Carcinoma, Hepatocellular; Case-Control Studies; Cyclin D1; Down-Regulation; Female; Gene Expression; Hepatitis C, Chronic; Humans; Inhibitor of Apoptosis Proteins; Liver Cirrhosis; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Grading; Proto-Oncogene Proteins c-myc; Survivin; Up-Regulation; Wnt Signaling Pathway

Hepatocellular carcinoma (HCC) is a common cancer worldwide with an aggressive and highly proliferative activity. Studies had confirmed that HCC cell proliferation is associated with the cell cycle's G1 phase, but the detailed molecular mechanism has not been thoroughly elucidated to date. Eukaryotic translation elongation factor 1A1 (eEF1A1) is an evolutionarily conserved elongation factor protein and is involved in tumor cell proliferation. However, which phase of the cell cycle is regulated by eEF1A1 to influence cell proliferation in HCC and its detailed molecular mechanism remain unclear. In this study, we observed that eEF1A1 influences HCC cell proliferation by regulating the cell cycle's G1 phase. In addition, eEF1A1 influences G1 phase by regulating cyclin D1 expression, promoting HCC cell proliferation both in vitro and in vivo. Moreover, our results indicated that eEF1A1 regulates cyclin D1 expression through STAT1 signaling. STAT1 increases the transcriptional activity of cyclin D1 by binding to the cyclin D1 promoter. Taken together, these findings enabled us to identify a novel mechanism by which eEF1A1 regulates the cell cycle's G1 phase to promote tumor proliferation by regulating cyclin D1 expression through STAT1 signaling in HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Humans; Liver Neoplasms; Peptide Elongation Factor 1; Signal Transduction; STAT1 Transcription Factor

To investigate gender-specific liver estrogen receptor (ER) expression in normal subjects and patients with hepatitis C virus (HCV)-related cirrhosis and hepatocellular carcinoma (HCC).. Liver tissues from normal donors and patients diagnosed with HCV-related cirrhosis and HCV-related HCC were obtained from the NIH Liver Tissue and Cell Distribution System. The expression of ER subtypes, ERα and ERβ, were evaluated by Western blotting and real-time RT-PCR. The subcellular distribution of ERα and ERβ was further determined in nuclear and cytoplasmic tissue lysates along with the expression of inflammatory [activated NF-κB and IκB-kinase (IKK)] and oncogenic (cyclin D1) markers by Western blotting and immunohistochemistry. The expression of ERα and ERβ was correlated with the expression of activated NF-κB, activated IKK and cyclin D1 by Spearman's correlation.. Both ER subtypes were expressed in normal livers but male livers showed significantly higher expression of ERα than females (. Gender differences were observed in ERα expression in normal livers. Alterations in ER subtype expression observed in diseased livers may influence gender-related disparity in HCV-related pathogenesis.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Nucleus; Cyclin D1; Cytoplasm; Disease Susceptibility; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Hepacivirus; Hepatitis C, Chronic; Humans; I-kappa B Kinase; Liver; Liver Neoplasms; Male; Middle Aged; NF-kappa B; Phosphorylation; Sex Factors

The tripartite motif containing (TRIM) family plays crucial roles in tumor development and progression. However, little is known about the function and mechanism of TRIM11 in hepatocellular carcinoma (HCC).. The expression levels of TRIM11 were examined by real-time PCR, Western blot and Immunohistochemical (IHC) staining. TRIM11 knockdown cells were produced by lentivirus infection, and functional assays, such as MTT, colony formation assay, migration and invasion assays and a xenograft tumor model were used to investigate the role of TRIM11 in HCC. We also determined the effect of TRIM11 on p53 signaling and its downstream molecules.. We found that TRIM11 mRNA and protein levels were significantly increased in HCC tissues as compared with normal tissues; increased levels correlated with poor patient survival. By loss- and gain-of-function investigations, knockdown of TRIM11 suppressed cell proliferation, migration, invasion in vitro and tumor growth in vivo. Moreover, TRIM11 negatively regulated p53 expression. Knockdown of p53 abrogated the in vitro and in vivo biological functions of TRIM11 shRNA in HCC cells.. These data show that TRIM11 exerts its oncogenic effect in HCC by downregulating p53 both in vitro and in vivo. Our data provide new insights into the pathogenesis of HCC and indicate that TRIM11 may serve as a new therapeutic target for HCC treatment.

Topics: Animals; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Epithelial-Mesenchymal Transition; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous; Tripartite Motif Proteins; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases; Up-Regulation

Interleukin (IL)-20 is a proinflammatory cytokine involved in rheumatoid arthritis, atherosclerosis, and osteoporosis. However, the role of IL-20 in hepatocellular carcinoma (HCC) is unclear. We explored the function of IL-20 in HCC. Tumor tissue samples were analyzed the expression of IL-20 and cyclin D1 by using immunohistochemistry staining and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. To examine the role of anti-IL-20 monoclonal antibody (7E) in tumor growth, BALB/c mice was injected with ML-1 cells and treated with 7E. HCC tumor tissue expressed higher levels of IL-20 than did non-tumor tissue. High IL-20 expression in HCC was correlated with poor overall survival (relative risk:>3). IL-20 and cyclin D1 expression were also highly correlated in HCC patient specimens and 3 human HCC cell lines. IL-20 also increased cell proliferation and migration, and it regulated matrix metalloproteinase (MMP)-13, tumor necrosis factor (TNF)-α, cyclin D1, and p21

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents, Immunological; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; China; Cyclin D1; Female; Humans; Immunohistochemistry; Interleukins; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Middle Aged; Real-Time Polymerase Chain Reaction; Retrospective Studies

Hepatocellular carcinoma (HCC) is the most common cancer in the world. MicroRNAs (miRNAs) are a type of small noncoding RNA that can regulate the expression of target genes under physiological and pathophysiological conditions. Aberrant expression of microRNA-935 (miR-935) has been reported in cancer studies. However, its expression and mechanism in HCC remain unclear. In our study, we found that miR-935 was upregulated in liver cancer tissues and cells. Overexpression of miR-935 in liver cells promoted cell proliferation, tumorigenesis, and cell cycle progression, whereas inhibition of miR-935 reduced cell proliferation, tumorigenicity, and cell cycle progression. These changes in the properties of HCC cells were associated with upregulation of two well-known cellular G1/S transitional regulators: cyclin D1 and c-Myc. Additionally, we identified SOX7 as a direct target of miR-935. Overexpression of miR-935 inhibited SOX7 expression but promoted the levels of c-Myc and cyclin D1, which promotes cell proliferation and tumorigenesis; knockdown of miR-935 increased SOX7 level and inhibited c-Myc and cyclin D1 expression, whereas SOX7 silencing could promote cell proliferation, cell motility, and invasiveness in vitro. Our findings suggest that miR-935 represents a biomarker and a potential new target in HCC progression by suppressing SOX7 expression.

Topics: Apoptosis; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; G1 Phase; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; MicroRNAs; Proto-Oncogene Proteins c-myc; S Phase; SOXF Transcription Factors; Up-Regulation

Metabolic dysregulation is one of the most common and recognizable features of cancer. Triosephosphate isomerase 1 (TPI1), which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde-3-phosphate (G3P) during glycosis and gluconeogenesis, is a crucial enzyme in the carbohydrate metabolism. However, the biological function and mechanism of TPI1 in cancer remain largely unknown. In this study, we have found that TPI1 expression was greatly decreased in clinical HCC samples, positively correlated with overall survival, and negatively associated with histological differentiation, tumor size and organ metastasis. Forced expression of TPI1 in HCC cells inhibited cell growth, migration, and invasion in vitro. Consistently, knockdown of TPI1 by shRNA promoted cell growth, migration and invasion. Moreover, overexpression of TPI1 led to slowed tumor growth and decreased tumor weight in vivo. Furthermore, cell cycle arrest was induced by TPI1 overexpression. These phenotypes were associated with altered expression of β-catenin, Vimentin, P53, P27 and CyclinD1. Therefore, our data suggested that TPI1 functioned as a tumor suppressor in HCC and might serve as a potential therapeutic target for the treatment of HCC.

Topics: Aged; Animals; beta Catenin; Carcinogenesis; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Transplantation; Proliferating Cell Nuclear Antigen; RNA, Small Interfering; Triose-Phosphate Isomerase; Tumor Suppressor Protein p53; Vimentin

Protein phosphatase 1γ (PP1γ), as a member of the protein phosphatase 1 family, may be involved in regulation of multiple cellular processes, such as mitosis, cell survival, and apoptosis. However, little is known about the underlying mechanisms by which PP1γ regulates hepatocellular carcinoma development.. We investigated the expression profile of PP1γ in hepatocellular carcinoma (HCC) cell lines and human HCC specimens, as well as its potential prognostic significance in HCC.. PP1γ expression profile was detected in 94 HCC specimens using immunohistochemistry. PP1γ levels in HCC cells were downregulated by small interfering RNA (siRNA) transfection. Cell cycle progression and proliferation status of HCC cells and the effectiveness of doxorubicin were evaluated by flow cytometry and CCK-8 assay. The levels of PP1γ, CyclinD1, PCNA, Mdmx, p53, p21, and active caspase-3 were evaluated by Western blot analysis.. PP1γ was upregulated in tumorous specimens, compared with adjacent nontumorous tissues. Univariate and multivariate survival analyses were conducted to determine the prognostic significance of PP1γ in HCC. The expression pattern of PP1γ was positively correlated with tumor size, histological grade, Ki-67 expression, and poor prognosis in HCC. In addition, depletion of PP1γ by siRNA could inhibit cell proliferation, resulted in G1 phase arrest, and attenuated resistance to doxorubicin in Huh7 cells.. PP1γ is upregulated in HCC cell lines and HCC specimens, promotes cancer cell proliferation through regulation of p53, and may be a potential target for treatment of HCC.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Doxorubicin; Drug Resistance, Neoplasm; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Neoplasm Grading; Nuclear Proteins; Prognosis; Proliferating Cell Nuclear Antigen; Proportional Hazards Models; Protein Phosphatase 1; Proto-Oncogene Proteins; RNA, Small Interfering; Tumor Stem Cell Assay; Tumor Suppressor Protein p53; Young Adult

Dysregulated expression of long noncoding RNAs has been reported in many types of cancers, indicating that it may play a critical role in tumorigenesis. The long noncoding RNA highly up-regulated in liver cancer (HULC) was first characterized in hepatocellular carcinoma. However, the detailed mechanisms of HULC remain unclear. Here, we demonstrate a novel mechanism by which long noncoding RNA plays oncogenic roles through modulating the phosphorylation status of its interaction protein. First, we validated the markedly increased expression levels of HULC in hepatocellular carcinoma tissues compared to their adjacent noncancerous tissues. Furthermore, up-regulation of HULC was correlated with grading and overall survival. Meanwhile, HULC could promote cell proliferation, migration, and invasion in vitro and inhibit cisplatin-induced apoptosis. Moreover, we show that HULC specifically binds to Y-box binding protein 1 (YB-1) protein both in vitro and in vivo. YB-1 is a major component of translationally inactive messenger ribonucleoprotein particles which keeps mRNA in a silent state. Our study further demonstrated that HULC could promote the phosphorylation of YB-1 protein, which leads to the release of YB-1 from its bound mRNA. As a consequence, translation of silenced oncogenic mRNAs would be activated, including cyclin D1, cyclin E1, and matrix metalloproteinase 3. In addition, we found that HULC promotes the phosphorylation of YB-1 protein mainly through extracellular signal-regulated kinase.. We demonstrate that HULC promotes the phosphorylation of YB-1 through the extracellular signal-regulated kinase pathway, in turn regulates the interaction of YB-1 with certain oncogenic mRNAs, and consequently accelerates the translation of these mRNAs in the process of tumorigenesis. (Hepatology 2017;65:1612-1627).

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; China; Cyclin D1; Cyclin E; Extracellular Signal-Regulated MAP Kinases; Female; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Male; Matrix Metalloproteinase 3; Middle Aged; Oncogene Proteins; Phosphorylation; RNA, Long Noncoding; Y-Box-Binding Protein 1

Hepatocellular carcinoma (HCC) is a common and fatal malignancy of the liver. Sorafenib is a small molecule multikinase inhibitor that acts against different cancer cell lines and is used for the treatment of HCC. However, some advanced HCC patients fail to respond to sorafenib, and those who do lack a meaningful clinical benefit. Interferon-lambda 3 (IFN-λ3) is a type III interferon with antiviral, antiproliferative, and immunomodulatory functions. Here, we evaluated the use of IFN-λ3 as an adjuvant treatment with sorafenib in HCC. In the present study, CCK-8 and colony formation assay results showed that treatment with a combination of IFN-λ3 and sorafenib suppresses the viability of HepG2 and SMMC7721 liver cancer cell lines more than treatment with either alone. In addition, flow cytometry results confirmed that treatment with a combination of IFN-λ3 and sorafenib promotes the loss of mitochondrial membrane potential and induces the production of ROS more than treatment with either alone. Furthermore, using a subcutaneous SMMC7721 tumor model, treatment with a combination of IFN-λ3 and sorafenib significantly reduced the tumor growth/volume and induced apoptosis compared to treatment with sorafenib alone. These results show that combined treatment with IFN-λ3 and sorafenib facilitates a synergistic effect on suppressing HCC cancer growth and promoting cell apoptosis in vitro and in vivo. Thus, IFN-λ3 in combination with sorafenib might prove to be a useful adjunctive strategy for the clinical treatment of HCC.

Topics: Animals; Apoptosis; Carcinogenesis; Carcinoma, Hepatocellular; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Drug Synergism; Female; Humans; Interferons; Interleukins; Liver Neoplasms; Membrane Potential, Mitochondrial; Mice, Nude; Niacinamide; Phenylurea Compounds; Reactive Oxygen Species; Sorafenib

DNA replication is a central procedure of cell proliferation, whereas aberrant DNA replication is indicated to be a driving force of oncogenesis. Minichromosome maintenance complex component 7 (MCM7) plays an essential role in initiating DNA replication. To investigate the potential oncogenic properties and prognostic value of MCM7 in hepatocellular carcinoma (HCC), we conducted immunohistochemistry staining of MCM7 in 153 HCC samples and found that MCM7 high expression level was associated with worse overall survival (OS) of HCC patients. Mechanistically, knockdown of MCM7 significantly inhibited cellular proliferation in vitro and HCC tumorigenicity in vivo. Cyclin D1 was proved to be regulated by MCM7-MAPK signaling pathway. Clinically, high expression of both MCM7 and cyclin D1 exhibited a relatively high sensitivity and specificity to predict worse outcome of HCC patients. Taken together, our results suggest that MCM7-cyclin D1 pathway may participate in cancer progression and serve as a biomarker for prognosis in HCC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; DNA Replication; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Middle Aged; Minichromosome Maintenance Complex Component 7; Nuclear Proteins; Prognosis; Signal Transduction; Young Adult

Hepatocellular carcinoma (HCC) is the most prevalent primary tumour of the liver. About a third of these tumours presents activating mutations of the β-catenin gene. The molecular pathogenesis of HCC has been elucidated, but mortality remains high, and new therapeutic approaches, including treatments based on microRNAs, are required. We aimed to identify candidate microRNAs, regulated by β-catenin, potentially involved in liver tumorigenesis.. We used a mouse model, in which β-catenin signalling was overactivated exclusively in the liver by the tamoxifen-inducible and Cre-Lox-mediated inactivation of the Apc gene. This model develops tumours with properties similar to human HCC.. We found that miR-34a was regulated by β-catenin, and significantly induced by the overactivation of β-catenin signalling in mouse tumours and in patients with HCC. An inhibitor of miR-34a (locked nucleic acid, LNA-34a) exerted antiproliferative activity in primary cultures of hepatocyte. This inhibition of proliferation was associated with a decrease in cyclin D1 levels, orchestrated principally by HNF-4α, a target of miR-34a considered to act as a tumour suppressor in the liver. In vivo, LNA-34a approximately halved progression rates for tumours displaying β-catenin activation together with an activation of caspases 2 and 3.. This work demonstrates the key oncogenic role of miR-34a in liver tumours with β-catenin gene mutations. We suggest that patients diagnosed with HCC with β-catenin mutations could be treated with an inhibitor of miR-34a. The potential value of this strategy lies in the modulation of the tumour suppressor HNF-4α, which targets cyclin D1, and the induction of a proapoptotic programme.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Cyclin D1; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; MicroRNAs; Mutation

Rab5a was reported to be overexpressed in human malignancy and associated with the malignant phenotype. To data, its expression pattern and biological function in hepatocellular carcinoma (HCC) have not been studied. We analyzed Rab5a protein expression in 98 cases of HCC tissues and four HCC cell lines. We found that Rab5a expression was upregulated in HCC tissues and cell lines. Rab5a overexpression correlated with TNM stage and nodal metastasis (p < 0.05). To confirm the biological function of Rab5a in HCC cell lines, Rab5a siRNA was employed in SK-Hep-1 cell line and plasmid transfection was performed in Huh7 cell line. CCK-8 assay showed that Rab5a depletion blocked cell growth rate while Rab5a overexpression facilitated proliferation. Transwell and migration assay showed that Rab5a positively regulated cell invasion and migration. To explore the molecular mechanism underlying the biological effects of Rab5a, we checked several signaling pathways and found that Rab5a overexpression upregulated cyclin D1, cyclin E expression, FAK (Tyr397), and AKT (Ser473) phosphorylation. Blockage of FAK using inhibitor PF573228 abolished the role of Rab5a on cyclin D1. In conclusion, Rab5a is overexpressed in human HCC and contributes to cancer cell proliferation and invasion through regulation of FAK and AKT signaling.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; rab5 GTP-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction

5-Hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived hepatocellular carcinoma (HCC) cells but the detailed intracellular mechanism is unknown. As Wnt/β-catenin signalling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β-catenin signalling by 5-HT. The expression of various 5-HT receptors was studied by quantitative real-time polymerase chain reaction (qPCR) in HCC cell lines as well as in 33 pairs of HCC tumours and corresponding adjacent non-tumour tissues. Receptors 5-HT1D (21/33, 63.6%), 5-HT2B (12/33, 36.4%) and 5-HT7 (15/33, 45.4%) were overexpressed whereas receptors 5-HT2A (17/33, 51.5%) and 5-HT5 (30/33, 90.1%) were reduced in HCC tumour tissues. In vitro data suggests 5-HT increased total β-catenin, active β-catenin and decreased phosphorylated β-catenin protein levels in serum deprived HuH-7 and HepG2 cells compared to control cells under serum free medium without 5-HT. Activation of Wnt/β-catenin signalling was evidenced by increased expression of β-catenin downstream target genes, Axin2, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS) by qPCR in serum-deprived HCC cell lines treated with 5-HT. Additionally, biochemical analysis revealed 5-HT disrupted Axin1/β-catenin interaction, a critical step in β-catenin phosphorylation. Increased Wnt/β-catenin activity was attenuated by antagonist of receptor 5-HT7 (SB-258719) in HCC cell lines and patient-derived primary tumour tissues in the presence of 5-HT. SB-258719 also reduced tumour growth in vivo. This study provides evidence of Wnt/β-catenin signalling activation by 5-HT and may represent a potential therapeutic target for hepatocarcinogenesis.

Topics: Animals; Axin Protein; beta Catenin; Carcinogenesis; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Female; Glutamate-Ammonia Ligase; Hep G2 Cells; Humans; Intercellular Signaling Peptides and Proteins; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Phosphorylation; Piperidines; Real-Time Polymerase Chain Reaction; Receptors, Serotonin; Serotonin; Serotonin Antagonists; Sulfonamides; Wnt Signaling Pathway

The Wnt signaling pathway is activated in hepatocellular carcinoma (HCC). This study investigated the effects of FH535, an inhibitor of the Wnt signaling pathway, on the proliferation and motility of HCC cells. HLF cells and PLC/PRF/5 cells, HCC cells, were subjected to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay with the addition of FH535. RNA was isolated from the cells and subjected to real-time quantitative PCR. Hematoxylin and eosin (H&E) staining was performed to analyze apoptosis. A scratch assay was performed to analyze cell motility. Cell proliferation significantly decreased (P<0.05). The expression levels of cyclin D1 significantly decreased in both cell lines (P<0.05). Pyknotic nuclei were observed in the cells cultured with FH535 (50 µM). In the scratch assay, the distance between the growing edges of cells and the scratched line significantly decreased with the addition of FH535 at 50 µM (P<0.05). The expression levels of matrix metalloproteinase 9 significantly decreased at 50 µM (P<0.05). FH535 suppressed the proliferation of HCC cells by downregulating the expression of cyclin D1 and by inducing apoptosis. Further, it suppressed cell motility by downregulating the expression of matrix metalloproteinase.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; Sulfonamides; Wnt Signaling Pathway

The compound 3-bromopyruvate (3BP) is an analogue of pyruvate, which is the final product of glycolysis that enters the citric acid cycle. The present study aimed to investigate the suppressive effects of 3BP on the proliferation and motility of hepatocellular carcinoma (HCC) cells. HLF and PLC/PRF/5 cells were cultured with 3BP and subjected to an MTS assay. Apoptosis was analyzed by hematoxylin and eosin staining. Cell motility was analyzed using a scratch assay. Real-time quantitative polymerase chain reaction (PCR) was performed to determine the expression levels of cyclin D1 and matrix metalloproteinase (MMP)9. Proliferation of both cell lines was significantly suppressed by 3BP at 100 µM (P<0.05). The expression level of cyclin D1 was decreased after 3BP treatment at 100 µM in both cell lines (P<0.05). Pyknotic nuclei were observed in the cells cultured with 3BP at 100 µM. These results revealed that 3BP suppressed cell proliferation, decreased the expression of cyclin D1, and induced apoptosis in HCC cells. 3BP significantly suppressed motility in both cell lines (P<0.05). The expression level of MMP9 was significantly decreased (P<0.05). 3BP suppressed the proliferation and motility of HCC cells by decreasing the expression of cyclin D1 and MMP9.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; Pyruvates

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cyclin D1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Immunoblotting; Ki-67 Antigen; Liver Neoplasms; Membrane Proteins; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA Transport

DYC-279 is a newly synthesized compound. In this study, we revealed that DYC-279 could inhibit the proliferation of HepG2 cells in a dose- and time-dependent manner using the CCK-8 test. FACS showed that DYC-279 induced a G2/M arrest and apoptosis in a dose-dependent manner. Western blot demonstrated that DYC-279-induced G2/M arrest effect was correlated with the inhibition of cyclin-dependent kinase 1 activity, including a concomitant downregulation of cyclinD1 and cdc2 and upregulation of cyclinB1 in HepG2 cells. DYC-279 also significantly increased the ratio of Bax/Bcl-2, and stimulated the released of cytochrome c into cytosol and also activated caspase-9 and caspase-3, suggesting DYC-279 induced apoptosis via mitochondrial apoptotic pathway. These data support that DYC-279 has anticancer properties in HepG2 cells and may be used as a novel effective reagent in the treatment of human liver cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; CDC2 Protein Kinase; Cell Proliferation; Cell Survival; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinases; Cytochromes c; Hep G2 Cells; Humans; Liver Neoplasms; Piperazines; Proto-Oncogene Proteins c-bcl-2; Triazoles

Previous studies have demonstrated the aberrant expression and oncogenic role of B-cell CLL/lymphoma-3 (BCL-3) in human malignancies. However, the clinical significance of BCL-3 and its biological function in human hepatocellular carcinoma (HCC) remain unknown. In the present study, the expression levels of BCL-3 protein and mRNA in 90 pairs of HCC and matched non-tumor tissues were analyzed using immunohistochemistry and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). We found that the expression levels of BCL-3 protein and mRNA in HCC tissues were significantly higher than those in the matched tumor-adjacent tissues. In addition, positive expression of BCL-3 was associated with adverse clinicopathological characteristics of the HCC patients including hepatitis B virus (HBV) infection, tumor size, cirrhosis and advanced tumor-node-metastasis (TNM) stage. Moreover, HCC patients with positive expression of BCL-3 had significantly decreased 5-year overall survival and recurrence-free survival. Importantly, BCL-3 expression was an independent prognostic factor for indicating the survival of the HCC patients. Functionally, BCL-3 knockdown markedly inhibited cell viability, proliferation and cell cycle progression in HepG2 cells, while BCL-3 overexpression promoted these cellular processes in Huh7 cells. Accordingly, in vivo experiments indicated that BCL-3 knockdown prominently suppressed the tumor growth of HepG2 cells in nude mice. Mechanistically, we revealed that the expression of cyclin D1 was decreased after BCL-3 knockdown in the HepG2 cells and was increased after BCL-3 overexpression in the Huh7 cells. Cyclin D1 silencing was found to abrogate the functional effects of BCL-3 on cellular processes in Huh7 cells. Taken together, our data suggest that BCL-3 may serve as a promising biomarker and an effective therapeutic target of HCC.

Topics: Animals; B-Cell Lymphoma 3 Protein; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatitis B; Humans; Liver Neoplasms; Male; Mice; Neoplasm Transplantation; Prognosis; Proto-Oncogene Proteins; Survival Analysis; Transcription Factors; Up-Regulation

The purpose of this study was to investigate the effect of ST2825, an inhibitor of myeloid differentiation factor 88 (MyD88), on the proliferation and apoptosis of human hepatocellular carcinoma (HCC) cells as well as the potential mechanism and clinical significance of ST2825 in the treatment of HCC. Immunohistochemical staining with an MyD88 antibody was performed on tissues from 80 human HCC patients and adjacent normal tissues. In the in vitro experiment, human HCC HepG-2 cells cultured in vitro were divided into the following groups: blank, control (1% DMSO), low-dose (2 μM), medium-dose (10 μM), and high-dose ST2825 (20 μM). Cell apoptosis was detected by the Annexin V-FITC assay, and HepG-2 cell proliferation was detected by the MTT assay. The expression of IκB, p65, cyclin D1, caspase-3, and bcl-2 in the cells after a 48-h treatment was assayed by western blot analysis. MyD88 expression in the HCC tissue was significantly higher than that in the adjacent normal tissue (P < 0.05). The proliferation and apoptosis rates of control HCC cells displayed no significant differences compared with those of the blank group (P > 0.05). Compared with the control, ST2825 significantly inhibited the proliferation of and promoted the apoptosis of HCC cells. Moreover, ST2825 significantly decreased bcl-2 expression, increased cleaved caspase-3 expression (P < 0.05), and reduced p65 nuclear expression (P < 0.05) in a dose-dependent manner. ST2825 inhibits the proliferation of and promotes the apoptosis of HCC cells, thereby suggesting that ST2825 may be a new drug for HCC treatment.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Heterocyclic Compounds, 2-Ring; Humans; Liver Neoplasms; Myeloid Differentiation Factor 88; Proto-Oncogene Proteins c-bcl-2; Spiro Compounds

The present study was designed to investigate the effect of cantharidin on cell proliferation, ability of selfrenewal, cell cycle arrest and induction of apoptosis in HepG2 hepatocellular carcinoma stem cells (HCSCs). It was observed that cantharidin treatment exhibited dose- and time-dependent inhibitory effect on the viability of HCSCs. The inhibition of cell viability by cantharidin in HepG2 CD133+ and parental cells was significant at the concentration 5 and 15 µM, respectively after 48 h. Cantharidin treatment inhibited the self-renewal ability of the HCSCs and the expression of β-catenin and cyclin D1. Flow cytometry revealed that cantharidin treatment at 5 µM concentration significantly increased the cell population in G2/M phase and decreased the population in the G1 phase. Cantharidin treatment in the HCSCs for 48 h increased expression of histone H2AX, Myt1, cyclin A2, cyclin B1, p53 and cdc2 (Tyr15) phosphorylation significantly compared to the parental cells. Exposure of the HCSCs to cantharidin for 48 h at a concentration of 5 µM caused a significant increase in the proportion of apoptotic cells. Therefore, cantharidin is a promising agent for the hepatocellular carcinoma treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cantharidin; Carcinoma, Hepatocellular; Cell Proliferation; Cell Self Renewal; Cyclin D1; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Lithium Chloride; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells

microRNAs (miRNAs) dysregulation is widely involved in cancer progression and contributed to sustained cell proliferation by directly targeting multiple targets. Therefore, better understanding the underlying mechanism of miRNA in carcinogenesis may improve diagnostic and therapeutic strategies for malignancy. In our study, we found that mir-765 is upregulated in both hepatocellular carcinoma (HCC) cell lines and tissues, compared to human normal liver cell line and adjacent non-cancerous tissues, respectively. Overexpression of mir-765 increased HCC cells proliferation and tumorigenicity, whereas inhibition of mir-765 reverses this effect. Furthermore, we demonstrated that INPP4B as a direct target of mir-765 and ectopic expression of mir-765 repressed INPP4B expression, resulting in upregulation of p-AKT, Cyclin D1, and downregulation of p-FOXO3a, p21 expression in HCC. Strikingly, we found that silencing the expression of INPP4B is the essential biological function of miR-765 during HCC cell proliferation. Collectively, our findings reveal that miR-765 is a potential onco-miR that participates in carcinogenesis of human HCC by suppressing INPP4B expression, and might represent a potential therapeutic target for HCC patients.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; Phosphoric Monoester Hydrolases; Proto-Oncogene Proteins c-akt

Hepatitis C virus (HCV) core protein plays an important role in the development of hepatocellular carcinoma. octamer-binding protein 4 (OCT4) is critically essential for the pluripotency and self-renewal of embryonic stem cells. Abnormal expression of OCT4 has been detected in several human solid tumors. However, the relationship between HCV core and OCT4 remains uncertain. In the present study, we found that HCV core is capable of upregulating OCT4 expression. The effect of HCV core-induced OCT4 overexpression was abolished by RNAi-mediated scilencing of HCV core. In addition, HCV core-induced OCT4 overexpression resulted in enhanced cell proliferation and cell cycle progression. Inhibition of OCT4 reduced the CCND1 expression and induced G0/G1 cell cycle arrest. Furthermore, OCT4 protein directly binds to CCND1 promoter and transactivates CCND1. These findings suggest that HCV core protein regulates OCT4 expression and promotes cell cycle progression in hepatocellular carcinoma providing new insight into the mechanism of hepatocarcinogenesis by HCV infection.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin D1; G1 Phase; Hep G2 Cells; Hepacivirus; Hepatitis C; Humans; Liver Neoplasms; Octamer Transcription Factor-3; Promoter Regions, Genetic; Resting Phase, Cell Cycle; Transcriptional Activation; Up-Regulation; Viral Core Proteins

Regulated intramembrane proteolysis of epithelial cell adhesion molecule (EpCAM) results in shedding of the extracellular domain (EpEX) and release of the intra-cellular domain (EpICD) into the cytoplasm. Released EpICD associates with FHL2, β-catenin and Lef-1 to form a nuclear complex and triggers oncogenic signaling. This study was conducted to examine the nuclear expression of EpICD in hepatocellular carcinoma (HCC) and to assess the role of EpICD in HCC. EpICD immunoexpression was examined in 100 cases of HCC using tissue microarrays and correlated with clinicopathological parameters. We also examined the role of EpICD in HCC using EpICD cDNA transfected HCC cell line and EpCAM silenced HCC cell line by small interfering RNA (siRNA). Nuclear expression of EpICD was observed in 19 of 100 (19%) cases. Nuclear expression of EpICD significantly correlated with nuclear expression of β-catenin, and Ki-67 labeling index. In addition, nuclear expression of EpICD was associated with higher histologic grade and advanced T category. Forced overexpression of EpICD in the HCC cell significantly increased the cell proliferation, migration and invasion. The overexpression of EpICD also increased the expression levels of the active form of β-catenin and c-myc and cyclin D1. In contrast, downregulation of EpCAM by siRNA decreased the cell proliferation, migration, invasion and the expression of active form of β-catenin, c-myc and cyclin D1. Our present data suggest that EpICD plays important roles in HCC progression by modulating expression of target genes of EpCAM.

Topics: Adult; Aged; beta Catenin; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Cell Nucleolus; Cell Proliferation; Cyclin D1; Epithelial Cell Adhesion Molecule; Female; Hep G2 Cells; Humans; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Proto-Oncogene Proteins c-myc; RNA, Small Interfering

We explored the anti-cancer capacity of (-)-oleocanthal in human hepatocellular carcinoma (HCC). (-)-Oleocanthal inhibited proliferation and cell cycle progression and induced apoptosis in HCC cells in vitro and suppressed tumor growth in an orthotopic HCC model. (-)-Oleocanthal also inhibited HCC cell migration and invasion in vitro and impeded HCC metastasis in an in vivo lung metastasis model. ( )-Oleocanthal acted by inhibiting epithelial-mesenchymal transition (EMT) through downregulation Twist, which is a direct target of STAT3. (-)-Oleocanthal also reduced STAT3 nuclear translocation and DNA binding activity, ultimately downregulating its downstream effectors, including the cell cycle protein Cyclin D1, the anti-apoptotic proteins Bcl-2 and survivin, and the invasion-related protein MMP 2. Overexpression of constitutively active STAT3 partly reversed the anti cancer effects of (-)-oleocanthal, which inhibited STAT3 activation by decreasing the activities of JAK1 and JAK2 and increasing the activity of SHP-1. These data suggest that (-)-oleocanthal may be a promising candidate for HCC treatment.

Topics: Aldehydes; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cyclin D1; Cyclopentane Monoterpenes; Down-Regulation; Epithelial-Mesenchymal Transition; Humans; Inhibitor of Apoptosis Proteins; Janus Kinase 1; Janus Kinase 2; Liver Neoplasms; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Nuclear Proteins; Olive Oil; Phenols; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Survivin; Twist-Related Protein 1; Xenograft Model Antitumor Assays

Exome and whole-genome sequencing studies have drawn attention to the role of somatic mutations in SWI/SNF chromatin remodeling complexes in the carcinogenesis of hepatocellular carcinoma (HCC). Here, we explored the molecular mechanisms underlying the biological roles of AT-rich interactive domain 2 (ARID2) in the pathogenesis of HCC. We found that ARID2 expression was significantly downregulated in HCC tissues compared with non-tumorous tissues. Restoration of ARID2 expression in hepatoma cells was sufficient to suppress cell proliferation and tumor growth in mice, whereas ARID2 knockdown contributed to the enhancement of cellular proliferation and tumorigenicity. Suppression of ARID2 expression accelerated G1/S transition associated with upregulation of cyclin D1, cyclin E1, CDK4, and phosphorylation of the retinoblastoma protein (Rb). Furthermore, we demonstrated that ARID2 physically interacts with E2F1 and decreases binding of E2F1/RNA Pol II to the promoters of CCND1 and CCNE1. Taken together, these results demonstrate that ARID2 suppresses tumor cell growth through repression of cyclin D1 and cyclin E1 expression, thereby retarding cell cycle progression and cell proliferation in hepatoma cells. These findings highlight the potential role of ARID2 as a tumor growth suppressor in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromatin Assembly and Disassembly; Cyclin D1; Cyclin E; Disease Progression; Heterografts; Humans; Liver Neoplasms; Mice; Mice, Nude; Oncogene Proteins; Transcription Factors

MafB, a member of the Maf transcription factor family, plays a key role in the regulation of pancreatic alpha and beta cell differentiation. However, its function in the control of cancer cell proliferation remains unknown.. The mRNA and protein expression levels of MafB in hepatocellular carcinoma tissues and adjacent non-tumor normal specimens were determined by real-time RT-PCR and Western blot, respectively. Report assay was performed to determine whether the regulation of Cyclin D1 by MafB is at the transcriptional level. The binding of MafB to the Cyclin D1 promoter was determined by Chromatin Immunoprecipitation (ChIP) assays. To determine the potential oncogenic effects of MafB in vivo, HepG2 cells transfected with adenovirus containing empty vector or MafB were injected subcutaneously to the skin under the front legs of the nude mice.. In the current study, we showed that MafB was markedly up-regulated in hepatocellular carcinoma (HCC) tissues and cells. Enforced overexpression of MafB enhanced, while its deficiency inhibited HCC cell proliferation. Mechanistically, Cyclin D1, an important regulator of cell cycle progression, was identified as a direct transcriptional target of MafB. Consistently, knockdown of Cyclin D1 largely attenuated the proliferative roles of MafB in HCC cells. Importantly, MafB overexpression significantly promoted cancer cell growth in mice.. Collectively, our results identified a novel HCC regulatory pathway involving MafB and Cyclin D1, the dysfunction of which drives proliferative character in HCC.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; MafB Transcription Factor; Male; Mice, Inbred BALB C; Mice, Nude; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transplantation, Heterologous; Up-Regulation

To assess the distribution of proteins coded by genes reported as relevant for the molecular classification of hepatocellular carcinoma (HCC).. In this retrospective cross-sectional study, the following clinicopathological data were analyzed in 80 autopsied HCC patients: sex, age, ethnicity, alcohol intake, infection with hepatitis B and/or C virus, infection with human immunodeficiency virus, prior treatment, basic and immediate causes of death, liver weight, presence of cirrhosis, number and size of nodules, gross pattern, histological grade and variants, architectural pattern, invasion of large veins, and presence and location of extrahepatic metastases. The protein products of genes known to be involved in molecular pathogenesis of HCC, including epidermal growth factor receptor (EGFR), MET, keratin 19 (K19), vimentin, beta-catenin, mechanistic target of rapamycin (mTOR), extracellular signaling-related kinase (ERK)1, ERK2, Ki67, cyclin D1, caspase 3 and p53, were detected by immunohistochemistry on tissue microarrays. The expression levels were scored and statistically assessed for correlation with HCC parameters.. Infection with hepatitis C virus was identified in 49% of the 80 autopsy patients, cirrhosis in 90%, advanced tumors in 95%, and extrahepatic metastases in 38%. Expression of K19, p53 and ERK1 correlated to high-grade lesions. Expression of ERK1, nuclear beta-catenin, cyclin D1 and ERK2 correlated to higher rates of cell proliferation as determined by Ki67. Expression of MET, EGFR (> 0) and caspase 3 correlated with lower histological grades. Expression of EGFR correlated to that of caspase 3, and overexpression of EGFR (≥ 200/300) was observed in low-grade tumors more frequently (grades 1 and 2: 67% vs grade 3: 27% and grade 4: 30%). Expression of ERK1 was associated with that of K19 and vimentin, whereas expression of ERK2 was associated with that of cyclin D1, MET and membrane beta-catenin. Expression of vimentin was strongly correlated with that of K19.. Expression of K19, p53, ERK1, ERK2, vimentin and nuclear beta-catenin was related to higher-grade markers, as opposed to expression/overexpression of EGFR, MET and caspase 3.

Topics: Adult; Aged; Aged, 80 and over; Autopsy; beta Catenin; Brazil; Carcinoma, Hepatocellular; Case-Control Studies; Caspase 3; Cross-Sectional Studies; Cyclin D1; ErbB Receptors; Female; Hepatitis B, Chronic; Hepatitis C, Chronic; HIV Infections; Humans; Immunohistochemistry; Keratin-19; Ki-67 Antigen; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-met; Retrospective Studies; Tissue Array Analysis; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Vimentin

The harmful effects of bile acid accumulation occurring during cholestatic liver diseases have been associated with oxidative stress increase and endothelial nitric oxide synthase (NOS-3) expression decrease in liver cells. We have previously reported that glycochenodeoxycholic acid (GCDCA) down-regulates gene expression by increasing SP1 binding to the NOS-3 promoter in an oxidative stress dependent manner. In the present study, we aimed to investigate the role of transcription factor (TF) AP-1 on the NOS-3 deregulation during GCDCA-induced cholestasis. The cytotoxic response to GCDCA was characterized by 1) the increased expression and activation of TFs cJun and c-Fos; 2) a higher binding capability of these at position -666 of the NOS-3 promoter; 3) a decrease of the transcriptional activity of the promoter and the expression and activity of NOS-3; and 4) the expression increase of cyclin D1. Specific inhibition of AP-1 by the retinoid SR 11302 counteracted the cytotoxic effects induced by GCDCA while promoting NOS-3 expression recovery and cyclin D1 reduction. NOS activity inhibition by L-NAME inhibited the protective effect of SR 11302. Inducible NOS isoform was no detected in this experimental model of cholestasis. Our data provide direct evidence for the involvement of AP-1 in the NOS-3 expression regulation during cholestasis and define a critical role for NOS-3 in regulating the expression of cyclin D1 during the cell damage induced by bile acids. AP-1 appears as a potential therapeutic target in cholestatic liver diseases given its role as a transcriptional repressor of NOS-3.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cyclin D1; Down-Regulation; Genes, Reporter; Glycochenodeoxycholic Acid; Hep G2 Cells; Humans; Liver Neoplasms; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Oxidative Stress; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Retinoids; Transcription Factor AP-1; Up-Regulation

20(S)-ginsenoside Rh2 [(S)Rh2] possesses potential to prevent cancer in vitro as well as in vivo, but the underlying mechanism is still unknown. First, we infected HepG2 cells with lentivirus which carries β‑catenin. We detected the pharmacological effects of (S)Rh2 on HepG2 and HepG2‑β‑catenin cells and found that the IC50 of (S)Rh2 exposure on HepG2-β-catenin cells was higher than HepG2 cells. Flow cytometry (FCM) indicated that (S)Rh2 could be arrested in G0/G1 phase and induce early apoptosis in HepG2 and HepG2‑β‑catenin cells. Second, ELISA kit was used to check the activity of glycogen synthase kinase‑3β (GSK‑3β), which was upregulated by (S)Rh2. GSK‑3β inhibitor BIO, was used to verify that (S)Rh2 activated GSK‑3β. PCR and western blotting results indicated that (S)Rh2 could degrade the expression of β‑catenin, which combined with TCF in the nucleus and activate transcription of Wnt target genes, such as Bax, Bcl‑2, cyclin D1, MMP3, which were checked by chromatin immunoprecipitation (ChIP), PCR and western blotting. The results showed that the expression of Bax mRNA and proteins increased, while the cyclin D1, Bcl‑2, MMP3 mRNA and proteins were downregulated in HepG2 and HepG2‑β‑catenin cells which was induced by (S)Rh2. By contrast, with the HepG2-β-catenin + (S)Rh2 group, the expression of other mRNA and proteins in HepG2 + (S)Rh2 group changed significantly. In vivo, experiments were performed using a nude mouse xenograft model to investigate the (S)Rh2 effect. So these results suggested that (S)Rh2 could suppress proliferation, promote apoptosis and inhibit metastasis of HepG2, decrease weight of tumor by downregulating β‑catenin through activating GSK‑3β and the pharmacological effect of (S)Rh2 on HepG2 cells might be weakened by overexpression of β‑catenin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Ginsenosides; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Polymerase Chain Reaction; Xenograft Model Antitumor Assays

Tylophorine analog DCB-3503 is a potential anticancer and immunosuppressive agent that suppresses the translation of cellular regulatory proteins, including cyclin D1, at the elongation step. However, the molecular mechanism underlying this phenomenon remains unknown. This study demonstrates that DCB-3503 preferentially binds to heat shock cognate protein 70 (HSC70), which is a determinant for cyclin D1 translation by binding to the 3'-untranslated region (3' UTR) of its mRNA. DCB-3503 allosterically regulates the ATPase and chaperone activities of HSC70 by promoting ATP hydrolysis in the presence of specific RNA binding motifs (AUUUA) of cyclin D1 mRNA. The suppression of cyclin D1 translation by DCB-3503 is not solely caused by perturbation of the homeostasis of microRNAs, although the microRNA processing complex is dissociated with DCB-3503 treatment. This study highlights a novel regulatory mechanism of protein translation with AUUUA motifs in the 3' UTR of mRNA by HSC70, and its activity can be allosterically modulated by DCB-3503. DCB-3503 may be used to treat malignancies, such as hepatocellular carcinoma or breast cancer with elevated expression of cyclin D1.

Topics: 3' Untranslated Regions; Allosteric Regulation; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; HSC70 Heat-Shock Proteins; Humans; Indolizines; Liver Neoplasms; Phenanthrenes; Protein Binding; Protein Processing, Post-Translational; Tumor Cells, Cultured

Forkhead box P1 (FOXP1) belongs to a family of winged-helix transcription factors that are involved in the processes of cellular proliferation, differentiation, metabolism, and longevity. FOXP1 can affect cell proliferation and migratory ability in hepatocellular carcinoma (HCC) in vitro. However, little is known about the mechanism of FOXP1 in the proliferation of HCC cells. This study aimed to further explore the function of FOXP1 on the proliferation of HCC cells as well as the relevant mechanism involved. Western blot analysis, tumor xenograft models, and flow cytometry analysis were performed to elucidate the function of FOXP1 in the regulation of cell proliferation in human HCC. We observed that silencing FOXP1 significantly suppressed the growth ability of HCC cells both in vitro and in vivo. In addition, knockdown of FOXP1 induced G1/S phase arrest, and the expression of total and phosphorylated Rb (active type) as well as the levels of E2F1 were markedly decreased at 24 h; however, other proteins, including cyclin-dependent kinase (CDK) 4 and 6 and cyclin D1 did not show noticeable changes. In conclusion, downregulation of FOXP1 inhibits cell proliferation in hepatocellular carcinoma by inducing G1/S phase cell cycle arrest, and the decrease in phosphorylated Rb is the main contributor to this G1/S phase arrest.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinases; Down-Regulation; E2F1 Transcription Factor; Forkhead Transcription Factors; G1 Phase Cell Cycle Checkpoints; Humans; Liver Neoplasms; Repressor Proteins; S Phase Cell Cycle Checkpoints

Human liver cancer is one of the most frequently diagnosed cancers worldwide. The development of resistance to therapy limits the application against the disease. To improve treatment, new effective anticancer agents are constantly pursued. Previously, we reported that an indolylquinoline, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), is effective in suppressing the growth of human lung cancer by impairing mitochondria functions. The present study revealed that EMMQ inhibited cell growth and induced apoptosis in liver cancer cells, but not in normal cells. This study demonstrated that EMMQ induced DNA damage by activating p53 and γ-H2AX and cell arrest by suppressing cyclin D1 and CDK2. Damaged DNA injured mitochondrial functions by lowering the membrane potential and producing reactive oxygen species. The subsequent mitochondrial cytochrome c release attenuated pro-survival signals and increased apoptotic characteristics. Introduction of p53 shRNA abrogated drug effects by reducing DNA damage while maintaining mitochondria integrity. In brief, the study demonstrates that the effectiveness of EMMQ accentuated apoptosis of hepatocarcinoma cells by activating p53. Based on these collective findings, the study offered a new perspective of EMMQ that was shown to be a promising candidate to treat liver cancer.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cytochromes c; DNA Damage; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Histones; Humans; Indoles; Liver; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Quinaldines; Quinolines; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCF

Topics: Active Transport, Cell Nucleus; Animals; Antineoplastic Agents, Phytogenic; Berberine; beta-Transducin Repeat-Containing Proteins; Carcinoma, Hepatocellular; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; RNA, Small Interfering; Signal Transduction; Ubiquitin; Xenograft Model Antitumor Assays

The molecular cochaperone CDC37 regulates the activities of multiple protein kinases, and is an attractive broad-spectrum target in many types of cancers in which it is over-expressed. This study investigates the antitumour effects of inhibiting CDC37 in human hepatocellular carcinoma (HCC).. A total of 91 patients were enrolled for CDC37 mRNA detection by using quantitative real-time PCR. Cell proliferation, gene expression changes and tumourigenicity were determined by targeting CDC37 using RNA interference in human hepatoma cell lines.. We confirmed the significant over-expression of CDC37 transcript and protein in HBV-associated HCC patients. Using a CDC37-specific small oligo-siRNA, we silenced CDC37 expression in HepG2 and Huh7 hepatoma cell lines, and observed inhibition of in vitro cell proliferation, cell cycle arrest at the G1 phase, and enhanced apoptosis. Specifically, we found concomitant down-regulation of Cyclin D1, CDK4, and pRB (S807/811 and S795) upon CDC37 suppression, which could mediate the arrest of cell cycle progression at the G1 phase. Gene expression profiling further identified several genes involved in cell proliferation, cell cycle progression, and apoptosis that are regulated by CDC37 suppression. Huh7 cells with stable knockdown of CDC37 showed decreased in vitro colony formation ability, and significantly slowed xenograft growth in vivo.. On the basis of the observed antitumour effects of inhibiting CDC37 expression, we propose that CDC37 is a promising therapeutic target in HCC. Its ability to regulate multiple pathways makes it potentially valuable in treating the heterogeneous subtypes of this malignancy.

Topics: Adolescent; Adult; Aged; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Proliferation; Chaperonins; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice, Nude; Middle Aged; Retinoblastoma Protein; RNA Interference; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Tumor Burden; Young Adult

Hepatocellular carcinoma (HCC) arises in a setting of chronic inflammation induced by inflammatory cytokines, such as nuclear factor-kappaB (NF-κB). HCC is a male-predominant cancer that can be attenuated by estradiol (E2) in vitro and in vivo. Although 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) has been implicated as an estradiol-inactivating enzyme, and its promoter sequence contains two putative NF-κB elements: it is currently unknown whether HSD17B4 is the link between inflammation, estradiol and proliferation in hepatoma cells. In this study, HepG2 cells were used to investigate the role of HSD17B4 in the proliferation of liver cancer cells treated with the NF-κB activator, tumor necrosis factor-alpha (TNF-α), with the inhibitor of NF-κB activation, pyrrolidinedithiocarbamate (PDTC), or with a related specific siRNA. We demonstrated that the human HSD17B4 gene is a target for NF-κB activation in inflammation-stimulated HepG2 cells. HSD17B4 is up-regulated via the binding of activated NF-κB to the distal NF-κB-responsive element via TNF-α stimulation, which then promotes cell proliferation by decreasing the levels of E2 and enhancing the expression of interleukin 6 (IL-6), cyclin D1 and proliferating cell nuclear antigen (PCAN). These results from HepG2 cells are consistent with the observation that HSD17B4 is highly expressed and activated NF-κB is highly co-localized with the NF-κB-responsive element of HSD17B4 in liver tumor tissues from HCC patients. Our findings indicate for the first time that HSD17B4 plays an important role in aggravated HCC progression and provides a novel therapeutic target for HCC.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Estradiol; Hep G2 Cells; Humans; Interleukin-6; Liver Neoplasms; Male; NF-kappa B; Peroxisomal Multifunctional Protein-2; Proliferating Cell Nuclear Antigen; Proline; Promoter Regions, Genetic; Thiocarbamates; Tumor Necrosis Factor-alpha; Up-Regulation

Hepatitis B virus core promoter (CP) mutations can increase risk of hepatocellular carcinoma. The CP region overlaps with the HBV X (HBx) gene, which has been associated with hepatocarcinogenesis. The cyclin kinase inhibitor P53 is an important regulator of cell cycle progression. We determined whether HBx mutants that result from mutations in the CP deregulate P53.. A HBx combination (combo) mutant with changes in the CP region that corresponded to A1762T/G1764A (TA), T1753A, and T1768A was constructed and expressed in L-02 and Hep3B cells. The effects of CP mutations on expression and degradation of P53, and the effects on cell cycle progression and proliferation were analyzed.. The combo mutant decreased levels of P53 and increased cyclin D1 expression, accelerated P53 degradation in L-02 cells, accelerated cell cycle progression, and increased expression of S-phase kinase-associated protein 2 (Skp2) in L-02 and Hep3B cells. Silencing of Skp2 abrogated the effects of CP mutations on P53 expression. The kinetics of P53 expression correlated with changes in cell cycle distribution.. The HBx mutant with a combination of CP mutations can up-regulate Skp2, which then down-regulates P53 via ubiquitin-mediated proteasomal degradation, increasing the risk of hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Viral; Cyclin D1; Gene Expression Regulation, Neoplastic; Genotype; Hepatitis B; Hepatitis B virus; Humans; Liver Neoplasms; Mutation; Phenotype; Proteasome Endopeptidase Complex; Proteolysis; RNA Interference; S-Phase Kinase-Associated Proteins; Time Factors; Trans-Activators; Transfection; Tumor Suppressor Protein p53; Ubiquitination; Viral Core Proteins; Viral Regulatory and Accessory Proteins

Kinesin spindle protein (KSP) plays a critical role in mitosis. Inhibition of KSP function leads to cell cycle arrest at mitosis and ultimately to cell death. The aim of this study was to suppress KSP expression by specific small-interfering RNA (siRNA) in Hep3B cells and evaluate its anti-tumor activity.. Three siRNA targeting KSP (KSP-siRNA #1-3) and one mismatched-siRNA (Cont-siRNA) were transfected into cells. Subsequently, KSP mRNA and protein levels, cell proliferation, and apoptosis were examined in both Hep3B cells and THLE-3 cells. In addition, the chemosensitivity of KSP-siRNA-treated Hep3B cells with doxorubicin was also investigated using cell proliferation and clonogenic survival assays.. The expression of endogenous KSP at both mRNA and protein levels in Hep3B cells was higher than in THLE-3 cells. In Hep3B cells, KSP-siRNA #2 showed a further downregulation of KSP as compared to KSP-siRNA #1 or KSP-siRNA #3. It also exhibited greater suppression of cell proliferation and induction of apoptosis than KSP-siRNA #1 or KSP-siRNA #3; this could be explained by the significant downregulation of cyclin D1, Bcl-2, and survivin. In contrast, KSP-siRNAs had no or lower effects on KSP expression, cell proliferation and apoptosis in THLE-3 cells. We also noticed that KSP-siRNA transfection could increase chemosensitivity to doxorubicin in Hep3B cells, even at low doses compared to control.. Reducing the expression level of KSP, combined with drug treatment, yields promising results for eradicating hepatocellular carcinoma (HCC) cells in vitro. This study opens a new direction for liver cancer treatment.

Topics: Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Doxorubicin; Drug Resistance, Neoplasm; Humans; Inhibitor of Apoptosis Proteins; Kinesins; Liver Neoplasms; Mitosis; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Survivin

The Hepatitis B virus X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). It has been suggested that the transcriptional activation of cyclin D1 by HBx is implicated in the development of HCC. However, numerous studies have shown that overexpression of cyclin D1 alone is not sufficient to drive oncogenic transformation. Herein, we investigated whether HBx can stabilize cyclin D1 and induce cyclin D1 protein nuclear accumulation, and thereby accelerate hepatocarcinogenesis. The effects of HBx on cyclin D1 stabilization were assessed in cell-based transfection, Western blot, immunoprecipitation, immunocytofluorescence staining, and flow-cytometric assays. The results demonstrated that ectopic expression of HBx in HCC cells could extend the half-life of cyclin D1 protein from 40-60 minutes to 80-110 minutes. HBx stabilized cyclin D1 primarily in the S phase of the cell cycle, in a manner dependent on the inactivation of GSK-3β, which was mediated by ERK activation. HBx also prompted the nuclear accumulation of cyclin D1, and cotransfection of the constitutively active mutant of GSK-3β along with HBx could reverse the nuclear accumulation and subsequent cell proliferation induced by HBx. Further, a positive correlation between HBx and nuclear cyclin D1 level was established in HCC specimens detected by an immunohistochemical assay. Taken together, our results indicated that HBx could stabilize and increase cyclin D1 nuclear accumulation through ERK-mediated inactivation of GSK-3β. This HBx-induced cyclin D1 upregulation might play an important role in HCC development and progression.

Topics: Active Transport, Cell Nucleus; Animals; Carcinoma, Hepatocellular; Cell Nucleus; Cell Transformation, Viral; Cells, Cultured; Cyclin D1; Disease Progression; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Liver Neoplasms; Mice; NIH 3T3 Cells; Protein Stability; Protein Transport; Trans-Activators; Viral Regulatory and Accessory Proteins

The G870A polymorphism in the exon 4/intron 4 boundary of CCND1 gene is thought to influence the generation of two mRNAs (cyclin D1a and cyclin D1b). The "A" allele codes for a truncated variant, cyclin D1b, which may have higher transforming activity. Herein, the tumor relevance of G870A polymorphism, the association between cyclin D1 variant expression and G870A genotype, and the oncogenic potential of cyclin D1 variants in HBV-related hepatocellular carcinoma (HCC) were examined. We found that there is no significant difference of G870A distribution among the HCC, chronic HBV (CHB) infection, cirrhotic CHB, and healthy control groups. Stratification analysis revealed that in younger patients (ages ≤ 50), cirrhotic CHB patients with AA genotype had an increased risk of developing HCC with odds ratio of 1.943 (95 % CI 1.022-3.694, p = 0.0411) as compared with AG/GG genotypes. The two variants were both transcripted from "A" and "G" alleles, and neither cyclin D1a nor D1b production was influenced by G870A genotype in HCC. The expression of both cyclins D1a and D1b decreased in HCC tissues (p = 0.003, p = 0.005), while increased in adjacent nontumor tissues as compared with normal liver tissues (p = 0.045, p = 0.034). Overexpression of cyclin D1a or D1b could promote the cell proliferation and cell-cycle progression in Huh-7 and LO2 cell lines. Collectively, our data suggest that G870A polymorphism has only very limited predictive value for HBV-related HCC. Both cyclins D1a and D1b could promote cell proliferation, which might contribute to the potential oncogenic role of cyclin D1 variants during the precancerous cirrhotic stage of hepatocarcinogenesis.

Topics: Adult; Alternative Splicing; Asian People; Carcinoma, Hepatocellular; Cyclin D1; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Hepatitis B virus; Humans; Liver Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3' UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Carcinoma, Hepatocellular; cdc25 Phosphatases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Down-Regulation; E2F2 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms, Experimental; Mice; MicroRNAs; RNA Interference; RNA, Messenger; Xenograft Model Antitumor Assays

Cullin 4B (CUL4B) is a component of the Cullin 4B-Ring E3 ligase (CRL4B) complex that functions in proteolysis and in epigenetic regulation. CUL4B possesses tumor-promoting properties and is markedly upregulated in many types of human cancers. To determine the role of CUL4B in liver tumorigenesis, we generated transgenic mice that expressed human CUL4B in livers and other tissues and evaluated the development of spontaneous and chemically-induced hepatocellular carcinomas. We observed that CUL4B transgenic mice spontaneously developed liver tumors at a high incidence at old ages and exhibited enhanced DEN-induced hepatocarcinogenesis. There was a high proliferation rate in the livers of CUL4B transgenic mice that was accompanied by increased levels of Cdk1, Cdk4 and cyclin D1 and decreased level of p16. The transgenic mice also exhibited increased compensatory proliferation after DEN-induced liver injury, which was accompanied by activation of Akt, Erk, p38 and NF-κB. We also found that Prdx3 was downregulated and that DEN induced a higher level of reactive oxygen species in the livers of transgenic mice. Together, our results demonstrate a critical role of CUL4B in hepatocarcinogenesis in mice.

Topics: Animals; Carcinoma, Hepatocellular; CDC2 Protein Kinase; Cell Proliferation; Cell Transformation, Neoplastic; Cullin Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Enzyme Activation; Liver; Liver Neoplasms; MAP Kinase Signaling System; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; NF-kappa B; Peroxiredoxin III; Promoter Regions, Genetic; Reactive Oxygen Species

MicroRNAs (miRNAs) contribute to a wide variety of human diseases by regulating gene expression, leading to imbalances in gene regulatory networks. To discover novel hepatocellular carcinoma (HCC)-related miRNA-target axes and to elucidate their functions, we here performed a systematic investigation combining biological data acquisition and integration, miRNA-target prediction, network construction, functional assay and clinical validation. As a result, a total of 117 HCC differentially expressed miRNAs were identified, and 728 high confident target genes of these miRNAs were collected. Then, the interaction network of target genes was constructed and 221 key nodes with topological importance in the network were identified according to their topological features including degree, node-betweenness, closeness and K-coreness. Among these key nodes, Cyclin D1 had the highest node-betweenness, implying its bottleneck role in the network. Luciferase reporter assay confirmed that miRNA-19a, which was one of HCC downregulated miRNAs, directly targeted Cyclin D1 in HCC cells. Moreover, miR-19a might play inhibitory roles in HCC malignancy via regulating Cyclin D1 expression. Further clinical evidence also highlighted the prognostic potential of miR-19a/Cyclin D1 axis in HCC. In conclusion, this systematic investigation provides a framework to identify featured miRNAs and their target genes which are potent effectors in the occurrence and development of HCC. More importantly, miR-19a/Cyclin D1 axis might have promising applications as a therapeutic target and a prognostic marker for patients with HCC.

Topics: Aged; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Databases, Nucleic Acid; Female; Gene Regulatory Networks; Humans; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Prognosis

Cyclophilin J (CYPJ) is a new member of the peptidyl-prolyl cis/trans-isomerase (PPIase) identified with upregulated expression in human glioma. However, the biological function of CYPJ remained unclear. We aimed to study the role of CYPJ in hepatocellular carcinoma (HCC) carcinogenesis and its therapeutic potential. We determined the expression of CYPJ in HCC/adjacent normal tissues using Western blot, Northern blot and semi-quantitative RT-PCR, analyzed the biochemical characteristics of CYPJ, and resolved the 3D-structure of CYPJ/Cyclosporin A (CsA) complex. We also studied the roles of CYPJ in cell cycle, cyclin D1 regulation, in vitro and in vivo tumor growth. We found that CYPJ expression was upregulated in over 60% HCC tissues. The PPIase activity of CYPJ could be inhibited by the widely used immunosuppressive drug CsA. CYPJ was found expressed in the whole cell of HCC with preferential location at the cell nucleus. CYPJ promoted the transition of cells from G1 phase to S phase in a PPIase-dependent manner by activating cyclin D1 promoter. CYPJ overexpression accelerated liver cell growth in vitro (cell growth assay, colony formation) and in vivo (xenograft tumor formation). Inhibition of CYPJ by its inhibitor CsA or CYPJ-specific RNAi diminished the growth of liver cancer cells in vitro and in vivo. In conclusion, CYPJ could facilitate HCC growth by promoting cell cycle transition from G1 to S phase through the upregulation of cyclin D1. Suppression of CYPJ could repress the growth of HCC, which makes CYPJ a potential target for the development of new strategies to treat this malignancy.

Topics: Animals; Carcinoma, Hepatocellular; Chlorocebus aethiops; COS Cells; Cyclin D1; Cyclophilins; Cyclosporine; G1 Phase; Gene Expression Regulation, Neoplastic; HEK293 Cells; HeLa Cells; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Protein Structure, Tertiary; S Phase; Up-Regulation; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the most common tumor types, and is the third leading cause of cancer mortalities worldwide. A large number of patients with HCC are diagnosed at a late stage when the curative treatment of surgical resection and liver transplantation are no longer applicable. Sorafenib has been proved to improve overall survival in advanced HCC; however, drug resistance is common. The present study reported that the CSN5 is correlated with sorafenib resistance of the HCC cell line HepG2/S. Following silencing of CSN5, resistance to sorafenib was reversed, and multi-drug‑resistance proteins, including as adenosine triphosphate binding cassette (ABC)B1, ABCC2 and ABCG2 as well as CDK6, cyclin D1 and B‑cell lymphoma 2 were downregulated. In addition, it was demonstrated that the integrin beta-1, transforming growth factor‑β1 and nuclear factor‑κB pathways were modified by CSN5.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Carcinoma, Hepatocellular; COP9 Signalosome Complex; Cyclin D1; Cyclin-Dependent Kinase 6; Drug Resistance, Neoplasm; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hep G2 Cells; Humans; Integrin beta1; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; NF-kappa B; Niacinamide; Peptide Hydrolases; Phenylurea Compounds; RNA, Small Interfering; Signal Transduction; Sorafenib; Transforming Growth Factor beta1

The anti-cancer activities of Thonningianin A on the HepG-2 human hepatocellular carcinoma cell line were evaluated by MTT assay, flow cytometry, quantitative real-time PCR and western blotting. Results showed that Thonningianin A effectively inhibited the proliferation of HepG-2 cells by inducing apoptosis, as evidenced by increase in the sub-G1 cell population, DNA fragmentation, and increase in the content of reactive oxygen species. Activation of caspase-9 and the subsequent activation of caspase-3 indicated that Thonningianin A-induced apoptosis is caspase-dependent. Thonningianin A also disrupted the mitochondrial membrane potential (Δψm) and down-regulated the Bcl-xL mRNA expression in HepG-2 cells. Thonningianin A induced cell cycle arrest by changing the cyclin D1 and CDK4 mRNA expression levels. Moreover, western blotting showed that Thonningianin A significantly down-regulated the NF-kappa-B cell survival pathway, along with up-regulation of the expression level of phosphorylated P38 and down-regulation of the expression level of phosphorylated ERK. The anti-cancer activity of Thonningianin A was confirmed by the characteristic patterns of DNA fragmentation and cell cycle arrest, suggesting that Th A is an effective antitumor ingredient in natural plant foods, and is worthy of further study.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Hep G2 Cells; Humans; Hydrolyzable Tannins; Liver Neoplasms

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. It is indispensable to understanding molecular mechanisms of HCC progression and to developing clinically useful biomarkers for this disease.. In this article, we examined whether HOXC8 was associated with the poor prognosis of hepatocellular carcinoma and explored the possible underlying mechanism.. The HOXC8 and Ki67 expression levels in 86 patients with hepatocellular carcinoma were examined using immunohistochemistry. HOXC8 levels in HCC cells were downregulated by siRNA transfection. The cycle progression and cell proliferation status of HCC cells and the oxaliplatin effectiveness were evaluated by flow cytometry and CCK-8 assay. HOXC8, CyclinD1, PCNA, Nkd2, and cleaved caspase-3 levels were detected by western blot.. HOXC8 was upregulated in HCC tissues, compared with adjacent non-tumor ones. HOXC8 expression levels in 86 patients with hepatocellular carcinoma were positively correlated with histological grade. Univariate and multivariate survival analysis revealed that HOXC8 was a significant predictor for overall survival of HCC patients. HOXC8 siRNA knockdown delayed the G1-S phase transition, inhibited cell proliferation, and attenuated resistance to oxaliplatin.. HOXC8 promoted HCC proliferation and predicted poor prognosis. Furthermore, upregulated HOXC8 expression was associated with oxaliplatin resistance in hepatocellular carcinoma.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Antineoplastic Agents; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Carrier Proteins; Caspase 3; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Homeodomain Proteins; Humans; Kaplan-Meier Estimate; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Neoplasm Grading; Organoplatinum Compounds; Oxaliplatin; Proliferating Cell Nuclear Antigen; Proportional Hazards Models; RNA Interference; Signal Transduction; Time Factors; Transfection; Treatment Outcome; Up-Regulation; Young Adult

To investigate the cell cycle of Huh-7 cells affected by I148M polymorphism of PNPLA3 gene and the possible mechanisms.. Huh-7 cells which could respectively overexpress PNPLA3 wild type and I148M variant were cultured and Huh-7 cells with zero load plasmids were used as matched control, Flow cytometry was conducted to detect the cell cycles of these 3 type of Huh-7 cells and western blot and realtime fluorescence quantitative PCR were applied to investigate the expression of regulatory factors (Cyclin D1 and p53) of cell cycle. t-test was used in statistical analysis.. Cell cycle phase distribution was presented by the proportion of cells in each phases (%), compared with the control group, the cell cycle phase distribution (G1 phase 59.27 ± 0.15, G2/M phase 24.23 ± 0.31, S phases 16.50 ± 0.26) had no differences in wild type group (G1 phase 58.53 ± 0.35, G2/M phase 24.87 ± 0.60, S phases 16.60 ± 0.26; Probability value less than 0.05). While between variant type group and wild type group, G1 phase was significantly decreased (variant type group G phase 38.37 ± 0.21, Probability value less than 0.05), S phase and G2/M phase were increased (variant type group S phase 27.47 ± 0.35, P less than 0.05; G2/M phase 34.17 ± 0.15, P less than 0.05), respectively. compared with control group, the relative expression of P53 mRNA in variant type group was significantly upregulated (control group 1.06 ± 0.41, variant type group 6.54 ± 0.34; Probability value less than 0.05) and there was no statistical significance in wild type group (1.66 ± 0.30, P more than 0.05); Cyclin D1 expression showed no statistical significance in any of these three groups, control group 1.00 ± 0.10, wild type group 1.06 ± 0.03, variant type group, 1.11 ± 0.04; P > 0.05).. I148M polymorphism of PNPLA3 gene affects cell cycles of Huh-7 cells via up-regulatating P53.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cyclin D1; Flow Cytometry; Humans; Lipase; Liver Neoplasms; Membrane Proteins; Polymorphism, Genetic

SYF2, also known as p29/NTC31/CBPIN, encodes a nuclear protein that interacts with Cyclin D-type binding-protein 1. SYF2 has been reported to be involved in pre-mRNA splicing and cell cycle regulation. In the present study, we observed that SYF2 was obviously upregulated in HCC tumor tissues and cell lines, and its level was positively correlated with the tumor grade and Ki-67 expression, as well as poor prognosis of HCC. In vitro, using serum starvation-refeeding experiment, our results suggested that SYF2 was upregulated in proliferating HCC cells, and was positive correlated with the expression of PCNA and Cyclin D1. In addition, depletion of SYF2 decreased PCNA and Cyclin D1 levels. Accordingly, interference of SYF2 resulted in cells cycle arrest at G1/S phase in Huh7 HCC cells. Furthermore, we found that SYF2 might interact with Cyclin D1 and could confer doxorubicin resistance in HCC cells. These findings revealed that SYF2 might play a regulatory role in the proliferation of HCC cells. In summary, SYF2 may be a novel prognostic marker and serve as a potential therapeutic target in HCC.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Doxorubicin; Drug Resistance, Neoplasm; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Middle Aged; Nuclear Proteins; Proliferating Cell Nuclear Antigen; RNA Interference; RNA-Binding Proteins; Signal Transduction; Survival Analysis; Time Factors; Transfection; Up-Regulation; Young Adult

The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray® Human Protein Microarrays, we identified glycogen synthase kinase 3β (GSK-3β) and the orphan nuclear receptor (Nur77) as potential interaction partners of NDRG1. These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3β and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3β and Nur77 to allow β-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased β-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3β-9ser (|”‚ R | = 0.28, p = 0.01), Nur77 (|”‚ R | = 0.42, p < 0.001), and β-catenin (| R |= 0.32, p = 0.003) expressions. In conclusion, we identified GSK-3β and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of β-catenin and subsequent downstream signaling mediated by β-catenin in HCC cells, and provides potential targets for future therapeutic interventions.

Topics: Animals; Antineoplastic Agents; beta Catenin; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Doxycycline; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Mice, Nude; Microscopy, Fluorescence; Niacinamide; Nuclear Receptor Subfamily 4, Group A, Member 1; Phenylurea Compounds; Protein Binding; Proteolysis; RNA Interference; Sorafenib; Tumor Burden; Xenograft Model Antitumor Assays

Condensed-bicyclic triazolo-thiadiazoles were synthesized via an efficient "green" catalyst strategy and identified as effective inhibitors of PTP1B in vitro. The lead compound, 6-(2-benzylphenyl)-3-phenyl-[1,2,4]triazolo[3][1,3,4]thiadiazole (BPTT) was most effective against human hepatoma cells, inhibits cell invasion, and decreases neovasculature in HUVEC and also tumor volume in EAT mouse models. This report describes an experimentally unidentified class of condensed-bicyclic triazolo-thiadiazoles targeting PTP1B and its analogs could be the therapeutic drug-seeds.

Topics: Animals; Antineoplastic Agents; Benzofurans; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cell Movement; Chromones; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Human Umbilical Vein Endothelial Cells; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Mice; Models, Molecular; Neoplasm Invasiveness; Neovascularization, Pathologic; Poly(ADP-ribose) Polymerases; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; STAT3 Transcription Factor; Structure-Activity Relationship; Survivin; Thiadiazoles; Triazoles

This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 (low) and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96(®)Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 - RUNX3 (low), the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line; Cell Proliferation; Cisplatin; Core Binding Factor Alpha 3 Subunit; Cyclin D1; Drug Resistance, Viral; Hep G2 Cells; Hepacivirus; Hepatocytes; Host-Pathogen Interactions; Humans; Liver Neoplasms; Nuclear Receptor Subfamily 4, Group A, Member 1; Smad7 Protein; Viral Core Proteins

Currently, sorafenib is the only available chemotherapeutic agent for advanced hepatocellular carcinoma (HCC), but it cannot be used in patients with liver cirrhosis (LC) or thrombocytopenia. In these cases, sorafenib is likely effective if given in combination with treatments that increase the number of platelets, such as thrombopoietin (TPO) receptor agonists. Increasing the platelet count via TPO treatment resulted in reduction of LC. Eltrombopag (EP), a TPO receptor agonist, has been reported to have antitumor effects against certain cancers, despite their lack of TPO receptor expression. We hypothesized that EP may possess antitumor activity against HCC in addition to its ability to suppress hepatic fibrosis by increasing the platelet count. In the present study, the antitumor activity of EP was examined by assessing the inhibition of cell proliferation and then ascertaining the ability of iron supplementation to reverse these effects in HepG2, Hep3B and Huh7 cells. In addition, a cell cycle assay was performed using flow cytometry, and signal transduction was evaluated by analyzing cell cycle-related protein expression. The results of EP were compared with those of the most common iron chelator, deferoxamine (DFO). The combined effect of EP and sorafenib was also assessed. The results revealed that EP exerts antitumor activity in HCC that is mediated by the modulation of intracellular iron content. EP suppressed the expression of the cell cycle-related protein cyclin D1 and elicited cell cycle arrest in the G0/G1 phase. The activity of EP was comparable to that of DFO in HCC, and EP did not compete with sorafenib at low concentrations. In conclusion, our findings suggest that EP is a good candidate chemotherapeutic agent for the treatment of HCC in patients with LC and thrombocytopenia.

Topics: Apoptosis; Benzoates; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Synergism; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Hydrazines; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Pyrazoles; Receptors, Thrombopoietin; Sorafenib

Viscum album var (VAV) also known as mistletoe, has long been categorized as a traditional herbal medicine in Asia. In addition to its immunomodulating activities, mistletoe has also been used in the treatment of chronic hepatic disorders in China and Korea. There are numerous reports showing that VAV possesses anti-cancer effects, however influence on human hepatocarcinoma has never been elucidated. In the present study, hot water extracts of VAV was evaluated for its potential anti-cancer effect in vitro. SK-Hep1 cells were treated with VAV (50-400 ug/ml) for both 24 and 48 hours then cell viability was measured by cell counting kit-8 (CCK-8). Flow cytometry analysis was used to measure the proportion of SK-Hep1 in the different stages of cell cycle. RT-PCR and Western blot analysis were conducted to measure expression of cell cycle arrest related genes and proteins respectively. VAV dose dependently inhibited the proliferation of SK-Hep1 cells without any cytotoxicity with normal Chang liver cell (CCL-13). Flow cytometry analysis showed that VAV extract inhibited the cell cycle of SK-Hep1 cells via G1 phase arrest. RT-PCR and Western blot analysis both revealed that cyclin dependent kinase 2 (Cdk2) and cyclin D1 gene expression were significantly down regulated while p21 was upregulated dose dependently by VAV treatment. Combined down regulation of Cdk2, Cyclin D1 and up regulation of p21 can result in cell death. These results indicate that VAV showed evidence of anti-cancer activity through G1 phase cell cycle arrest in SK-Hep1 cells.

Topics: Anticarcinogenic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression; Hot Temperature; Humans; Liver Neoplasms; Plant Extracts; Up-Regulation; Viscum album; Water

Aflatoxin-B1 (AFB1), a hepatocarcinogenic mycotoxin, was demonstrated to induce the high rate of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in the regulation of several biological processes in HCC. However, the function of miRNAs in AFB1-induced HCC has received a little attention. Here, we applied Illumina deep sequencing technology for high-throughout profiling of microRNAs in HepG2 cells lines after treatment with AFB1. Analysis of the differential expression profile of miRNAs in two libraries, we identified 9 known miRNAs and 1 novel miRNA which exhibited abnormal expression. KEGG analysis indicated that predicted target genes of differentially expressed miRNAs are involved in cancer-related pathways. Down-regulated of Drosha, DGCR8 and Dicer 1 indicated an impairment of miRNA biogenesis in response to AFB1. miR-34a was up-regulated significantly, down-regulating the expression of Wnt/β-catenin signaling pathway by target gene β-catenin. Anti-miR-34a can significantly relieved the down-regulated β-catenin and its downstream genes, c-myc and Cyclin D1, and the S-phase arrest in cell cycle induced by AFB1 can also be relieved. These results suggested that AFB1 might down-regulate Wnt/β-catenin signaling pathway in HepG2 cells by up-regulating miR-34a, which may involve in the mechanism of liver tumorigenesis.

Topics: Aflatoxin B1; beta Catenin; Carcinoma, Hepatocellular; Cluster Analysis; Cyclin D1; DEAD-box RNA Helicases; Down-Regulation; Hep G2 Cells; Humans; Liver Neoplasms; MicroRNAs; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; Ribonuclease III; RNA-Binding Proteins; RNA, Messenger; S Phase Cell Cycle Checkpoints; Sequence Analysis, RNA; Up-Regulation; Wnt Signaling Pathway

Liver X receptors (LXRs), including LXRα and LXRβ isoforms, have important roles in the metabolic regulation of glucose, cholesterol and lipid. Moreover, activation of LXRs also represses the expression of cyclin D1 and cyclin B1, and thus suppresses the proliferation of multiple cancer cells, but the relevant mechanism is not well known. Forkhead box M1 (FOXM1) is a proliferation-specific member of forkhead box family, which is highly expressed in proliferating normal cells and numerous cancer cells. FOXM1 directly activates transcription of cyclin D1 and cyclin B1, resulting in the enhancement of cell cycle progression and cell proliferation. However, it is unclear whether LXRs are involved in the regulation of FOXM1. In this study, we demonstrated that specific LXRs agonists downregulated expression of FOXM1, cyclin D1 and cyclin B1 in hepatocellular carcinoma (HCC) cells, which led to cell cycle and cell proliferation arrest. Knockdown of FOXM1 significantly alleviated LXRs activation-mediated cell cycle arrest and cell growth suppression. Reporter assays showed that the activation of LXRs significantly reduced the transcriptional activity of FOXM1 promoter. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that LXRα but not LXRβ could bind to an inverted repeat IR2 (-52CCGTCAcgTGACCT-39) in the promoter region of FOXM1 gene. Moreover, the xenograft tumor growth and the corresponding FOXM1 expression in nude mice were dramatically repressed by LXRs agonists. Taken together, we conclude that LXRα but not LXRβ functions as a transcriptional repressor for FOXM1 expression. The pathway 'LXRα-FOXM1-cyclin D1/cyclin B1' is a novel mechanism by which LXRs suppress the proliferation of HCC cells, suggesting that the pathway may be a novel target for HCC treatment.

Topics: Animals; Base Sequence; Benzoates; Benzylamines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Disease Models, Animal; Down-Regulation; Forkhead Box Protein M1; Forkhead Transcription Factors; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hepatocytes; Heterografts; Humans; Liver Neoplasms; Liver X Receptors; Mice; Molecular Sequence Data; Orphan Nuclear Receptors; Promoter Regions, Genetic; Protein Binding; Transcription, Genetic

Hepatocellular carcinoma (HCC) is a highly lethal cancer with increasing worldwide incidence, and there are few therapeutics options available for patients with HCC. Thus, novel therapeutic targets for this disease are desperately needed. Chronic infection with hepatitis B virus (HBV) is the major risk factor for the development of HCC, while hepatitis B virus X protein (HBx) is essential for HBV-associated HCC. Based on our previous studies showing that HBx promoted hepatocarcinogenesis of the human non-tumor hepatic cell line L02 and activated Notch1 signaling, Notch1 short hairpin RNA (shRNA) was utilized to inhibit Notch1 mRNA in the present study. We observed that Notch1 shRNA inhibited cell proliferation together with decreased activity of the Notch1 pathway in vitro, and also markedly suppressed tumor formation of L02/HBx cells in a BALB/c nude mouse model in vivo. Furthermore, the blockade of Notch1 was capable of arresting the cell cycle in the G0/G1 phase through the downregulation of CyclinD1, CDK4, E2F1 and the upregulation of p21 and Rb, while all of these factors were involved in the CyclinD1/CDK4 pathway. Inhibition of Notch1 by shRNA markedly promoted the apoptosis of L02/HBx cells via the caspase-9-caspase-3 pathway. These data suggest that inhibition of Notch1 impairs the growth of human HBx-transformed L02 cells, and Notch1 may be a putative therapeutic target for human HBx-associated HCC.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; E2F1 Transcription Factor; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Hepatitis B virus; Hepatitis B, Chronic; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Receptor, Notch1; Retinoblastoma Protein; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Trans-Activators; Viral Regulatory and Accessory Proteins; Xenograft Model Antitumor Assays

Dichlorodiphenyltrichloroethane (DDT) is a persistent organic pollutant, involved in the progression of many cancers, including liver cancer. However, the underlying mechanism(s) of DDT, especially how low doses DDT cause liver cancer, is poorly understood. In this study, we evaluated the impact of p,p'-DDT on the growth of hepatocellular carcinoma using both in vitro and in vivo models. The present data indicated that the proliferation of HepG2 cells was strikingly promoted after exposed to p,p'-DDT for 4 days. In addition, reactive oxygen species (ROS) content was significantly elevated, accompanied with inhibitions of γ-glutamylcysteine synthetase (γ-GCS) and superoxide dismutase (SOD) activities. Interestingly, the levels of β-catenin and its downstream target genes (c-Myc and CyclinD1) were significantly up-regulated, and co-treatment of NAC, the ROS inhibitor, inhibited these over-expressed proteins. Moreover, the p,p'-DDT-stimulated proliferation of HepG2 cells could be reversed after NAC or β-catenin siRNA co-treatment. Likewise, p,p'-DDT treatment increased the growth of tumor in nude mice, stimulated oxidative stress and Wnt/β-catenin pathway. Our study indicates that low doses p,p'-DDT exposure promote the growth of hepatocellular carcinoma via Wnt/β-catenin pathway which is activated by oxidative stress. The finding suggests an association between low dose DDT exposure and liver cancer growth.

Topics: Animals; Antioxidants; beta Catenin; Carcinogens; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; DDT; Dose-Response Relationship, Drug; Glutamate-Cysteine Ligase; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Oxidative Stress; Proto-Oncogene Proteins c-myc; Reactive Oxygen Species; Superoxide Dismutase; Time Factors; Tumor Burden; Wnt Signaling Pathway

The multifunctional RNA-binding protein, CUGBP1, regulates splicing, stability and translation of mRNAs. Previous studies have shown that CUGBP1 is expressed at high levels in the liver, although its role in hepatocellular carcinoma is unknown. Our aim was to determine if CUGBP1 could regulate hepatocellular carcinoma growth.. Expression levels of CUGBP1 were analyzed in 70 hepatic carcinoma and 20 normal hepatic tissue samples by immunohistochemistry (IHC). Using lentivirus-mediated short hairpin RNA (shRNA), CUGBP1 expression in human hepatocellular carcinoma HepG2 cells was knocked-down. The effect of CUGBP1 on hepatic cancer cell growth was investigated.. CUGBP1 was expressed in 85.7% hepatocellular carcinoma specimens compared with 50% in normal liver specimens. CUGBP1 silencing remarkably decreased the proliferation of HepG2 cells, as determined by MTT assay. Flow cytometry analysis showed that knock-down of CUGBP1 led to G0/G1 phase cell cycle arrest, accompanied by sub-G1 accumulation. Moreover, depletion of CUGBP1 resulted in downregulation of cyclin B1 and upregulation of cyclin D1.. These results suggest that CUGBP1 is essential for the growth of hepatocellular carcinoma cells. Knockdown of CUGBP1 might be a potential therapeutic approach for human hepatocellular carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; CELF1 Protein; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Female; Gene Knockdown Techniques; Hep G2 Cells; Humans; Liver Neoplasms; Male; Middle Aged; RNA-Binding Proteins; RNA, Small Interfering

Cyclin D1, encoded by the gene CCND1, is a regulatory protein in the cell cycle transition from G1 phase to S phase. A common polymorphism (G870A) in the exon 4 of CCND1 gene affects splicing of the CCND1 transcript and may cause uncontrollable cellular growth. Therefore, the CCND1 G870A polymorphism may influence an individual's susceptibility to the development of certain tumors. The present study was performed to test the association between G870A polymorphism in the CCND1 gene and hepatocellular carcinoma (HCC) risk in a Chinese population. We extracted the peripheral blood samples from 220 patients with HCC and 220 age- and gender-matched healthy controls. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct DNA sequencing were performed to detect the polymorphism. The CCND1 genotype distribution among HCC patients was not significantly different from that among healthy controls (P=0.08). Compared with the wild-type GG genotype, neither the variant AA genotype nor the variant genotypes containing the A allele were associated with risk of HCC. However, stratification analysis by HBV carrier status revealed that the variant genotypes containing the A allele were associated with a significantly increased risk of HCC among HBsAg-positive individuals (adjusted OR=3.87; 95 % CI=1.12, 13.30). These results suggest that the CCND1 G870A polymorphism may increase the risk of HBV-related HCC in the Chinese population.

Topics: Adult; Aged; Asian People; Carcinoma, Hepatocellular; Cyclin D1; Female; Genetic Predisposition to Disease; Hepatitis B; Humans; Liver Neoplasms; Male; Middle Aged; Polymorphism, Genetic; Risk

The therapeutic potential of baicalein against hepatoma cells was evaluated in vitro and in vivo. In cell viability assays, baicalein showed significant cytotoxicity against the hepatocellular carcinoma cell lines H22, Bel-7404, and Hep G2 and moderate cytotoxicity against immortalized human hepatocytes. Baicalein induced G0/G1-phase arrest in hepatocellular carcinoma cells, inhibited AKT, and promoted the degradation of β-catenin and cyclin D1 without activation of GSK-3β. Furthermore, baicalein significantly inhibited H22 xenograft tumor growth without causing obvious adverse effects on weight or liver and spleen weight indexes in ICR mice. Immunohistochemical analysis showed that the inhibition of tumor growth in baicalein-treated mice was associated with decreased AKT, β-catenin, and cyclin D1 expression ex vivo. Our data indicate that baicalein might regulate cyclin D1 transcription via a β-catenin-dependent mechanism, leading to cell cycle arrest at G0/G1 phase and impaired cancer cell proliferation. These results suggest that baicalein is a potential candidate for the treatment of hepatocellular carcinoma.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flavanones; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Liver Neoplasms; Male; Mice, Inbred ICR; Proto-Oncogene Proteins c-akt; Xenograft Model Antitumor Assays

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

Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168-245 nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers.

Topics: Alternative Splicing; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cytoplasm; Down-Regulation; F-Box Proteins; Humans; Liver; Liver Neoplasms; Mice; NIH 3T3 Cells; Protein Isoforms; Proteolysis

Rsf-1 (HBXAP) was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of Rsf-1 and its clinical significance in human hepatocellular carcinoma (HCC) have not been reported. In the present study, we analyzed the expression pattern of Rsf-1 in human HCC tissues and found that Rsf-1 was overexpressed in 41.1 % of HCC specimens. There was a significant association between Rsf-1 overexpression and tumor stage (p = 0.0322), AFP (p = 0.0184), and tumor relapse (p = 0.0112). Furthermore, Rsf-1 overexpression correlated with poor overall survival in HCC patients (p < 0.001). Rsf-1 overexpression could serve as an independent predictor for poor recurrence-free survival (p = 0.0079). Small interfering RNA (siRNA) knockdown in SK-Hep-1 cells with high endogenous Rsf-1 expression inhibited cell proliferation and colony formation, with downregulation of cyclin E protein. In conclusion, Rsf-1 is overexpressed in HCCs and serves as a novel tumor marker. Rsf-1 contributes to hepatocellular carcinoma cell growth through regulation of cell cycle proteins.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromatin Assembly and Disassembly; Cyclin D1; Female; Humans; Liver Neoplasms; Male; Middle Aged; Nuclear Proteins; Prognosis; Trans-Activators

Sperm-associated antigen 9 (SPAG9) was reported as a novel biomarker for several cancers and associated with the malignant behavior of cancer cells. However, its expression pattern and biological role in human hepatocellular carcinoma (HCC) have not been reported. In the present study, we analyzed SPAG9 expression in human HCC tissues by immunohistochemistry and found that SPAG9 overexpression is correlated with tumor stage (p < 0.001), tumor multiplicity (p = 0.019), tumor size (p = 0.034), AFP levels (p = 0.006), and tumor relapse (p = 0.0017). Furthermore, SPAG9 overexpression is correlated with poor overall survival (p < 0.001) and relapse-free survival (p = 0.002). Transfection of SPAG9 small interfering RNA (siRNA) was performed in Bel-7402 cell line. Colony formation and MTT showed that SPAG9 siRNA knockdown inhibited HCC cell proliferation. We also found that SPAG9 depletion could increase cell apoptosis. In addition, the level of cyclin D1 and cyclin E protein expression was downregulated after siRNA treatment. In conclusion, SPAG9 is overexpressed in human HCC and serves as a prognostic marker. SPAG9 contributes to cancer cell growth through regulation of cyclin proteins.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Disease Progression; Female; Humans; Liver Neoplasms; Male; Middle Aged; Prognosis; Proportional Hazards Models

Activation of the Wnt/β-catenin pathway has been observed in at least 1/3 of hepatocellular carcinomas (HCC), and a significant number of these have mutations in the β-catenin gene. Therefore, effective inhibition of this pathway could provide a novel method to treat HCC. The purposed of this study was to determine whether FH535, which was previously shown to block the β-catenin pathway, could inhibit β-catenin activation of target genes and inhibit proliferation of Liver Cancer Stem Cells (LCSC) and HCC cell lines. Using β-catenin responsive reporter genes, our data indicates that FH535 can inhibit target gene activation by endogenous and exogenously expressed β-catenin, including the constitutively active form of β-catenin that contains a Serine37Alanine mutation. Our data also indicate that proliferation of LCSC and HCC lines is inhibited by FH535 in a dose-dependent manner, and that this correlates with a decrease in the percentage of cells in S phase. Finally, we also show that expression of two well-characterized targets of β-catenin, Cyclin D1 and Survivin, is reduced by FH535. Taken together, this data indicates that FH535 has potential therapeutic value in treatment of liver cancer. Importantly, these results suggest that this therapy may be effective at several levels by targeting both HCC and LCSC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Molecular Targeted Therapy; Neoplastic Stem Cells; Sulfonamides; Survivin; Transcriptional Activation; Wnt Signaling Pathway

The human sulfatase 1 (hSulf-1) gene encodes an endosulfatase that functions to inhibit the heparin-binding growth factor signaling, including the basic fibroblast growth factor (bFGF)-mediated pathway, by desulfating the cell surface heparan sulfate proteoglycans (HSPGs). bFGF could stimulate cell cycle progression and inhibit cell apoptosis, this biological effect can be reversed by hSulf-1. However, molecular mechanisms have not been fully reported. In the current study, by reactivation of hSulf-1 expression and function in the hSulf-1-negative hepatocellular carcinoma (HCC) cell lines and HCC xenograft tumors, we found that hSulf-1 blocked the bFGF effect on the promotion of cell cycle and inhibition of apoptosis. The bFGF-stimulated activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways was suppressed by hSulf-1, which led to a decreased expression of the target genes Cyclin D1 and Survivin, then finally induced cell cycle arrest and apoptosis in HCC cells. Our data suggested that hSulf-1 may be a suitable target for cancer therapy.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Survival; Cyclin D1; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Sulfotransferases; Survivin; Tumor Burden; Xenograft Model Antitumor Assays

Macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, plays important roles in cancer-related biological processes. However, few studies have focused on the clinical relevance of MIF and cyclin D1 expression in hepatocellular carcinoma cells (HCCs). In this study, MIF and cyclin D1 expression levels in HCC tissues and cell lines were significantly upregulated compared with adjacent normal tissues or a normal liver cell line. In HCC specimens, MIF expression positively correlated with cyclin D1 expression. Additionally, MIF and cyclin D1 expression positively correlated with tumor size. MIF knockdown inhibited the proliferation of PLC and HepG2 cells and promoted apoptosis. However, small interfering RNA (siRNA) against MIF did not influence the cell cycle in these cells. In an in vivo xenograft model, MIF knockdown reduced the tumor growth rate. The expression levels of Bcl-2, p-caspase-3, BIM and Bax were upregulated, while the expression levels of cyclin D1, p-Akt and p-ERK were downregulated in MIF-knockdown cells. These findings indicate that MIF siRNA reduces proliferation and increases apoptosis in HCC cells. MIF knockdown inhibits the expression of growth-related proteins and induces the expression of apoptosis-related proteins, supporting a role for MIF as a novel therapeutic target for HCC.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Hep G2 Cells; Humans; Liver Neoplasms; Macrophage Migration-Inhibitory Factors; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; RNA, Small Interfering; Xenograft Model Antitumor Assays

The aim of the present study was to examine the expression and significance of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), β‑catenin and cyclin D1 in hepatocellular carcinoma (HCC). A total of 24 samples of HCC and adjacent normal tissues were analyzed. The expression of Pin1, β‑catenin and cyclin D1 in HCC were detected using immunohistochemistry, western blot analysis, polymerase chain reaction and immunofluorescence. The expression of Pin1, β‑catenin and cyclin D1 in HCC tissues were significantly higher than that in adjacent tissues. Pin1 was not markedly expressed in the adjacent normal tissues, while expression in the cytoplasm and nucleus of HCC cells was high. However, β‑catenin and cyclin D1 only revealed a weak expression in the cytoplasm and nucleus of HCC cells. Immunoprecipitation analyses demonstrated two clear bands at 19 and 34 kDa, and a brown band at 55 kDa as expected. Immunofluorescence analysis of HCC cells indicated that Pin1 was present in the cytoplasm and nucleus, and β‑catenin and cyclin D1 were present in the nucleus. In conclusion, the present study indicated that Pin1, β‑catenin and cyclin D1 were highly expressed in HCC. Therefore, detection of the expression of Pin1, β‑catenin and cyclin D1 may be useful for the development of novel diagnostic and treatment strategies for HCC.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Humans; Immunohistochemistry; Immunoprecipitation; Liver; Liver Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; RNA, Messenger

Metastasis is one of the major causes of cancer-related death. It is a complex biological process involving multiple genes, steps, and phases. It is also closely connected to many biological activities of cancer cells, such as growth, invasion, adhesion, hematogenous metastasis, and lymphatic metastasis. Fucoidan derived from Undaria pinnatifida sporophylls (Ups-fucoidan) is a sulfated polysaccharide with more biological activities than other fucoidans. However, there is no information on the effects of Ups-fucoidan on tumor invasion and metastasis. We used the mouse hepatocarcinoma Hca-F cell line, which has high invasive and lymphatic metastasis potential in vitro and in vivo, to examine the effect of Ups-fucoidan on cancer cell invasion and metastasis. Ups-fucoidan exerted a concentration- and time-dependent inhibitory effect on tumor metastasis in vivo and inhibited Hca-F cell growth, migration, invasion, and adhesion capabilities in vitro. Ups-fucoidan inhibited growth and metastasis by downregulating vascular endothelial growth factor (VEGF) C/VEGF receptor 3, hepatocyte growth factor/c-MET, cyclin D1, cyclin-dependent kinase 4, phosphorylated (p) phosphoinositide 3-kinase, p-Akt, p-extracellular signal regulated kinase (ERK) 1/2, and nuclear transcription factor-κB (NF-κB), and suppressed adhesion and invasion by downregulating L-Selectin, and upregulating protein levels of tissue inhibitor of metalloproteinases (TIMPs). The results suggest that Ups-fucoidan suppresses Hca-F cell growth, adhesion, invasion, and metastasis capabilities and that these functions are mediated through the mechanism involving inactivation of the NF-κB pathway mediated by PI3K/Akt and ERK signaling pathways.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Liver Neoplasms, Experimental; Lymphatic Metastasis; Male; MAP Kinase Signaling System; Mice; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Plant Extracts; Polysaccharides; Proto-Oncogene Proteins c-met; Receptors, Vascular Endothelial Growth Factor; Undaria; Vascular Endothelial Growth Factor A

Small molecules that restore the expression of growth-inhibitory microRNAs (miRNA) downregulated in tumors may have potential as anticancer agents. miR34a functions as a tumor suppressor and is downregulated or silenced commonly in a variety of human cancers, including hepatocellular carcinoma (HCC). In this study, we used an HCC cell-based miR34a luciferase reporter system to screen for miR34a modulators that could exert anticancer activity. One compound identified as a lead candidate, termed Rubone, was identified through its ability to specifically upregulate miR34a in HCC cells. Rubone activated miR34a expression in HCC cells with wild-type or mutated p53 but not in cells with p53 deletions. Notably, Rubone lacked growth-inhibitory effects on nontumorigenic human hepatocytes. In a mouse xenograft model of HCC, Rubone dramatically inhibited tumor growth, exhibiting stronger anti-HCC activity than sorafenib both in vitro and in vivo. Mechanistic investigations showed that Rubone decreased expression of cyclin D1, Bcl-2, and other miR34a target genes and that it enhanced the occupancy of p53 on the miR34a promoter. Taken together, our results offer a preclinical proof of concept for Rubone as a lead candidate for further investigation as a new class of HCC therapeutic based on restoration of miR34a tumor-suppressor function.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Proliferation; Chalcones; Cyclin D1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; MicroRNAs; Small Molecule Libraries; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Sorafenib is the approved systemic drug of choice for advanced hepatocellular carcinoma (HCC), but has demonstrated limited benefits because of drug resistance. 2-Methoxyestradiol (2ME2) has been shown to be a promising anticancer drug against various types of cancers and acts by dysregulating hypoxia-inducible factor (HIF)-1. Hypoxic cancer cells are extremely resistant to therapies since they elicit strong survival ability due to the cellular adaptive response to hypoxia, which is controlled by HIF-1 and HIF-2. The present study has demonstrated that sorafenib downregulated the expression of HIF-1α, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways, resulting in upregulation of HIF-2α, which contributes to the insensitivity of hypoxic HCC cells to sorafenib. HIF-2α played a dominant role in regulating VEGF, thus sorafenib in turn increased the expression of VEGF (a downstream molecule of both HIF-1 and HIF-2) and cyclin D1 (a downstream molecule of HIF-2), but reduced the expression of LDHA (a downstream molecule of HIF-1), in hypoxic HCC cells. 2ME2 significantly reduced the expression of both HIF-1α and HIF-2α, and their downstream molecules, VEGF, LDHA and cyclin D1, rendering hypoxic HCC cells to increased sensitivity to 2ME2. 2ME2 also inhibited the nuclear translocation of HIF-1α and HIF-2α proteins, but had no effect on their mRNA expression. 2M2 synergized with sorafenib to suppress the proliferation and induction of apoptosis of HCC cells in vitro and in vivo, and inhibited tumoral angiogenesis. These results indicate that 2ME2 given in combination with sorafenib acts synergistically for treating HCC.

Topics: 2-Methoxyestradiol; Active Transport, Cell Nucleus; Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Drug Synergism; Estradiol; Hep G2 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Niacinamide; Phenylurea Compounds; RNA Interference; Signal Transduction; Sorafenib; Time Factors; Transfection; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

To observe the effect of bear bile powder (BBP) on the STAT3 pathway and its downstream target genes of nude mice hepatocellular carcinoma (HCC) xenograft, and to explore its mechanism for treating HCC.. The subcutaneous xenograft model was established using HepG2 cells. When the subcutaneous transplanted tumor was formed, naked mice were randomly divided into two groups, the BBP group and the control group. Mice in the BBP group were administered with BBP by gastrogavage, once daily for 3 consecutive weeks, while mice in the control group were administered with normal saline by gastrogavage, once daily for 3 consecutive weeks. The body weight and the tumor volume were measured once per week. By the end of medication, the tumor weight was weighed and the tumor inhibition ratio calculated. The apoptosis of the tumor tissue was detected by TdT-mediated dUTP nick end labeling (TUNEL). The expression of Bcl2-associated X protein (Bax), B cell lymphoma/eukemina-2 (Bcl-2), cyclin-dependent protein kinase (CDK4), cyclinD1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of signal transducers and transcription activators 3 (p-STAT3), proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, CDK4, and cyclinD1 were determined by immunohistochemistry.. BBP could inhibit the tumor volume and tumor weight, showing statistical difference when compared with the control group (P < 0.01). Results of TUNEL showed that BBP could significantly induce the apoptosis of hepatoma carcinoma cells. Results of RT-PCR showed that BBP could up-regulate the expression of Bax and down-regulate mRNA expression of Bcl-2, CDK4, and cyclinD1. Immunohistochemical results showed that BBP could up-regulate the expression of Bax and inhibit the protein expression of p-STAT3, PCNA, Bcl-2, CDK4, and cyclinD1.. BBP could induce the apoptosis of hepatoma carcinoma cells and inhibit their proliferation by regulating STAT3 pathway.

Topics: Animals; bcl-2-Associated X Protein; Bile; Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase 4; Drugs, Chinese Herbal; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Ursidae; Xenograft Model Antitumor Assays

Aberrant Notch signaling is observed in human hepatocellular carcinoma (HCC) and has been associated with the modulation of cell growth. However, the role of Notch signaling in HCC and its underlying mechanism remain elusive. RBP-J-interacting and tubulin-associated (RITA) mediates the nuclear export of RBP-J to tubulin fibers and downregulates Notch-mediated transcription. In this study, we found that RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. These changes led to growth inhibition and induced G0/G1 cell cycle arrest and apoptosis in SMMC7721 and HepG2 cells. Our findings indicate that RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; DNA-Binding Proteins; Down-Regulation; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Knockdown Techniques; Hep G2 Cells; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Liver Neoplasms; Neoplasm Proteins; RNA, Small Interfering; Signal Transduction; Transcription Factor HES-1; Transcription Factor RelA; Tubulin; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Up-Regulation

To study the effect of Biejiajian Pills on Wnt signal pathway and the mechanisms underlying its action to suppress the invasiveness of hepatocellular carcinoma.. HepG2 cells cultured in the serum of rats fed with Biejiajian Pills for 48 h were examined for β-catenin expression using immunofluorescence, β-catenin/TCF4 complex activity with luciferase, and expressions of the downstream proteins cyclin D1 and MMP-2 using qRT-PCR.. Biejiajian Pills-treated sera significantly reduced the expressions of cytoplasmic and nuclear β-catenin protein, cyclin D1 and MMP-2 proteins and lowered the activities of β-catenin/TCF4 complex.. Biejiajian Pills may serve as a potential anti-tumor agent, whose effect might be mediated by inhibiting the Wnt/β-catenin pathway.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Carcinoma, Hepatocellular; Cyclin D1; Drugs, Chinese Herbal; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Rats; Transcription Factor 4; Transcription Factors; Wnt Proteins; Wnt Signaling Pathway

The aims of this study are to explore the effects of epidermal growth factor (EGF) on hepatocyte proliferation in presence of plasma from patients with acute liver failure (ALF).. HepG2 cells were cultured with 50% plasma from patients with ALF for 6, 12, 24, 48 and 72h with or without different concentrations of EGF. Cell proliferation was determined by the MTT assay and intracellular cyclin D1, cyclin-dependent kinase 4 (CDK4) expressions were analyzed by western blotting.. The proliferation of HepG2 cells was significantly inhibited by treatment with plasma from patients with ALF from 12 to 72 h. Intracellular expression of cyclin D1 and CDK4 was also markedly down-regulated. 5ng/ml, 10ng/ml and 20ng/ml EGF dose dependently induced HepG2 proliferation in presence of plasma from normal control, but only 20ng/ml EGF showed a transient promoting effect on proliferation of HepG2 cells in presence of plasma from patients with ALF.. Plasma from patients with ALF inhibits HepG2 cell proliferation via downregulation of cyclin D1 and CDK4 expression. Plasma from patient with ALF dampens HepG2 cells to EGF induced proliferation response.

Topics: Adolescent; Adult; Apoptosis; Carcinoma, Hepatocellular; Case-Control Studies; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Hep G2 Cells; Humans; Liver Failure, Acute; Liver Neoplasms; Male; Middle Aged; Time Factors

Hepatocellular carcinoma (HCC) frequently recurs even after curative resection. The purpose of this study was to identify factors predictive for postoperative recurrence of HCC in patients who underwent curative resection using immunohistochemistry.. Expression of vascular endothelial growth factor (VEGF), E-cadherin and cyclin D1 in HCC tissue were analyzed for 133 HCC patients who underwent curative resection of tumors using immunohistochemical analysis. Relationships of expressions and disease-free survival of HCC were evaluated using univariate and multivariate analyses.. The average period of follow-up of the patients was 6.7 years. Multivariate analyses revealed that only strong expression of VEGF in HCC tissue was significantly associated with metastatic recurrence (p < 0.001, hazard ratio, HR, 3.32).. Evaluating VEGF in HCC tissue after surgical resection has predictive value for metastatic HCC recurrence. The ability to risk stratify should improve the treatment strategies after hepatectomy.

Topics: Cadherins; Carcinoma, Hepatocellular; Chi-Square Distribution; Cyclin D1; Disease-Free Survival; Female; Hepatectomy; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Neoplasm Recurrence, Local; Predictive Value of Tests; Proportional Hazards Models; Retrospective Studies; Vascular Endothelial Growth Factor A

OCT4 and BIRC5 are preferentially expressed in human cancer cells and mediate cancer cell survival and tumor maintenance. However, the molecular mechanism that regulates OCT4 and BIRC5 expression is not well characterized.. By manipulating OCT4 and BIRC5 expression in hepatocellular carcinoma (HCC) cell lines, the regulatory mechanism of OCT4 on BIRC5 and CCND1 were investigated.. Increasing or decreasing OCT4 expression could enhance or suppress BIRC5 expression, respectively, by regulating the activity of BIRC5 promoter. Because there is no binding site for OCT4 within BIRC5 promoter, the effect of OCT4 on BIRC5 promoter is indirect. An octamer motif for OCT4 in the CCND1 promoter has directly and partly participated in the regulation of CCND1 promoter activity, suggesting that OCT4 also could upregulated the expression of CCND1. Co-suppression of OCT4 and BIRC5 induced cancer cell apoptosis and cell cycle arrest, thereby efficiently inhibiting the proliferative activity of cancer cells and suppressing the growth of HCC xenogrfts in nude mice.. OCT4 can upregulate BIRC5 and CCND1 expression by increasing their promoter activity. These factors collusively promotes HCC cell proliferation, and co-suppression of OCT4 and BIRC5 is potentially beneficial for HCC treatment.

Topics: Analysis of Variance; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Octamer Transcription Factor-3; Promoter Regions, Genetic; Survivin; Up-Regulation

Studies of the transcriptional networks that regulate nuclear receptor-mediated proliferation of quiescent hepatocytes could lead to new information about liver growth and hepatoprotective strategies.. We used quantitative real-time PCR to analyze expression of neuron-derived orphan receptor 1 (Nor-1) and its target genes during liver regeneration after hepatectomy in mice, and in hepatocellular carcinoma (HCC) samples from patients. We used adenoviral vectors to express Nor-1 in normal liver (Ad/CMV/V5-Nor-1), or reduce its level with small hairpin RNAs (Ad/BLOCK-iT/Nor-1(small hairpin RNA)) after partial hepatectomy.. Levels of Nor-1 messenger RNA and protein, and transcription of Nor-1 target genes (Ccnd1 and Vcam-1), increased during the late priming and proliferative phases of liver regeneration after partial hepatectomy. Levels of NOR-1 messenger RNA and transcription of its target gene CCND1 and of the NOR-1 subfamily member NUR-77 also increased in human HCC samples compared with paired HCC-free tissue. Ad-Nor-1(small hairpin RNA) reduced the hepatocyte proliferation after hepatectomy. Overexpression of Nor-1 in normal livers of mice induced proliferation of quiescent hepatocytes independently of interleukin-6 and tumor necrosis factor-α signaling. In gene expression profile analysis, Nor-1 altered expression of genes involved in the cell cycle, proliferation, and tumorigenesis.. In mice, the orphan nuclear receptor Nor-1 activates proliferation of quiescent hepatocytes and is required for hepatocyte proliferation after partial hepatectomy. Nor-1 and its gene targets are also up-regulated in human HCC samples. Nor-1 activates a transcriptional program that induces hepatocyte proliferation independently of inflammatory signaling pathways.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Hepatectomy; Hepatocytes; Humans; Liver Neoplasms; Liver Regeneration; Male; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Nuclear Receptor Subfamily 4, Group A, Member 1; Receptors, Steroid; Receptors, Thyroid Hormone; RNA, Messenger; Up-Regulation; Vascular Cell Adhesion Molecule-1

Hepatocellular carcinoma (HCC) is one of the most common and lethal human malignancies. Lack of efficient therapy for advanced HCC is a pressing problem worldwide. This study aimed to determine the efficacy and mechanism of combined sorafenib and radiation therapy treatment for HCC.. HCC cell lines (PLC5, Huh-7, Sk-Hep1, and Hep3B) were treated with sorafenib, radiation, or both, and apoptosis and signal transduction were analyzed.. All 4 HCC cell lines showed resistance to radiation-induced apoptosis; however, this resistance could be reversed in the presence of sorafenib. Inhibition of phospho-STAT3 was found in cells treated with sorafenib or sorafenib plus radiation and subsequently reduced the expression levels of STAT3-related proteins, Mcl-1, cyclin D1, and survivin. Silencing STAT3 by RNA interference overcame apoptotic resistance to radiation in HCC cells, and the ectopic expression of STAT3 in HCC cells abolished the radiosensitizing effect of sorafenib. Moreover, sorafenib plus radiation significantly suppressed PLC5 xenograft tumor growth.. These results indicate that sorafenib sensitizes resistant HCC cells to radiation-induced apoptosis via downregulating phosphorylation of STAT3 in vitro and in vivo.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Chemoradiotherapy; Cyclin D1; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Niacinamide; Phenylurea Compounds; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; RNA Interference; Sorafenib; STAT3 Transcription Factor; Survivin

To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.. The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000(TM), and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay.. Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group; in the siRNA transfection group, the amount of hepatoma cells in G1 phase was more than that in the control group. The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group. The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.. Hypoxia increases the expression of HIF-1α, and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cobalt; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Gene Silencing; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Time Factors; Tumor Microenvironment; Vascular Endothelial Growth Factor A

There has been increased interest in the possible role of human cytomegalovirus (HCMV) in carcinogenesis during the last decade. HCMV seroprevalence was enhanced in patients with hepatocellular carcinoma (HCC) but a possible relationship between HCC and HCMV infection remained to be assessed. The aim of this work was to investigate the pro-tumor influence of HCMV on primary human hepatocytes (PHH) and HepG2 cells.. Following infection of PHH and HepG2 cells by two different strains of HCMV, we measured the production of IL-6 in culture supernatants by ELISA and the protein levels of STAT3, pSTAT3, JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western Blotting in infected and uninfected cells. Cell proliferation and transformation were investigated using Ki67Ag expression measurement and soft-agar colony formation assay respectively.. Infection of HepG2 cells and PHH by HCMV resulted in the production of IL-6 and the subsequent activation of the IL-6R-JAK-STAT3 pathway. HCMV increased the expression of cyclin D1 and survivin. Cell proliferation was enhanced in HepG2 and PHH infected with HCMV, despite a paradoxical overexpression of p53 and p21. More importantly, we observed the formation of colonies in soft agar seeded with PHH infected with HCMV and when we challenged the HepG2 cultures to form tumorspheres, we found that the HCMV-infected cultures formed 2.5-fold more tumorspheres than uninfected cultures.. HCMV activated the IL-6-JAK-STAT3 pathway in PHH and HepG2 cells, favored cellular proliferation, induced PHH transformation and enhanced HepG2 tumorsphere formation. Our observations raise the possibility that HCMV infection might be involved in the genesis of hepatocellular carcinoma.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytomegalovirus; Enzyme Activation; Hep G2 Cells; Hepatocytes; Humans; Inhibitor of Apoptosis Proteins; Interleukin-6; Janus Kinases; Liver Neoplasms; Mice; STAT3 Transcription Factor; Survivin; Tumor Suppressor Protein p53; Up-Regulation

Hepatitis B virus X protein (HBx) has been shown to induce hepatocarcinogenesis by disrupting the functions of intracellular molecules. Cyclin-dependent kinase inhibitor p21 (Cip1/WAF1), known as a tumour-suppressor gene, has been reported to have paradoxical function, that is, acting as an oncogene, particularly when expressed in the cytoplasm. The effects of HBx on the expression and function of p21 also remain controversial.. We attempted to investigate the role of HBx in the hepatocarcinogenic process, focusing on the association with this paradoxical function of p21. The results obtained were further verified with experiments using the antihepatocarcinogenic action of interferon (IFN)-β.. HBx transgenic mice (Xg) and HBx-transfected hepatoma cell lines were used. Intracellular localization of p21 was determined by Western blot analysis and immunofluorescence.. Xg and HBx-transfected cells exhibited increased expression of p21. Up-regulation of p21 was positively correlated with the expression of cyclin D1 and inactive phosphorylation of retinoblastoma protein (pRb). These HBx-induced cell proliferative responses were cancelled by knockdown of p21, which resulted in growth reduction in HBx-expressing cells, suggesting the oncogenic properties of HBx-induced p21. HBx induced accumulation of p21 in the cytoplasm, and activation of PKCα was involved. Finally, IFN-β-treated Xg liver, as well as hepatoma cells, showed a shift of cytoplasmic p21 to the nucleus, accompanied by the abrogation of HBx-induced oncogenic modulation.. Our results suggest that HBx induces hepatocarcinogenesis via PKCα-mediated overexpression of cytoplasmic p21 and IFN-β suppressed these molecular events by shifting p21 to the nucleus.

Topics: Active Transport, Cell Nucleus; Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Viral; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytoplasm; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Interferon-beta; Liver Neoplasms; Mice; Mice, Transgenic; Phosphorylation; Protein Kinase C-alpha; Retinoblastoma Protein; RNA Interference; Signal Transduction; Trans-Activators; Transfection; Up-Regulation; Viral Regulatory and Accessory Proteins

Signal transducer and activator of transcription 3 (STAT3) is persistently activated in cancer cells and contributes to malignant progression in various types of cancer. The Janus-activated kinase (JAK) family phosphorylates STAT3 in response to stimulation by cytokines or growth factors. The JAK1-STAT3 signaling pathway plays an important role in cell proliferation and apoptosis. Nitidine chloride (NC) is a benzophenanthridine alkaloid that has been reported as an antitumor agent due to its its inhibitory effects on topoisomerase I. Using a mouse xenograft model of hepatocellular carcinoma (HCC), this study aimed to evaluate the effects of NC on tumor growth in vivo and to elucidate the underlying mechanisms. The analysis of the effects of NC on apoptosis in HCC tumor xenografts in mice was carried out by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expression of Bcl-2, Bax, cyclin-dependent kinase (CDK)4, cyclin D1, p21 and proliferating cell nuclear antigen (PCNA) was analyzed by immunohistochemistry; and the protein expression of JAK1 and STAT3 was examined by western blot analysis. Our results revealed that treatment with NC decreased the tumor volume and tumor weight, suggesting that NC inhibits HCC cell growth in vivo. In addition, NC blocked the activation of JAK1-STAT3 in the tumor tissues, which in turn resulted in the induction of cancer cell apoptosis and the inhibition of proliferation. Consequently, treatment with NC downregulated the expression of cyclin D1, CDK4 and Bcl-2 and increased the level of p21 and Bax. Our data provide a molecular basis for the antitumor activity of NC.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Benzophenanthridines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 1; Liver Neoplasms; Mice; p21-Activated Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Tumor Burden; Xenograft Model Antitumor Assays

Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we used dual gene targeted siRNA (DGT siRNA) conjugate composed of NET-1 and VEGF siRNA sequences in the same backbone could inhibit growth and angiogenesis HCC. DGT siRNA showed a further down regulation on VEGF mRNA and protein levels compared with NET-1 siRNA or VEGF siRNA, but not on NET-1 expression. It also exhibited greater suppression on proliferation and trigger of apoptosis in HepG2 cells than NET-1 siRNA or VEGF siRNA; this could be explained by the significant down regulation of cyclin D1 and Bcl-2. A lower level of ANG2 mRNA and protein was detected in HUVEC cultured with supernatant of HepG2 cells treated with DGT siRNA than that of VEGF siRNA or NET-1 siRNA, resulting in much more inhibited angiogenesis of HUVEC. Tumor growth was inhibited and microvessel density dropped in the xenograft tumor models compared to the untreated controls. NET-1 and VEGF silencing play a key role in inhibiting hepatocellular cell proliferation, promoting apoptosis, and reducing angiogenesis. Simultaneous silencing of NET-1 and VEGF using DGT siRNA construct may provide an advantageous alternative in development of therapeutics for Hepatocellular carcinoma.

Topics: Angiopoietin-2; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Silencing; Hep G2 Cells; Human Umbilical Vein Endothelial Cells; Humans; Immunoprecipitation; Liver Neoplasms; Mice; Mice, Nude; Microvessels; Neovascularization, Pathologic; Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Small Interfering; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

It has been demonstrated that nuclear factor-kappa B (NF-κB), which is overactivated in hepatocellular carcinoma (HCC), plays important roles in the development of HCC. Recently, a group of dysregulated micro RNAs were reported to be involved in HCC progression. Further understanding of micro RNA-mediated regulation of NF-κB pathway may provide novel therapeutic targets for HCC. In this study, we found that miR-451 expression was markedly downregulated in HCC cells and tissues compared with immortalized normal liver epithelial cells and adjacent non- cancerous tissues, respectively. Upregulation of miR-451 inhibited, while downregulation of miR-451 promoted, the tumorigenicity of HCC cells both in vitro and in vivo. These changes in the properties of HCC cells were associated with deregulation of two well-known cellular G1/S transitional regulators, cyclin D1 and c-Myc, which are downstream targets of NF-κB pathway. Furthermore, we demonstrated that miR-451 upregulation led to downregulation of cyclin D1 and c-Myc through inhibition of NF-κB pathway initiated by direct targeting of the IKBKB 3'-untranslated region. Therefore, these results suggest that miR-451 downregulation plays an important role in promoting proliferation of HCC cells and may provide the basis for the development of novel anti-HCC therapies.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin D1; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; NF-kappa B; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

This study aims to investigate the using of bone marrow mesenchymal stem cells (BMSCs) genetically engineered to produce interferon-β (IFN-β) as a gene delivery system to treat hepatocellular carcinoma (HCC) in vitro and in vivo.. To measure the effects on tumour cell growth in vitro, IFN-β-producing BMSCs (BMSC/IFN-β) were co-cultured with the HCC cell line HepG2 and Huh7. Enzyme-linked immunosorbent assay (ELISA) was used to detect the IFN-β secretion in the BMSC culture condition medium (CM). The effect of BMSC/IFN-β on HCC cells proliferation was examined both in vitro and in vivo by using MTT, colony formation assay, BrdU staining, cell cycle analysis, and xenografted NOD/SCID mouse tumour model. To examine the impact of BMSC/IFN-β on the AKT/FOXO3a signalling, RT-PCR and western blotting were performed.. The BMSC/IFN-β cells can stably secrete high levels of IFN-β. Both MTT and colony forming assay showed that HCC cells had a lower growth rate when cultured in BMSC/IFN-β-CM as compared with that in BMSC/vector-CM or DMEM culture group. Co-culture with BMSC/IFN-β-CM dramatically decreased the percentages of cells with incorporated BrdUrd. In BMSC/IFN-β-CM-treated HCC cells, the proportion of G1-phase cells increased but it decreased in the S phase of the cell. The BMSC/IFN-β inhibited HCC growth in NOD/SCID mice and proved the survival period of these mice. Compared with the control group, p21 and p27 expression of hepatoma cells increased, whereas cyclin D1 and phosphorylation of Rb expression decreased when co-cultured with BMSC/IFN-β-CM. It was associated with suppression of Akt activity and enhanced transcriptional activity of FOXO3a.. The IFN-β gene-modified BMSCs can effectively inhibit the proliferation of HCC cells in vitro and in vivo through inhibiting AKT/FOXO3a pathway. These results indicate that BMSC/IFN-β are a powerful anticancer cytotherapeutic tool for HCC.

Topics: Animals; Bone Marrow Cells; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Drug Delivery Systems; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Interferon-beta; Liver Neoplasms; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Transcription, Genetic; Xenograft Model Antitumor Assays

Metformin is used as a first-line therapy for type 2 diabetes, with reports of its usefulness for the prevention and control of several types of cancers. This study investigated the effects of metformin on hepatocellular carcinoma (HCC). The human HCC cell lines HepG2 and PLC/PRF/5 were cultured and treated with metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of adenosine monophosphate (AMP)-activated protein kinase. Changes in cell viability and cell cycle distribution were evaluated by MTT and flow cytometry, respectively. Apoptosis was assessed by fluorescent-dye staining. An HCC model was established in 6- to 8-week-old BALB/c-nu mice by subcutaneous injection of PLC/PRF/5 cells. After 1 week, mice were treated intragastrically with metformin or vehicle. Tumor xenograft tissues were examined using immunohistochemistry for evaluation of the the expression of cyclin D1, p21CIP and p27KIP. HCC cells and tissues from the in vitro and in vivo experiments, respectively, were subjected to protein extraction and western blotting. We found that metformin treatment reduced HCC cell viability in a dose-dependent manner similar to AICAR treatment. In addition, metformin treatment induced HCC cell cycle arrest at G1/G0 phase and apoptosis. Intragastric treatment of the mouse PLC/PRF/5 cell xenograft model with metformin showed that metformin not only blocked tumor progression, but also reduced tumor morbidity. Treatment with metformin upregulated the expression of p21CIP and p27KIP, but downregulated cyclin D1 levels, both in vitro and in vivo. Metformin treatment also upregulated the expression of phosphorylated AMPK protein in xenograft tissues. These findings indicate that metformin warrants further evaluation as a novel therapeutic and preventive strategy against HCC.

Topics: Aminoimidazole Carboxamide; Animals; Carcinoma, Hepatocellular; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Metformin; Mice; Ribonucleotides; Xenograft Model Antitumor Assays

To investigate the effect of Oxaliplatin (L-OHP) on cell cycle in hepatocellular carcinoma cell line HepG2 and the involved mechanism.. Inhibitory effect of L-OHP on the proliferation of HepG2 cells was determined by MTT assay. Cell cycle distribution was shown by flow FCM. The expression levels of cyclinD1, CDK2, CDK4, p16, p21, p53 were detected by RT-PCR and Western blot.. MTT method revealed that L-OHP inhibited proliferation of hepatocellular carcinoma HepG2 cells in a dose- and time-dependent manner. L-OHP induced S cell cycle arrest in HepG2 cell; down-regulated the levels of CDK4, cyclinD1 and up-regulated the levels of p21, p53. There were no significant changes of CDK2 and p16 after L-OHP treatment.. L-OHP inhibits the proliferation of HepG2 cells by blocking cell at S stage, which may be resulted from the activity of CDK4, CyclinD1 and p21.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Hep G2 Cells; Humans; Liver Neoplasms; Organoplatinum Compounds; Oxaliplatin; Tumor Suppressor Protein p53

This study was conducted to evaluate the effect of mesenchymal stem cells (MSCs) and a novel curcumin derivative (NCD) on HepG2 cells (hepatoma cell line) and to investigate their effect on Notch1 signaling pathway target genes. HepG2 cells were divided into HepG2 control group, HepG2 cells treated with MSC conditioned medium (MSCs CM), HepG2 cells treated with a NCD, HepG2 cells treated with MSCs CM and NCD, and HepG2 cells treated with MSCs CM (CM of MSCs pretreated with a NCD). Expression of Notch1, Hes1, VEGF, and cyclin D1 was assessed by real-time, reverse transcription-polymerase chain reaction (RT-PCR) in HepG2 cells. In addition, HepG2 proliferation assay was performed in all groups. Notch1 and its target genes (Hes1 and cyclin D1) were downregulated in all treated groups with more suppressive effect in the groups treated with both MSCs and NCD. Also, treated HepG2 cells showed significant decrease in cell proliferation rate. These data suggest that modulation of Notch1 signaling pathway by MSCs and/or NCD can be considered as a therapeutic target in HCC.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Proliferation; Curcumin; Cyclin D1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Homeodomain Proteins; Humans; Liver Neoplasms; Mesenchymal Stem Cells; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1; Vascular Endothelial Growth Factor A

Curcumin from the rhizome of Curcuma longa (zingiberaceae) has been reported to be a chemopreventive agent that affects cell proliferation by arresting the cell cycle in G2 and modulating the wnt signaling pathway. We found that curcumin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma (HCC) cells in a concentration-dependent manner. We identified that curcumin interrupts wnt signaling by decreasing β-catenin activity, which in turn suppresses the expression of β-catenin target genes (c-myc, VEGF and cyclin D1). Our results from molecular simulation of curcumin binding to Dvl2 protein and from binding free energy calculations suggest that curcumin may prevent axin recruitment to cellular membrane in order to maintain the functional β-catenin destruction complex in normal cells. This results in β-catenin being unable to accumulate in the nucleus, depriving the protein of its ability to bind with lymphoid enhancer factor/T cell-specific transcription factor (Lef/Tcf) and repressing its activation of target gene transcription. This may be one mechanism through which curcumin inhibits proliferation and induces apoptosis of HCC cells.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Axin Protein; Axin Signaling Complex; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Curcumin; Cyclin D1; Dishevelled Proteins; Down-Regulation; Enzyme Inhibitors; Humans; Liver Neoplasms; Molecular Docking Simulation; Phosphoproteins; Protein Binding; Proto-Oncogene Proteins c-myc; TCF Transcription Factors; Transcription, Genetic; Vascular Endothelial Growth Factor A; Wnt Proteins; Wnt Signaling Pathway

Obesity is associated with increased risk in hepatocellular carcinoma (HCC) development and mortality. An important disease control strategy is the prevention of obesity-related hepatic inflammation and tumorigenesis by dietary means. Here, we report that apo-10'-lycopenoic acid (APO10LA), a cleavage metabolite of lycopene at its 9',10'-double bond by carotene-9',10'-oxygenase, functions as an effective chemopreventative agent against hepatic tumorigenesis and inflammation. APO10LA treatment on human liver THLE-2 and HuH7 cells dose dependently inhibited cell growth and upregulated sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase that may suppress hepatic carcinogenesis. This observed SIRT1 induction was associated with decreased cyclin D1 protein, increased cyclin-dependent kinase inhibitor p21 protein expression, and induced apoptosis. APO10LA supplementation (10 mg/kg diet) for 24 weeks significantly reduced diethylnitrosamine-initiated, high fat diet (HFD)-promoted hepatic tumorigenesis (50% reduction in tumor multiplicity; 65% in volume) and lung tumor incidence (85% reduction) in C57Bl/6J mice. The chemopreventative effects of APO10LA were associated with increased hepatic SIRT1 protein and deacetylation of SIRT1 targets, as well as with decreased caspase-1 activation and SIRT1 protein cleavage. APO10LA supplementation in diet improved glucose intolerance and reduced hepatic inflammation [decreased inflammatory foci, TNFα, interleukin (IL)-6, NF-κB p65 protein expression, and STAT3 activation] in HFD-fed mice. Furthermore, APO10LA suppressed Akt activation, cyclin D1 gene, and protein expression and promoted PARP protein cleavage in transformed cells within liver tumors. Taken together, these data indicate that APO10LA can effectively inhibit HFD-promoted hepatic tumorigenesis by stimulating SIRT1 signaling while reducing hepatic inflammation.

Topics: Alkylating Agents; Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Carotenoids; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Diet, High-Fat; Diethylnitrosamine; Fatty Acids, Unsaturated; Female; Glucose Tolerance Test; Humans; Inflammation; Liver Neoplasms; Lycopene; Mice; Mice, Inbred C57BL; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

A malfunction of RXRα due to phosphorylation is associated with liver carcinogenesis, and acyclic retinoid (ACR), which targets RXRα, can prevent the development of hepatocellular carcinoma (HCC). Activation of PI3K/Akt signaling plays a critical role in the proliferation and survival of HCC cells. The present study examined the possible combined effects of ACR and LY294002, a PI3K inhibitor, on the growth of human HCC cells.. This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells.. ACR and LY294002 preferentially inhibited the growth of HLF cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR and 5 μM LY294002, in which the concentrations used are less than the IC₅₀ values of these agents, synergistically inhibited the growth of HLF, Hep3B, and Huh7 human HCC cells. These agents when administered in combination acted cooperatively to induce apoptosis in HLF cells. The phosphorylation of RXRα, Akt, and ERK proteins in HLF cells were markedly inhibited by treatment with ACR plus LY294002. Moreover, this combination also increased RXRE promoter activity and the cellular levels of RARβ and p21(CIP1), while decreasing the levels of cyclin D1.. ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromones; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Hepatocytes; Humans; Liver Neoplasms; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Retinoid X Receptor alpha; Retinoid X Receptor beta; Tretinoin

The cell division cycle-associated 7-like protein (CDCA7L) is a recently-identified target gene of c-Myc which can also interact with c-Myc. It is known to be upregulated in many tumors, however, its role in tumor progression remains unclear. We investigated the role of CDCA7L expression in hepatocellular carcinoma (HCC). We confirmed that CDCA7L is strongly upregulated in human HCC, and demonstrated that ectopic overexpression of CDCA7L promotes HCC cell proliferation and colony formation. Conversely, knockdown of CDCA7L inhibits these malignant phenotypes. In an in vivo model, subcutaneous transplantation of the tumor in nude mice showed that overexpression of CDCA7L can accelerate the tumor growth rate. Mechanistic analyses indicated that CDCA7L was able to activate the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and regulate the cell cycle, thus promoting HCC progression. Collectively, these findings show that CDCA7L plays a role in promoting the development of HCC and may constitute a potential therapeutic target in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, Nude; Neoplasm Transplantation; Repressor Proteins; RNA Interference; RNA, Small Interfering; Up-Regulation; Xenograft Model Antitumor Assays

Src homology 2 domain containing (SHC) is a proto-oncogene which mediates cell proliferation and carcinogenesis in human carcinomas. Here, the SHC SH2-domain binding protein 1 (SHCBP1) was first established to be up-regulated in human hepatocellular carcinoma (HCC) tissues by array-base comparative genome hybridization (aCGH). Meanwhile, we examine and verify it by quantitative real-time PCR and western blot. Our current data show that SHCBP1 was up-regulated in HCC tissues. Overexpression of SHCBP1 could significantly promote HCC cell proliferation, survival and colony formation in HCC cell lines. Furthermore, knockdown of SHCBP1 induced cell cycle delay and suppressed cell proliferation. Furthermore, SHCBP1 could regulate the expression of activate extracellular signal-regulated kinase 1/2 (ERK1/2) and cyclin D1. Together, our findings indicate that SHCBP1 may contribute to human hepatocellular carcinoma by promoting cell proliferation and may serve as a molecular target of cancer therapy.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MAP Kinase Signaling System; Proto-Oncogene Mas; Shc Signaling Adaptor Proteins; Up-Regulation

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in diverse cancers, which contributes to the proliferation and survival of cancer cells by upregulating apoptosis inhibitors and cell cycle regulators. Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator of STAT pathways and is frequently silenced in many types of cancers. In this study, we used oncolytic adenoviral vector to deliver SOCS1 gene (AdCN305-SOCS1) to treat hepatocellular carcinoma (HCC). Our data showed that SOCS1 was downregulated in HCC cells by hypermethylation. AdCN305-SOCS1 was found selectively replicated, which led to SOCS1 overexpression in HCC cells. Infection of HCC cells with AdCN305-SOCS1 resulted in inhibition of STAT3 phosphorylation and downregulation of survivin, cyclin D1, Bcl-xL and C-myc. AdCN305-SOCS1 exhibited strong cytotoxicity to HCC cells by inducing apoptosis in vitro and in vivo. This study suggests that transfer of SOCS1 by an oncolytic adenovirus may be a potent antitumor approach for cancer therapy.

Topics: Adenoviridae; Apoptosis; bcl-X Protein; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; DNA Methylation; DNA Replication; Gene Expression Regulation, Neoplastic; Genetic Vectors; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Oncolytic Viruses; Phosphorylation; Proto-Oncogene Proteins c-myc; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Survivin

Metabolic syndrome (MS) is an emerging risk factor in hepatocellular carcinoma (HCC). HCC related to MS may occur either in advanced fibrosis or before the development of cirrhosis, suggesting involvement of different molecular pathways according to the features of background liver.. To investigate genomic aberrations in HCC related to MS in order to identify new target genes involved in liver carcinogenesis.. Chromosomal aberrations of HCC obtained from 20 patients with MS (HCC/MS) were studied by comparative genomic hybridisation and compared with HCC related to hepatitis C virus (HCV) infection (HCC/HCV, n=10) and, within the group of HCC with MS, according to the condition of the background liver (presence or absence of significant fibrosis).. Among the most frequent chromosomal alterations observed in HCC, 6p21.1 amplification had a higher incidence in HCC/MS than in HCC/HCV (60% vs 20%, p<0.01). Advanced fibrosis/cirrhosis in the peritumoral liver was the only clinicopathological factor associated with the 6p21.1 amplicon in HCC/MS. Increased expression of cullin7 (CUL7), a gene located at the 6p21.1 locus, was demonstrated in HCC with the 6p21.1 amplicon, in parallel with a decrease in cyclin D1 expression. CUL7 downregulation using siRNA transfection in hepatoma cell lines induced significant cyclin D1 expression (by promoting its degradation), decreased cell proliferation and increased apoptosis.. This study demonstrates specific genomic alterations in HCC/MS and points to CUL7 as a novel gene potentially involved in liver carcinogenesis associated with MS, the amplification of which might influence cell proliferation.

Topics: Aged; Aged, 80 and over; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosomes, Human, Pair 6; Cullin Proteins; Cyclin D1; Female; Gene Expression; Hepatitis C; Humans; Immunohistochemistry; Liver Cirrhosis; Liver Neoplasms; Male; Metabolic Syndrome; Middle Aged; Nucleic Acid Hybridization; Real-Time Polymerase Chain Reaction

Liver cancers, including hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and fibrolamellar HCCs (FL-HCCs) are among the most common cancers worldwide and are associated with a poor prognosis. Investigations of genes important in liver cancers have focused on Sal-like protein 4 (SALL4), a member of a family of zinc finger transcription factors. It is a regulator of embryogenesis, organogenesis, pluripotency, can elicit reprogramming of somatic cells, and is a marker of stem cells. We found it expressed in normal murine hepatoblasts, normal human hepatic stem cells, hepatoblasts and biliary tree stem cells, but not in mature parenchymal cells of liver or biliary tree. It was strongly expressed in surgical specimens of human HCCs, CCs, a combined hepatocellular and cholangiocarcinoma, a FL-HCC, and in derivative, transplantable tumor lines in immune-compromised hosts. Bioinformatics analyses indicated that elevated expression of SALL4 in tumors is associated with poor survival of HCC patients. Experimental manipulation of SALL4's expression results in changes in proliferation versus differentiation in human HCC cell lines in vitro and in vivo in immune-compromised hosts. Virus-mediated gene transfer of SALL4 was used for gain- and loss-of-function analyses in the cell lines. Significant growth inhibition in vitro and in vivo, accompanied by an increase in differentiation occurred with down-regulation of SALL4. Overexpression of SALL4 resulted in increased cell proliferation in vitro, correlating with an increase in expression of cytokeratin19 (CK19), epithelial cell adhesion molecules (EpCAM), and adenosine triphosphate (ATP)-binding cassette-G2 (ABCG2).. SALL4's expression is an indicator of stem cells, a prognostic marker in liver cancers, correlates with cell and tumor growth, with resistance to 5-FU, and its suppression results in differentiation and slowed tumor growth. SALL4 is a novel therapeutic target for liver cancers.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; DNA-Binding Proteins; Humans; In Vitro Techniques; Liver; Liver Neoplasms; Male; Mice; Mice, SCID; Prognosis; Transcription Factors; Transplantation, Heterologous

Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, we compared the expression of interleukin (IL)-6 and cyclin D1 (CCND1) with the IL-6-induced homeobox gene ISX (intestine-specific homeobox) in 119 paired specimens of HCCs and adjacent normal tissues and also in paired specimens from 11 patients with non-HCCs. In pathologic analysis, ISX exhibited a tumor-specific expression pattern and a high correlation to patient survival time, tumor size, tumor number, and progression stage. Enforced expression of ISX accelerated cell proliferation and tumorigenic activity in hepatoma cells through CCND1 induction. In contrast, short hairpin RNA-mediated attenuation of ISX in hepatoma cells decreased cell proliferation and malignant transformation in vitro and in vivo. A high positive correlation existed in human hepatoma tumors between ISX and CCND1 expression. Together, our results highlight ISX as an important regulator in hepatoma progression with significant potential as a prognostic and therapeutic target in HCCs.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; E2F1 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Homeodomain Proteins; Humans; Interleukin-6; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; RNA Interference; Transcription Factors

A better understanding of human hepatocellular carcinoma (HCC) pathogenesis at the molecular level will facilitate the discovery of tumor-initiating events. Transcriptome sequencing revealed that adenosine-to-inosine (A→I) RNA editing of AZIN1 (encoding antizyme inhibitor 1) is increased in HCC specimens. A→I editing of AZIN1 transcripts, specifically regulated by ADAR1 (encoding adenosine deaminase acting on RNA-1), results in a serine-to-glycine substitution at residue 367 of AZIN1, located in β-strand 15 (β15) and predicted to cause a conformational change, induced a cytoplasmic-to-nuclear translocation and conferred gain-of-function phenotypes that were manifested by augmented tumor-initiating potential and more aggressive behavior. Compared with wild-type AZIN1 protein, the edited form has a stronger affinity to antizyme, and the resultant higher AZIN1 protein stability promotes cell proliferation through the neutralization of antizyme-mediated degradation of ornithine decarboxylase (ODC) and cyclin D1 (CCND1). Collectively, A→I RNA editing of AZIN1 may be a potential driver in the pathogenesis of human cancers, particularly HCC.

Topics: Active Transport, Cell Nucleus; Adenosine Deaminase; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Liver Neoplasms; Male; Mice; Ornithine Decarboxylase; RNA Editing; RNA-Binding Proteins

Deregulated cell cycle can contribute to the unscheduled proliferation in cancer cells. Overexpression of cell cycle regulators CDK4 and Cyclin D1 has been reported in many cancers. The aim of this study is to determine the clinical implications of CDK4 and Cyclin D1 in hepatocellular carcinoma (HCC). The levels of mRNA and protein were analyzed by quantitative real-time RT-PCR and immunohistochemistry, respectively, in 59 paired HCC and the neighboring noncancer tissues. The relationship between CDK4 and Cyclin D1 expression, clinicopathological parameters, and prognosis was investigated. Our data demonstrated that the mRNA level of CDK4 was up-regulated (p = 0.019), while that of Cyclin D1 was down-regulated (p = 0.002), in HCC. Immunohistochemical data confirmed that CDK4 protein was increased in 73 % and Cyclin D1 protein was decreased in 66 % of HCC samples. Overexpression of CDK4 was correlated with HBV (p = 0.054, borderline significant), tumor size (p = 0.014), and stage (p = 0.010). The Kaplan-Meier survival curves showed that high CDK4 was correlated with a poor survival rate (I vs. II, p < 0.001; I vs. III, p < 0.001). Univariate analysis showed that tumor size (p = 0.002), stage (p = 0.021), and high CDK4 score (I vs. II-III, p < 0.001) were significant prognostic factors. Multivariate analysis showed that tumor size (p = 0.007) and high CDK4 score (I vs. II-III, p < 0.001) were independent factors for overall survival of HCC. The expression of Cyclin D1 was not correlated with CDK4 expression, tumor grades, survival rate, and any clinicopathological parameters. CDK4 could provide a clinical prognostic marker for HCC progression.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Liver Neoplasms; Male; Middle Aged; Neoplasm Grading; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Young Adult

βKlotho is a regulator in multiple metabolic processes, while its role in cancer remains unclear. We found the expression of βKlotho was down-regulated in human hepatocellular carcinoma tissues compared with that in paired adjacent non-tumourous liver tissues. Hepatoma cells also showed decreased expression of βKlotho compared with normal hepatocyte cells. Reintroduction of βKlotho into hepatoma cells inhibited their proliferation. The anti-proliferative effect of βKlotho might be linked with G1 to S phase arrest, which was mediated by Akt/GSK-3β/cyclin D1 signaling, since forced expression βKlotho reduced the phosphorylation level of Akt and GSK-3β and induced down-regulation of cyclin D1. Furthermore, βKlotho overexpression could inhibit tumorgenesis, while constitutively activated Akt could override the suppressive effects of βKlotho in vivo. These data suggest βKlotho suppresses tumor growth in hepatocellular carcinoma.

Topics: Adult; Aged; Animals; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Glucuronidase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Klotho Proteins; Liver Neoplasms; Male; Mice; Middle Aged; Proto-Oncogene Proteins c-akt; Signal Transduction; Transplantation, Heterologous

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.

Topics: Acetylcysteine; Antineoplastic Agents; Antioxidants; Apoptosis Regulatory Proteins; Autophagy; bcl-X Protein; Beclin-1; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Gossypol; Hep G2 Cells; HMGB1 Protein; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Membrane Proteins; Microtubule-Associated Proteins; Phosphorylation; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) incidence rates are increasing in many parts of the world. HCC's limited treatment remedies and the poor prognosis emphasize the importance in developing an effective chemoprevention for this disease. Here, we investigated the molecular mechanisms involved in the chemoprevention of silymarin in N-nitrosodiethylamine (NDEA)-induced rat model of HCC. Liver of the rats treated with NDEA showed higher proliferation index and glycoconjugates. NDEA treatment also increased the level of anti-apoptotic proteins with simultaneous decrease in the level of pro-apoptotic proteins along with increased accumulation of Cytochrome c in mitochondria. The carcinogenic insult also increased microsomal phase I metabolizing enzymes with a simultaneous decrease in the Phase II detoxifying enzyme glutathione-S-transferase (GST). Whereas dietary silymarin administration along with NDEA treatment significantly decreased the proliferation and down regulated the expression of anti-apoptotic proteins with simultaneously increased expression of pro-apoptotic proteins along with the release of Cytochrome c to cytosol there by activating the intrinsic apoptotic pathway. Silymarin administration also decreased the level of glycoproteins and activated the phase II detoxifying enzyme GST. These results demonstrate that suppression of HCC by silymarin in vivo involves inhibition of proliferation, activation of apoptosis, and efficient detoxification.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Cells, Cultured; Cyclin D1; Dietary Supplements; Drug Screening Assays, Antitumor; Glycoproteins; Hexosamines; Hexoses; Liver; Liver Neoplasms, Experimental; Male; Membrane Potential, Mitochondrial; Metabolic Detoxication, Phase II; Microtubule-Associated Proteins; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Sialic Acids; Silymarin; Survivin; Tumor Suppressor Protein p53

MicroRNAs (miRNAs) have been implicated in the maintenance of the cancer stem cell (CSC) phenotype via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Here, we compared the miRNA profiles of CD133+ and CD133- primary hepatocellular carcinoma (HCC) subpopulations and found upregulation of 5 miRNAs in CD133- subpopulations, including hsa-miR-150, which may be involved in maintenance of the CD133+ liver CSC phenotype. We also show that miR-150 interacts with the 3'UTR of c-Myb mRNA and overexpression of miR-150 downregulates c-Myb protein levels. Furthermore, overexpression of miR-150 lead to a significant reduction of CD133+ cells, accompanied by significant inhibition of cell growth and tumorsphere formation. In addition, overexpression of miR-150 induces cell cycle arrest and apoptosis in CD133+ cells. Consistent with the outcome of cell cycle arrest and cell apoptosis, Western blotting results demonstrate that the cell cycle regulator cyclin D1 and cell survival regulator Bcl-2 are decreased in cells transfected with miR-150. Collectively, our findings demonstrate for the first time that miR-150 may be involved in liver CSC self-renewal, potentially via modulation of the downstream target c-Myb.

Topics: 3' Untranslated Regions; AC133 Antigen; Adult; Animals; Antigens, CD; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Growth Processes; Cell Line, Tumor; Cell Survival; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Glycoproteins; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Middle Aged; Neoplastic Stem Cells; Peptides; Proto-Oncogene Proteins c-myb; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation; Xenograft Model Antitumor Assays

Hypoxia-inducible-1α (HIF-1α) expression was intimately correlated with apoptosis and proliferation of cancer cells. However, conclusions of different studies on the effects of HIF-1α expression on cell apoptosis and cell proliferation of hepatoma cells remain controversial. In view of the current status, we reassess its roles and possible mechanism in hepatoma cells. In order to acquire more convincing and reliable results, we used a Tet-on system to stably and effectively regulate HIF-1α expression in the HepG2 cells in vitro. In our study we not only confirmed some common conclusions of previous studies, but also acquired some different and significant results that HIF-1α facilitates cell proliferation and cell cycle through influencing the expression of cyclin A and cyclin D, and suppresses cell apoptosis through inducing the expression of survivin and Bcl-2. These results further enrich our knowledge on the role of HIF-1α expression on cell apoptosis and cell proliferation of hepatoma cells.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin D1; Cyclin E; Doxycycline; Enzyme Activation; Gene Expression Regulation; Genes, bcl-2; Hep G2 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Survivin

Hepatocellular carcinoma (HCC) often displays resistance to recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Dovitinib, a multiple tyrosine kinase inhibitor, and tigatuzumab, a novel humanized anti-human death receptor 5 (DR5) agonistic antibody, are both under clinical investigations in HCC. Here, we report that dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through inhibition of signal transducers and activators of transcription 3 (STAT3). Our data indicate that HCC cells showed significant resistance to TRAIL- and tigatuzumab-induced apoptosis. The combination of dovitinib and tigatuzumab restored the sensitivity of HCC cells to TRAIL- and tigatuzumab-induced apoptosis. Dovitinib down-regulated phospho-STAT3 (Tyr705) (p-STAT3) and subsequently reduced the protein levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in TRAIL-treated HCC cells. Knockdown of STAT3 by RNA-interference overcame apoptotic resistance to TRAIL in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effect of dovitinib on TRAIL-induced apoptosis. Importantly, silencing SHP-1 by RNA-interference reduced the effects of dovitinib and TRAIL on p-STAT3 and apoptosis, whereas co-treatment of TRAIL and dovitinib increased the activity of SHP-1. Moreover, in vivo the combination of tigatuzumab and dovitinib inhibited Huh-7 xenograft tumor growth. In conclusion, dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through a novel machinery: SHP-1 dependent STAT3 inhibition.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzimidazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Male; Mice; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Protein Kinase Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Quinolones; Receptors, TNF-Related Apoptosis-Inducing Ligand; STAT3 Transcription Factor; Survivin; TNF-Related Apoptosis-Inducing Ligand

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although some epidemiologic and etiologic differences between Asian and Western HCC are known, detailed comparative studies with pathologic correlations have not been performed.. Paraffin sections of resected HCC specimens from Memorial Sloan-Kettering Cancer Center and Korea University Medical Center were used to construct tissue microarrays. Immunohistochemical staining of microarray sections was performed using antibodies against markers of proliferation and regulators of cell cycle. Patient data were correlated with staining results.. When comparing both cohorts, significant differences were found in expression of p53 and MDM2. In the Asian group, more frequent positive staining for p53 (24%) was observed compared with the American group (9%; P = 0.037). For MDM2, 26% of American cases stained positive compared with 2% of Asian cases (P = 0.0003). No significant differences were found in expression of Ki67, p21, p27, cyclin D1, or bcl2. Female gender, vascular invasion, and lack of viral hepatitis infection correlated with positive MDM2 staining.. These data likely correlate with differences in molecular pathogenesis of HCC based on racial and regional differences. These findings may have implications in choice of molecular targeted therapies based on patient ethnicity.

Topics: Adult; Aged; Asian People; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression Regulation, Neoplastic; Hepatitis B; Hepatitis C; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Paraffin; Prognosis; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Republic of Korea; Risk Factors; Tissue Array Analysis; Tumor Suppressor Protein p53; United States

Amplification of broad regions of 8q is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC), suggesting the existence of oncogenes in addition to MYC at 8q24.21. In this report we examine the potential role of the candidate amplified oncogene serum and glucocorticoid kinase 3 (SGK3) at 8q13.1 in HCC pathogenesis. We found amplification and overexpression of SGK3 was frequently detected in clinical HCC specimens and that SGK3 genomic activation was significantly associated with poor outcome of patients (P = 0.028). Functionally, we found that overexpression of SGK3 in HCC cells increased cell cycle progression through G(1), cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. In contrast, RNA interference (RNAi) silencing of SGK3 inhibited its oncogenic effects. We provide evidence that SGK3 promotes HCC growth and survival through inactivating glycogen synthase kinase 3 beta and Bcl-2-associated death promoter, respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1 signaling, has more significance than overexpression of AKT in predicting poor outcome in HCC patients. Taken together, our findings in the present study suggests that the SGK3 pathway may function in parallel with the AKT pathway and undergoes an AKT-independent signaling pathway in the pathogenesis of HCC. Further characterization of SGK3 may provide a prognostic biomarker for HCC outcome prediction and a novel therapeutic target in HCC treatment.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromosomes, Human, Pair 8; Cyclin D1; G1 Phase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Liver Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; S Phase

Growing evidence indicates that the deregulation of microRNAs (miRNAs) contributes to the tumorigenesis. We previously revealed that microRNA-520b (miR-520b) was involved in the complement attack and migration of breast cancer cells. In this report, we show that miR-520b is an important miRNA in the development of hepatocellular carcinoma (HCC). Our data showed that the expression levels of miR-520b were significantly reduced in clinical HCC tissues and hepatoma cell lines. We observed that the introduction of miR-520b dramatically suppressed the growth of hepatoma cells by colony formation assays, 5-ethynyl-2-deoxyuridine (EdU) incorporation assays and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, ectopic expression of miR-520b was able to inhibit the growth of hepatoma cells in nude mice. Further studies revealed that the mitogen-activated protein kinase kinase kinase 2 (MEKK2) and cyclin D1 were two of direct target genes of miR-520b. Silencing of MEKK2 or cyclin D1 was able to inhibit the growth of hepatoma cells in vitro and in vivo, which is consistent with the effect of miR-520b overexpression on the growth of hepatoma cells. In addition, miR-520b significantly decreased the phosphorylation levels of c-Jun N-terminal kinase (p-JNK, a downstream effector of MEKK2) or retinoblastoma (p-Rb, a downstream effector of cyclin D1). In conclusion, miR-520b is able to inhibit the growth of hepatoma cells by targeting MEKK2 or cyclin D1 in vitro and in vivo. Our findings provide new insights into the role of miR-520b in the development of HCC, and implicate the potential application of miR-520b in cancer therapy.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Liver Neoplasms; MAP Kinase Kinase Kinase 2; MAP Kinase Kinase Kinases; Mice; MicroRNAs

Although sEH inhibitors are well studied in inflammatory and cardiovascular diseases, their effects on gliomas are unclear. In this study, we investigated the effects of t-AUCB, a more potent and selective sEH inhibitor, on U251 and U87 human glioblastoma cell lines and the HepG2 human hepatocellular carcinoma cell line. Our results showed that t-AUCB efficiently inhibited sEH activities in all three cell lines (the inhibition rate was more than 80% in each) and suppressed U251 and U87 cell growth in a dose-dependent manner, but exhibited no cell growth inhibition on HepG2. We detected high levels of phosphorylated NF-κB-p65 (Ser536) in t-AUCB-treated U251 and U87 cells, and then found that the NF-κB inhibitor PDTC can completely abolish t-AUCB-induced growth inhibition. This indicated that t-AUCB suppresses U251 and U87 cell growth by activating NF-κB-p65. Moreover, we found that t-AUCB induces cell-cycle G0/G1 phase arrest by regulating Cyclin D1 mRNA and protein levels and CDC2 (Thr161) phosphorylation level. We propose to further test this promising reagent for its anti-glioma activity in clinical relevant orthotopic brain glioma models.

Topics: Apoptosis; Benzoates; Blotting, Western; Brain Neoplasms; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epoxide Hydrolases; Flow Cytometry; Glioblastoma; Humans; Liver Neoplasms; NF-kappa B; Phosphorylation; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Urea

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Despite improved diagnosis and treatment, the prognosis for HCC patients remains poor. The goal of this study was to identify key regulatory proteins and signaling pathways important for cell apoptosis and proliferation as biomarkers for prognostication and targeted therapy. Protein Pathway Array was applied to screen 38 signaling proteins and phosphoproteins in 12 paired HCC tumors and surrounding benign tissues and found that 20 of them, including XIAP, CDK4, CDK6, and Cyclin D1, were overexpressed in HCC tissues. Immunostaining results of XIAP, CDK4, and Cyclin D1 in an additional 59 HCC tissues showed that the expression of XIAP correlated with the expression of CDK4/Cyclin D1, and that the increased expression of these proteins correlated with poor overall survival in these patients. Further studies using the HCC Huh7 cell line transfected with XIAP siRNA or expression vector demonstrated that XIAP regulated the expression of CDK4, CDK6, and Cyclin D1 via NF-êB and PTEN pathways. Finally, inhibition of XIAP using embelin, a XIAP-specific small molecule, leads to an increased apoptosis and decreased cell proliferation via arrest at G1 phase. Taken together, XIAP is a central modulator regulating cell apoptosis and cell cycle progression. Therefore, XIAP together with cell cycle regulatory proteins can be used as prognostic markers and therapeutic targets.

Topics: Aged; Apoptosis; Benzoquinones; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Phosphorylation; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Tumor Cells, Cultured; X-Linked Inhibitor of Apoptosis Protein

EZH2/H3K27me3 and polycomb group complex (PcG) play a major role in regulating global gene expression including tumor suppressor genes. EZH2 is linked to cell cycle regulated EZH2 phosphorylation by CDK1, a mitotic kinase which increases in arrested mitosis compared to S phase. CDK1 phosphorylation of EZH2 accelerates the degradation of pEZH2. Phospho-EZH2 is subjected to ubiquitination. The half-like of pEZH2 is shorter when compared to total EZH2. In the present study, pEZH2 was found concentrated together with ubiquitin in the Mallory-Denk bodies (MDB) that were formed in hepatocytes in the livers of drug primed mice refed DDC and humans with alcoholic hepatitis or hepatocellular carcinoma. The cells that formed MDBs in the mice livers studied were associated with a growth advantage and a high proliferative index. However, the livers from patients with alcoholic hepatitis showed evidence of cell cycle arrest where PCNA, cyclin D1 and p27 positive nuclei were numerous but Ki-67 positive nuclei were scarce. It is concluded that MDB formation is linked to the cell cycle and global gene expression (i.e. loss of gene silencing) through its association with the regulation of the polycomb group PRC2/EZH2/H3K27me3 complex.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; DNA Methylation; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Hepatitis, Alcoholic; Hepatocytes; Histones; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Lysine; Mallory Bodies; Methylation; Mice; Microscopy, Electron; Polycomb Repressive Complex 2; Proliferating Cell Nuclear Antigen; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; S-Adenosylmethionine; Transcription Factors

To enhance tumor-targeting abilities and therapeutic efficiency, a monoclonal antibody-conjugated gene nanocomplex was herein designed. The biodegradable cationic polyethylenimine-grafted-α,β-poly(N-3-hydroxypropyl)-DL-aspartamide (PHPA-PEI) was used for complexing pDNA to form the PHPA-PEI/pDNA nanoparticle, and then 9B9 mAb, an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, was conjugated to produce the PHPA-PEI/pDNA/9B9 mAb (PP9mN) complex. The PP9mN complex with the diameter of around 300 nm at its optimal weight ratio could be uptaken effectively by SMMC-7721 cells. The cytotoxicity of the PP9mN complex was much lower than that of PEI 25 kD in SMMC-7721, HepG2, Bel-7404 and COS-7 cell lines. The PP9mN complex possessed the highly efficient in vitro gene delivery ability to the hepatocellular carcinoma cells. The in vivo gene expression indicated that PP9mN could target to the tumor tissues effectively. By using the therapeutic AChE gene, it was found that the PP9mN complexes significantly enhanced the anti-tumor effect on tumor-bearing nude mice. Such monoclonal antibody-conjugated gene complex should have great potential applications in liver cancer therapy.

Topics: Acetylcholinesterase; Animals; Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Cyclin D1; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Genetic Therapy; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Polyethyleneimine; Polymers

Alocasia macrorrhiza has been used as a folk medicine for cancer treatment in the Southwest of China.. The purpose of this study is to confirm the anticancer activity of aqueous extract of alocasia macrorrhiza against hepatic cancer and to elucidate its mechanism of action.. Human normal liver cells and hepatocellular carcinoma cells were tested in vitro for cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining apoptotic cells, apoptotic DNA fragmentation, and cell cycle distribution in response to alocasia macrorrhiza extract. The mRNA and protein expressions of PPARγ, Cyclin D1, Rb, P21, Bax, Bcl-2 and caspase-3 were detected through RT-PCR and Western blotting; the tumor growth inhibition in vivo was tested by oral administration of the extract.. Alocasia macrorrhiza aqueous extract exhibited proliferation inhibition and apoptosis effects on human hepatocellular carcinoma cells in vitro, inhibited hepatoma growth in vivo.. Alocasia macrorrhiza extract has potential cytotoxic and apoptotic effect on human hepatocellular carcinoma cells and inhibits hepatoma growth in vivo, its mechanism of action might be associated with the inhibition of DNA synthesis, cell cycle (G(0)/G(1)) arrest, apoptosis induction through up-regulation the mRNA and protein expressions of PPARγ, Rb, Bax and capase-3genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes.

Topics: Alocasia; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Cell Cycle; Cell Line; Cell Survival; Cyclin D1; DNA Fragmentation; Hepatocytes; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred ICR; Neoplasm Transplantation; Phytotherapy; Plant Extracts; PPAR gamma; Proto-Oncogene Proteins c-bcl-2; Tumor Burden

Hepatitis C virus (HCV) infection usually induces chronic hepatic inflammation, which favors the initiation and progression of hepatocellular carcinoma (HCC). Moreover, microRNA-155 (miR-155) plays an important role in regulating both inflammation and tumorigenesis. However, little is known about whether and how miR-155 provides the link between inflammation and cancer. In this study we found that miR-155 levels were markedly increased in patients infected with HCV. MiR-155 transcription was regulated by nuclear factor kappa B (NF-κB), and p300 increased NF-κB-dependent miR-155 expression. The overexpression of miR-155 significantly inhibited hepatocyte apoptosis and promoted cell proliferation, whereas miR-155 inhibition induced G(0) /G(1) arrest. Up-regulated miR-155 resulted in nuclear accumulation of β-catenin and a concomitant increase in cyclin D1, c-myc, and survivin. Gain-of-function and loss-of-function studies demonstrated that miR-155 promoted hepatocyte proliferation and tumorigenesis by increasing Wnt signaling in vitro and in vivo, and DKK1 (Wnt pathway inhibitor) overexpression inhibited the biological role of miR-155 in hepatocytes. Finally, adenomatous polyposis coli (APC), which negatively regulates Wnt signaling, was identified as the direct and functional target of miR-155.. HCV-induced miR-155 expression promotes hepatocyte proliferation and tumorigenesis by activating Wnt signaling. The present study provides a better understanding of the relationship between inflammation and tumorigenesis, and thus may be helpful in the development of effective diagnosis and treatment strategies against HCV-HCC.

Topics: Adenomatous Polyposis Coli Protein; Adult; Aged; Animals; Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chemokine CXCL10; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Hepacivirus; Hepatitis C, Chronic; Hepatocytes; Humans; Inhibitor of Apoptosis Proteins; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; NF-kappa B; p300-CBP Transcription Factors; Proto-Oncogene Proteins c-myc; RNA, Viral; Survivin; Transfection; Up-Regulation; Wnt Signaling Pathway

Primary liver cancer is one of the highly malignant tumours. The traditional surgery, chemotherapy and radiation therapy only established 6% of 5-year survival rate in HCC (hepatocellular carcinoma). Therefore there is an urgent need to develop new therapeutic strategies. HSP90 (heat shock protein 90) is one of the important molecular chaperones and was identified with high expression in the primary liver cancer. In this study, we evaluated the therapeutic effect of specific HSP90 inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxy geldanamycin) in HCC cells. The time and concentration effects of 17-DMAG were investigated in HCC cells. Cell proliferation was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and cell counting. Apoptosis was detected by flow cytometry with staining of Annexin V-FITC/PI (propidium iodide). The protein levels of survivin, cyclin D1, p53 and NF-κB (nuclear factor κB) were measured by Western blotting. 17-DMAG inhibited the proliferation of HCC cells in a time- and concentration-dependent manner. Treatment with 400 nmol/l 17-DMAG for 48 h significantly induced early-stage apoptosis (22.4%). Conversely, it induced less late-stage apoptosis (3.03%). The 5 mg/l of cisplatin induced significantly less early-stage apoptosis (6.5%), but similar proportion of late-stage apoptosis (4.89%) compared with 17-DMAG. Inhibition of HSP90 activity by 400 nmol/l 17-DMAG decreased protein levels of survivin, cyclin D1 and NF-κB protein levels, whereas increased p53 protein level. HSP90 plays a key role in HCC cell growth and survival through regulation of survivin, cyclin D1, p53 and nucleus NF-κB protein levels and the specific HSP90 inhibitor 17-DMAG can play a therapeutic role in HCC treatment.

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; HSP90 Heat-Shock Proteins; Humans; Inhibitor of Apoptosis Proteins; Lactams, Macrocyclic; Liver Neoplasms; NF-kappa B; Survivin; Tumor Suppressor Protein p53

Oncogenic activation of the Wnt/β-catenin signaling pathway is common in hepatocellular carcinoma (HCC). Our recent studies have demonstrated that SRY (sex determining region Y)-box 1 (SOX1) and secreted frizzled-related proteins are concomitantly promoter-hypermethylated, and this might lead to abnormal activation of the Wnt signaling pathway in HCC. SOX1 encodes a transcription factor involved in the regulation of embryonic development and cell fate determination. However, the expression and functional role of SOX1 in HCC remains unclear. In this study, we confirmed via quantitative methylation-specific polymerase chain reaction that SOX1 was frequently downregulated through promoter hypermethylation in HCC cells and tissues. Overexpression of SOX1 by a constitutive or inducible approach could suppress cell proliferation, colony formation, and invasion ability in HCC cell lines, as well as tumor growth in nonobese diabetic/severe combined immunodeficiency mice. Conversely, knockdown of SOX1 by withdrawal of doxycycline could partially restore cell proliferation and colony formation in HCC cells. We used a T cell factor (TCF)-responsive luciferase reporter assay and western blot analysis to prove that SOX1 could regulate TCF-responsive transcriptional activity and inhibit the expression of Wnt downstream genes. Furthermore, we used glutathione S-transferase pull-down, co-immunoprecipitation, and confocal microscopy to demonstrate that SOX1 could interact with β-catenin but not with the β-catenin/TCF complex. Moreover, restoration of the expression of SOX1 induces significant cellular senescence in Hep3B cells.. Our data show that a developmental gene, SOX1, may function as a tumor suppressor by interfering with Wnt/β-catenin signaling in the development of HCC.

Topics: Animals; Anti-Bacterial Agents; Antigens, CD; Cadherins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; DNA Methylation; Down-Regulation; Doxycycline; Genes, bcl-1; Genes, myc; Genes, Tumor Suppressor; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Proliferating Cell Nuclear Antigen; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); RNA, Messenger; Snail Family Transcription Factors; SOXB1 Transcription Factors; Transcription Factors; Wnt Signaling Pathway

The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2).. We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice.. Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/β-catenin signaling in Apc(lox/lox) animals.. Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Female; Hepatectomy; Homeostasis; Humans; LIM-Homeodomain Proteins; Liver; Liver Neoplasms; Liver Regeneration; Male; Mice; Mice, Transgenic; Muscle Proteins; Transcription Factors; Tumor Suppressor Protein p53

The goal of the present study was to investigate the potential correlation between the expression level of upregulator of cell proliferation (URGCP/URG4) and the prognosis of hepatocellular carcinoma (HCC), and to examine the biological function of URGCP/URG4 in the progression of HCC, to better understand its underlying molecular mechanism in hepatic tumorigenesis.. URGCP/URG4 expression was analyzed in 15 HCC cell lines, in 278 archived paraffin-embedded HCC sections, and in 10 pairs of fresh HCC tumor and para-tumor non-cancerous tissues using immunohistochemistry (IHC) and Western blotting analysis (WB). The effect of URGCP/URG4 on cell proliferation and tumorigenesis was examined in vitro and in vivo. WB and luciferase reporter analyses were performed to identify the effects of URGCP/URG4-overexpression or -knockdown on expression of cell cycle regulators and transcriptional activity of FOXO3a.. IHC results revealed an upregulation of URGCP/URG4 in all HCC cell lines and fresh HCC samples as compared with normal liver cells and para-tumor tissues, respectively. URGCP/URG4 was also expressed at a high level in 122 of the 278 (43.8%) archived HCC specimens. The expression level of URGCP/URG4 was significantly correlated with clinical staging and poor patient survival of HCC in the study cohort, and in various clinical subgroups. Strikingly, ectopic expression of URGCP/URG4 induced proliferation and anchorage-independent growth of HCC cells, while silencing of URGCP/URG4 had the opposite effect. Furthermore, URGCP/URG4 overexpression in HCC cells increased cellular entry into the G1/S transitional phase, associated with downregulation of p27(Kip1) and p21(Cip1) and upregulation of cyclin D1. These effects were accompanied by enhanced Akt activity and reduced FOXO3a transcriptional activity.. URGCP/URG4 plays an important role in promoting proliferation and tumorigenesis of HCC and may represent a novel prognostic biomarker and therapeutic target for this disease.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Down-Regulation; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Mice; Mice, SCID; Neoplasm Proteins; Prognosis; Protein Kinase Inhibitors; Transcription, Genetic; Up-Regulation

Hepatitis B virus X protein (HBx) is recognized as an oncogene in hepatocellular carcinoma (HCC). HBx regulates microRNA expression, including down-regulating miR-338-3p in LO2 cells. Here, we investigated miR-338-3p function in HBx-mediated hepatocarcinogenesis. In 23 HBV-infected HCC clinical patient tumor and adjacent non-tumor control tissues, 17 and 19 tumors expressed HBx mRNA and protein, respectively. When considered as a group, HBV-infected HCC tumors had lower miR-338-3p expression than controls; however, miR-338-3p was only significantly down-regulated in HBx-positive tumors, indicating that HBx inversely correlated with miR-338-3p. Functional characterization of miR-338-3p indicated that miR-338-3p mimics inhibited cell proliferation by inducing cell cycle arrest at the G1/S phase as assessed by EdU and cell cycle assays in HBx-expressing LO2 cells. CyclinD1, containing two putative miR-338-3p targets, was confirmed as a direct target using 3'-UTR luciferase reporter assays from cells transfected with mutated binding sites. Mutating the 2397-2403 nt binding site conferred the greatest resistance to miR-338-3p suppression of CyclinD1, indicating that miR-338-3p suppresses CyclinD1 at this site. Overall, this study demonstrates that miR-338-3p inhibits proliferation by regulating CyclinD1, and HBx down-regulates miR-338-3p in HCC. This newly identified miR-338-3p/CyclinD1 interaction provides novel insights into HBx-mediated hepatocarcinogenesis and may facilitate therapeutic development against HCC.

Topics: Base Sequence; Binding Sites; Carcinoma, Hepatocellular; Cell Line; Cell Proliferation; Cyclin D1; Down-Regulation; Hepatitis B, Chronic; Host-Pathogen Interactions; Humans; Liver Neoplasms; MicroRNAs; RNA Interference; Trans-Activators; Viral Regulatory and Accessory Proteins

Swainsonine, an extract from Astragalus membranaceus, exhibits broad inhibition of growth and pro-apoptotic activity in a number of tumor types. However, the underlying mechanism involved remains unclear. To investigate the effects and mechanisms of swainsonine on hepatocellular carcinoma (HCC), we performed experiments on HepG2, SMCC7721, Huh7 and MHCC97-H human hepatoma and HL-7702 human hepatocyte cells. We observed that swainsonine significantly inhibited the viability of human hepatoma cells in a dose- and time-dependent manner, but did not affect human hepatocytes. Due to their highly proliferative and tumorigenic nature, we selected MHCC97-H cells as a model system to examine. Swainsonine significantly inhibited MHCC97-H cell growth by causing cell cycle arrest at the G0/G1 phase and the induction of apoptosis. Blockage of G0/G1 phase was accompanied by a decrease in cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4) and an increase in the Cdk inhibitors p21 and p27. Furthermore, swainsonine enhanced the apoptosis of MHCC97-H cells with the induction of the upregulation of Bax and the downregulation of Bcl-2, whereas the expressionof Fas and Fas-L remained almost unchanged. These changes were accompanied by the enhanced cytoplasmic accumulation of nuclear factor κB (NF-κB) with a concomitant decrease in the nuclear fraction. Importantly, swainsonine also potentiated the cytotoxic effects of paclitaxel in vitro and in vivo, in part, by restricting the paclitaxel-induced nuclear accumulation of NF-κB. Taken together, these results suggest that swainsonine may be an important agent against HCC via directly inhibiting HCC cell growth and enhancing the responsiveness of HCC cells to paclitaxel.

Topics: Antineoplastic Agents; Apoptosis; Astragalus propinquus; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Drug Synergism; Fas Ligand Protein; fas Receptor; Hep G2 Cells; Humans; Liver Neoplasms; NF-kappa B; Paclitaxel; Swainsonine

Mounting evidence has shown that microRNAs (miRNAs) are implicated in carcinogenesis and can function as oncogenes or tumor suppressor genes in human cancers. Recent profile studies of miRNA expression have documented a deregulation of miRNA (miR-214) in hepatocellular carcinoma (HCC). However, its potential functions and underlying mechanisms in hepatocarcinogenesis remain largely unknown. Here, we confirmed that miR-214 is significantly downregulated in HCC cells and specimens. Ectopic overexpression of miR-214 inhibited proliferation of HCC cells in vitro and tumorigenicity in vivo. Further studies revealed that miR-214 could directly target the 3'-untranslated region (3'-UTR) of β-catenin mRNA and suppress its protein expression. Similar to the restoring miR-214 expression, β-catenin downregulation inhibited cell growth, whereas restoring the β-catenin expression abolished the function of miR-214. Moreover, miR-214-mediated reduction of β-catenin resulted in suppression of several downstream genes including c-Myc, cyclinD1, TCF-1, and LEF-1. These findings indicate that miR-214 serves as tumor suppressor and plays substantial roles in inhibiting the tumorigenesis of HCC through suppression of β-catenin. Given these, miR-214 may serve as a useful prognostic or therapeutic target for treatment of HCC.

Topics: 3' Untranslated Regions; Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Genes, myc; Genes, Tumor Suppressor; Hep G2 Cells; Humans; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Male; Mice; Mice, Nude; MicroRNAs; T Cell Transcription Factor 1

The human glycoprotein, stanniocalcin 2 (STC2) plays multiple roles in several tumor types, however, its function and clinical significance in hepatocellular carcinoma (HCC) remain unclear. In this study, we detected STC2 expression by quantitative real-time PCR and found STC2 was upregulated in HCC tissues, correlated with tumor size and multiplicity of HCC. Ectopic expression of STC2 markedly promoted HCC cell proliferation and colony formation, while silencing of endogenous STC2 resulted in a reduced cell growth by cell cycle delay in G0/G1 phase. Western blot analysis demonstrated that STC2 could regulate the expression of cyclin D1 and activate extracellular signal-regulated kinase 1/2 (ERK1/2) in a dominant-positive manner. Transwell chamber assay also indicated altered patterns of STC2 expression had an important effect on cell migration. Our findings suggest that STC2 functions as a potential oncoprotein in the development and progression of HCC as well as a promising molecular target for HCC therapy.

Topics: Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Case-Control Studies; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin D1; Glycoproteins; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

Hepatocellular carcinoma (HCC) is the most prevalent liver tumor and a deadly disease with limited therapeutic options. Dysregulation of cell signaling pathways is a common denominator in tumorigenesis, including hepatocarcinogenesis. The epidermal growth factor receptor (EGFR) signaling system is commonly activated in HCC, and is currently being evaluated as a therapeutic target in combination therapies. We and others have identified a central role for the EGFR ligand amphiregulin (AR) in the proliferation, survival and drug resistance of HCC cells. AR expression is frequently up-regulated in HCC tissues and cells through mechanisms not completely known. Here we identify the β-catenin signaling pathway as a novel mechanism leading to transcriptional activation of the AR gene in human HCC cells. Activation of β-catenin signaling, or expression of the T41A β-catenin active mutant, led to the induction of AR expression involving three specific β-catenin-Tcf responsive elements in its proximal promoter. We demonstrate that HCC cells expressing the T41A β-catenin active mutant show enhanced proliferation that is dependent in part on AR expression and EGFR signaling. We also demonstrate here a novel cross-talk of the EGFR system with fibroblast growth factor 19 (FGF19). FGF19 is a recently identified driver gene in hepatocarcinogenesis and an activator of β-catenin signaling in HCC and colon cancer cells. We show that FGF19 induced AR gene expression through the β-catenin pathway in human HCC cells. Importantly, AR up-regulation and EGFR signaling participated in the induction of cyclin D1 and cell proliferation elicited by FGF19. Finally, we demonstrate a positive correlation between FGF19 and AR expression in human HCC tissues, therefore supporting in clinical samples our experimental observations. These findings identify the AR/EGFR system as a key mediator of FGF19 responses in HCC cells involving β-catenin signaling, and suggest that combined targeting of FGF19 and AR/EGFR may enhance therapeutic efficacy.

Topics: Amphiregulin; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; EGF Family of Proteins; ErbB Receptors; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Mutation; Prognosis; Promoter Regions, Genetic; Signal Transduction; Transcription Factor 4; Transcription Factors

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K(d)) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x(L), Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; bcl-X Protein; Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytotoxins; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms, Experimental; Mice; Neoplasm Transplantation; Phosphorylation; Proto-Oncogene Proteins c-akt; Transplantation, Heterologous; Xenograft Model Antitumor Assays

We previously reported that the abnormal BTG2 expression was related to genesis/development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate the BTG2 expression in HCC compared with p53, cyclin D1, and cyclin E. For this purpose, modified diethylnitrosamine (DEN)-induced primary HCC rat model was established. Target proteins and mRNAs were measured by western blot and RT-PCR/northern blot, respectively. In rat liver, expression of BTG2 and other proteins was determined by western blot, and BTG2 mRNA in HCC/normal tissues was detected by high-flux tissue microarray (TMA) and in situ hybridization (ISH). BTG2 mRNA/protein expression was increased in fetal liver, 7701, and LO2 cell lines but decreased in HepG2 cells. BTG2/p53 were expressed early after DEN treatment, peaked at 5 weeks and decreased gradually thereafter. Cyclin-D1/Cyclin-E expression increased significantly with the tumor progression. BTG2 mRNA was expressed in 71.19% HCC by ISH and correlated with differentiation. Expression of p53/cyclin D1/cyclin E was positive in 82.35/94.12/76.47% BTG2 mRNA-negative tissues, respectively. BTG2 protein expression was lost in 32.2% (19/59) HCC tissues, and the mRNA/protein expression correlated significantly with the increasing tumor grade (P < 0.05). In conclusion, BTG2 expression is commonly impaired in HCC which may be a factor involved in deregulation of cyclin-D1/cyclin-E expression during hepatocarcinogenesis.

Topics: Adult; Aged; Aged, 80 and over; Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin E; Diethylnitrosamine; Disease Models, Animal; Female; Humans; Immediate-Early Proteins; Liver Neoplasms; Male; Middle Aged; Rats; Rats, Wistar; RNA, Messenger; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

We screened 124 genes that are amplified in human hepatocellular carcinoma (HCC) using a mouse hepatoblast model and identified 18 tumor-promoting genes, including CCND1 and its neighbor on 11q13.3, FGF19. Although it is widely assumed that CCND1 is the main driving oncogene of this common amplicon (15% frequency in HCC), both forward-transformation assays and RNAi-mediated inhibition in human HCC cells established that FGF19 is an equally important driver gene in HCC. Furthermore, clonal growth and tumorigenicity of HCC cells harboring the 11q13.3 amplicon were selectively inhibited by RNAi-mediated knockdown of CCND1 or FGF19, as well as by an anti-FGF19 antibody. These results show that 11q13.3 amplification could be an effective biomarker for patients most likely to respond to anti-FGF19 therapy.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromosomes, Human, Pair 11; Cyclin D1; Female; Fibroblast Growth Factors; Gene Amplification; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Genomics; Humans; Immunoblotting; Liver Neoplasms; Mice; Mice, Nude; Oncogene Proteins; RNA Interference; Tumor Burden; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC), a highly aggressive form of solid tumor, has been increasing in South East Asia. The lack of effective therapy necessitates the introduction of novel chemopreventive strategies to counter the substantial morbidity and mortality associated with the disease. Recently, we reported that dimethoxy flavone (DMF), a methylated flavone derived from chrysin, significantly suppressed the development of preneoplastic lesions induced by N-nitrosodiethylamine (DEN) in rats, although the mechanism of action was not known. In the present study, we have investigated the effects of DMF administration on gene expression changes related to the inflammation-mediated NF-kB pathway, Wnt pathway and apoptotic mediators in DEN-induced preneoplastic nodules. There was a significant increase in inflammatory markers like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and a decrease in apoptotic mediators like p53, caspase-3 and bax in DEN-treated rats when compared to the control group. Activation of NF-kB was noticed by an elevated expression of nuclear protein expression of NF-kB and cytoplasmic phospho-IkBαSer(32/36) in the same animals. Likewise, upregulation of canonical Wnt pathway was noticed by elevated expression of nuclear protein levels of phospho-β-cateninThr(393) and cytoplasmic casein kinase-2 (CK2), Dvl2 and cyclin D1 levels, along with a simultaneous decrease in expression of phospho-GSK3β(Ser9). Dietary DMF (100mg/kg) administration inhibited liver nodule incidence and multiplicity by 82% and 78%, respectively. DMF also reversed the activation of NF-kB and Wnt pathway as shown by the decrease in protein expression of several proteins. Results of the present investigation provide evidence that attenuation of Wnt pathway and suppression of inflammatory response mediated by NF-kB could be implicated, in part, in the chemopreventive effects of methylated flavone. Therefore, the present findings hold great promise for the utilization of DMF as an effective chemotherapeutic agent in treating early stages of liver cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Casein Kinase II; Caspase 3; Cyclin D1; Cyclooxygenase 2; Diethylnitrosamine; Disease Models, Animal; Flavones; Flavonoids; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; NF-kappa B; Nitric Oxide Synthase Type II; Precancerous Conditions; Rats; Rats, Wistar; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation; Wnt Proteins

To clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.. siRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.. The inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ± 0.10 vs. 0.73 ± 0.11 and 0.71 ± 0.07 respectively, P < 0.05). AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ± 0.35 vs. 3.47 ± 0.93 and 3.17 ± 0.78, P < 0.01). Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42)% vs. (70.58 ± 10.10)% and (69.83 ± 14.77)%, respectively, P < 0.01]. Cell cycle analysis by FACS showed that G(0)/G(1) cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ± 5.93)% vs. (57.05 ± 5.97)% and (52.99 ± 4.80)%, P < 0.05]. Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1(0.45 ± 0.21 vs. 1.25 ± 0.24 and 1.12 ± 0.15, respectively, P < 0.05)and CDK4 (0.55 ± 0.08 vs. 1.18 ± 0.32 and 1.10 ± 0.29, respectively, P < 0.05); the following genes were notably increased: p16(1.07 ± 0.23 vs. 0.36 ± 0.12 and 0.35 ± 0.13, respectively, P < 0.01)and p21(0.42 ± 0.12 vs. 0.17 ± 0.06 and 0.19 ± 0.08, respectively, P < 0.05). RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line. From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 and 0.58 ± 0.55, respectively, P < 0.05).. RhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Humans; Liver Neoplasms; Plasmids; rho GTP-Binding Proteins; rhoC GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Transfection

p28(GANK) (also known as PSMD10 or gankyrin) is a novel oncoprotein that is highly expressed in hepatocellular carcinoma (HCC). Through its interaction with various proteins, p28(GANK) mediates the degradation of the tumor suppressor proteins Rb and p53. Although p53 was reported to downregulate β-catenin, whether p28(GANK) is involved in the regulation of β-catenin remains uncertain. Here we report that both growth factors and Ras upregulate p28(GANK) expression through the activation of the phosphoinositide 3-kinase-AKT pathway. Upregulation of p28(GANK) expression subsequently enhanced the transcription activity of β-catenin. This effect was observed in p53-deficient cells, suggesting a p53-independent mechanism for the p28(GANK)-mediated activation of β-catenin. p28(GANK) overexpression also reduced E-cadherin protein levels, leading to increased release of free β-catenin into the cytoplasm from the cadherin-bound pool. Interestingly, exogenous expression of p28(GANK) resulted in elevated expression of the endogenous protein. We also observed that both β-catenin and c-Myc were transcriptional activators of p28(GANK), and a correlation between p28(GANK) overexpression and c-Myc, cyclin D1 and β-catenin activation in primary human HCC. Together, these results suggest that p28(GANK) expression is regulated by a positive feedback loop involving β-catenin, which may play a critical role in tumorigenesis and the progression of HCC.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; ras Proteins; Signal Transduction; Transcription, Genetic; Tumor Suppressor Protein p53

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. However, little is known regarding the molecular mechanism of HCC development and progression and effective therapeutic methods. Recently, the granulin-epithelin precursor (GEP) was reported as a novel growth factor that can control HCC cell proliferation. Using the CAPSID program, we designed three small interfering RNAs (siRNAs) targeting the GEP gene (GEP-siRNA1, 2 and 3) and examined their tumor regression and suppression effects on cell proliferation. GEP-siRNA1 exhibited the strongest anti-proliferative effect among the GEP-siRNAs, in a time-dependent manner. To increase the biostability of the siRNA, we also constructed a short hairpin RNA (shRNA) using an H1/TO promoter with the same sequence of GEP-siRNA1 (GEP-shRNA). GEP-shRNA decreased the expression levels of GEP and tumor cell growth via cell cycle arrest at the G2/M stage and down-regulation of the cell proliferation proteins cyclin D1 and α-tubulin. Furthermore, GEP-shRNA inhibited tumor growth significantly after intratumoral injection into tumor-bearing Balb/C nude mice. Taken together, these results represent the first therapeutic application of RNA interference to GEP, which is a promising target molecule for HCC treatment, as an approach for the suppression of HCC cell proliferation.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Growth Processes; Cyclin D1; Down-Regulation; G2 Phase; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Progranulins; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; Tubulin; Tumor Burden; Xenograft Model Antitumor Assays

P300 impacts the transcription of several genes involved in biological behavior of human malignancies including hepatocellular carcinomas (HCC). We found p300 is highly expressed in 47% of surgically resected HCC specimens by immunohistochemistry, which correlated with advanced TNM staging (P = 0.034), vascular invasion (P = 0.036), intrahepatic metastasis (P = 0.001) and shortened overall survival (P = 0.028). In vitro study, knocking down of p300 expression in hepatoma cells recovered E-cadherin expression, inhibited the translocation of beta (β)-catenin into the nuclei, decreased cyclin D1 activity and suppressed the migration/invasion of HCC cells. Furthermore, suppression of p300 led to down-regulation of epithelial-mesenchymal transition (EMT)-related molecules such as Snail, Twist and HIF-1 alpha. These observations suggest that p300 contributes to the EMT-related progression of HCCs.

Topics: Aged; Apoptosis; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cell Dedifferentiation; Cell Line, Tumor; Cyclin D1; Down-Regulation; E1A-Associated p300 Protein; Epithelial-Mesenchymal Transition; Female; Humans; Immunoblotting; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Neoplasm Staging; RNA, Small Interfering; Survival Analysis

A multikinase inhibitor of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK/ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co-treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1/2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl-2 family. ERK1/2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX-induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib-induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK/ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX-induced autophagy and thus promote cancer cell survival. The use of MEK/ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Autophagy; Benzenesulfonates; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Doxorubicin; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitochondria, Liver; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Phenylurea Compounds; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyridines; Sorafenib

FLZ is a synthetic novel squamosamide derivative and has previously been proved to be a potential drug for Parkinson's disease and Alzheimer's disease. FLZ has strong antioxidant activity, which implies that FLZ could eliminate excessive intracellular reactive oxygen species (ROS) in tumor cells and induce a pathway related to low cellular ROS levels, thereby inhibiting tumor cells proliferation. However, few reports have focused on the antitumor effects of FLZ. In this study, we investigated the antitumor efficacy of FLZ in HepG2 cells and the mechanism of cell growth inhibition. FLZ effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner; meanwhile, it was minimally toxic to normal cells. FLZ induced a significant decrease in oxidative stress through elimination of excessive intracellular ROS and strengthening of the glutathione antioxidant system. In addition, FLZ can effectively attenuate redundant [Ca(2+)](i), thereby avoiding uncontrolled amplification by Ca(2+)/ROS positive feedback. Furthermore, Western blot showed that FLZ inhibited phosphorylation of Akt and retinoblastoma protein (Rb), down-regulated the expressions of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), and enhanced the expression of CDK inhibitor p27(kip1), while did not affect CDK4 expression. These results suggest that FLZ has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the expression or activation of cell-cycle regulatory proteins, which are associated with decreased Ca(2+)/ROS levels, and indicate that FLZ is a potential liver cancer drug worthy of further research and development.

Topics: Acrylamides; Antineoplastic Agents; Apoptosis; Caffeic Acids; Calcium; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase; Glutathione; Hep G2 Cells; Humans; Liver Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Retinoblastoma Protein

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Chronic Disease; Cyclin D1; Hepatitis B virus; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Transgenic; Mutagenesis, Insertional; Mutation; Unfolded Protein Response; Virus Replication

HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol.

Topics: Animals; Antibodies; Antioxidants; Autophagy; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cyclin D1; Genome; Hepacivirus; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Mice, Transgenic; Mitochondria; Oxidation-Reduction; Time Factors; Up-Regulation

Peroxisome proliferator-activated receptor α (PPARα) regulates lipid metabolism in the liver. It is unclear, however, how this receptor changes in liver cancer tissue. On the other hand, mouse carcinogenicity studies showed that PPARα is necessary for the development of liver cancer induced by peroxisome proliferators, and the relationship between PPARα and the development of liver cancer have been the focus of considerable attention. There have been no reports, however, demonstrating that PPARα is involved in the development of human liver cancer.. The subjects were 10 patients who underwent hepatectomy for hepatocellular carcinoma. We assessed the expression of PPARα mRNA in human hepatocellular carcinoma tissue and non-cancerous tissue, as well as the expression of target genes of PPARα, carnitine palmitoyltransferase 1A and cyclin D1 mRNAs. We also evaluated glyceraldehyde 3-phosphate dehydrogenase, a key enzyme in the glycolytic system.. The amounts of PPARα, carnitine palmitoyltransferase 1A and glyceraldehyde 3-phosphate dehydrogenase mRNA in cancerous sections were significantly increased compared to those in non-cancerous sections. The level of cyclin D1 mRNA tends to be higher in cancerous than non-cancerous sections. Although there was a significant correlation between the levels of PPARα mRNA and cyclin D1 mRNA in both sections, however the correlation was higher in cancerous sections.. The present investigation indicated increased expression of PPARα mRNA and mRNAs for PPARα target genes in human hepatocellular carcinoma. These results might be associated with its carcinogenesis and characteristic features of energy production.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Case-Control Studies; Cyclin D1; Humans; Liver; Liver Neoplasms; PPAR alpha; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To investigate let-7c's effect on the proliferation of human hepatocellular carcinoma cell HCCLM3 by transient transfection and the mechanism inside.. Lipofectamine 2000 was used to transfect miRNAs into HCCLM3 cells. The cells were divided into three groups, let-7c group: let-7c was transfected, negative control group: negative control miRNA was transfected, blank control group: nothing was transfected. The proliferation of HCCLM3 cells was evaluated using Cell Counting Kit-8 (CCK-8). The cell cycles of each group were assayed by flow cytometry. Western blot and Real time PCR were used to analyze the protein and mRNA expressions of cyclin D1. Statistical analysis was performed with SPSS 17.0.. The absorbances of let-7c group were 0.70 ± 0.05, 0.77 ± 0.09 at 48 h and 72 h after transfection, lower than that of blank control group (0.97 ± 0.10, 1.21 ± 0.12) and negative control group (0.91 ± 0.07, 1.12 ± 0.09), 48 h: F = 14.431, P < 0.05, 72 h: F = 21.146, P < 0.05. The flow cytometry at 72 h after transfection revealed that let-7c increased the percentage of cells in G1 phase. The percentage of blank control group was 43.53% ± 0.86%, the negative control group was 44.82% ± 0.77%, and the let-7c group was 54.52% ± 0.13%, F = 240.739, P < 0.05. let-7c suppressed expressions of cyclin D1 at both protein and mRNA levels. The protein levels of cyclin D1 were 0.48 ± 0.09, 0.47 ± 0.06 and 0.23 ± 0.06 (F = 11.316, P < 0.05) in blank control group, negative control group and let-7c group, respectively. The mRNA levels were 1.03% ± 0.29%, 1.01% ± 0.11% and 0.63% ± 0.14% (F=6.315, P < 0.05) in the above three groups, respectively.. Let-7c can inhibit proliferation of HCCLM3 cells and increase the proportion of cells in G1 phase. The mechanism may be that let-7c represses the expressions of cyclin D1 at both protein and mRNA levels.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Liver Neoplasms; MicroRNAs; RNA, Small Interfering; Transfection

Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide. It is intrinsically resistant toward standard chemotherapy, making it imperative to develop novel selective chemotherapeutic agents. The Wnt/beta-catenin pathway plays critical roles in development and oncogenesis, and is dysregulated in HCC. Our study aims to evaluate the activity of 3 small molecule antagonists of the Tcf4/beta-catenin complex (PKF118-310, PKF115-584 and CGP049090) on HCC cell lines in vitro and in vivo. All 3 chemicals displayed dose-dependent cytotoxicity in vitro against all 3 HCC cell lines (HepG2, Hep40 and Huh7), but were at least 10 times less cytotoxic to normal hepatocytes (from 3 donors) by using ATP assay. In HepG2 and Huh7 cells, treatment with the antagonists decreased Tcf4/beta-catenin binding capability and transcriptional activity, associated with downregulation of the endogenous Tcf4/ beta-catenin target genes c-Myc, cyclin D1 and survivin. In HepG2 and Huh7 cells, treatment with the antagonists induced apoptosis and cell cycle arrest at the G1/S phase. All antagonists suppressed in vivo tumor growth in a HepG2 xenograft model, associated with apoptosis and reduced c-Myc, cyclin D1 and survivin expressions. Our results suggest that these 3 antagonists of the Tcf4/beta-catenin complex are potential chemotherapeutic agents which may offer a pathway specific option for the clinical management of HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Genes, myc; Hepatocytes; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Mice; Mice, Nude; Microtubule-Associated Proteins; Perylene; Pyrimidinones; Random Allocation; Signal Transduction; Survivin; Transcription Factor 4; Transcription Factors; Triazines; Wnt Proteins; Xenograft Model Antitumor Assays

IKK-NF-kappaB signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy. Therefore, in this study, we analyzed the expression of mRNAs encoding components and targets of NF-kappaB signaling including IKKalpha, IKKbeta, RANK, RANKL, OPG, CyclinD3, mammary serine protease inhibitor (Maspin), CyclinD1, c-FLIP, Bcl-xl, Stat3, Cip1 and Cip2 by real-time PCR in 40 patients with liver cancer. After statistical analysis, 7 indices including IKKalpha, IKKbeta, RANK, Maspin, c-FLIP, Cip2 and cyclinD1 were found to show significant differences between tumor tissue and its corresponding adjacent tissue. When IKKalpha and IKKbeta were downregulated in the hepatocellular carcinoma (HCC) cell lines of MHCC-97L and MHCC-97H in vitro, the numbers of BrdU positive cells were decreased in both IKKalpha and IKKbeta knockdown cells. Levels of apoptosis were also investigated in IKKalpha and IKKbeta knockdown cells. The growth of HCC was inhibited in the subcutaneous implantation model, and lung metastatogenesis was also significantly inhibited in the kidney capsule transplantation model. Downregulation of IKKalpha and IKKbeta in HCC cultured in vitro revealed that increased Maspin, OPG and RANKL expression was associated with metastasis of HCC. These findings were associated with downregulation of Bcl-XL and c-FLIP, which may be the reason for increased apoptosis. The therapeutic effect of IKKalpha and IKKbeta downregulation depends on extent of NF-kappaB inhibition and the malignant nature of the HCC. We anticipate that IKK-targeted gene therapy can be used in the treatment of HCC, a cancer that is notoriously resistant to radiation and chemotherapy.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Cyclin D1; Flow Cytometry; Gene Expression; Gene Expression Profiling; Humans; I-kappa B Kinase; Immunohistochemistry; Liver Neoplasms; Mice; Mice, Nude; NF-kappa B; Osteoprotegerin; RANK Ligand; Reverse Transcriptase Polymerase Chain Reaction; Serpins; Signal Transduction; Xenograft Model Antitumor Assays

The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry, occurring with diethylnitrosamine treatment, was independent of cyclin D1 levels and associated with the specific induction of E2F1. Combined, these studies demonstrate that RB loss has disparate effects on the response to unique tumorigenic stresses, which is reflective of distinct mechanisms of cell cycle entry.

Topics: Alkylating Agents; Animals; Carcinoma, Hepatocellular; Cell Cycle; Cyclin D1; Diethylnitrosamine; DNA Damage; DNA Replication; E2F1 Transcription Factor; Gene Deletion; Liver Neoplasms; Mice; Mice, Transgenic; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130

The E2F family member of transcription factors includes the atypical member E2F8, which has been little studied in cancer. We report that E2F8 is strongly upregulated in human hepatocellular carcinoma (HCC), where it was evidenced to contribute to oncogenesis and progression. Ectopic overexpression of E2F8 promoted cell proliferation, colony formation, and tumorigenicity, whereas E2F8 knockdown inhibited these phenotypes, as documented in Huh-7, Focus, Hep3B, and YY-8103 HCC cell lines. Mechanistic analyses indicated that E2F8 could bind to regulatory elements of cyclin D1, regulating its transcription and promoting accumulation of S-phase cells. Together, our findings suggest that E2F8 contributes to the oncogenic potential of HCC and may constitute a potential therapeutic target in this disease.

Topics: Animals; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; G1 Phase; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, SCID; Repressor Proteins; RNA, Small Interfering; S Phase; Transfection; Up-Regulation

The initiation and growth of hepatocellular carcinoma (HCC) are closely linked to chronic inflammation. Not only is cyclin D1 overexpressed, but it is also related to aggressive progression in HCC. However, the mechanism of expression cyclin D1, a cell-cycle regulator of paramount importance, in the tumor microenvironment remains unknown. Here, we investigated the mechanism of cyclin D1 expression induced by interleukin-6 (IL-6) and whether 3-[3,4-dihydroxy-phenyl]-acrylic acid 2-[3,4-dihydroxy-phenyl]-ethyl ester (CADPE), a derivate of caffeic acid, suppresses cyclin D1 expression. CADPE significantly inhibited IL-6-induced signal transducer and activator of transcription 3 (STAT3) activity in the Huh7 HCC cell line and attenuated IL-6-induced cyclin D1 transcription. Moreover, overexpression of constitutively active STAT3 increased cyclin D1 transcriptional activity and protein expression, whereas overexpression of a dominant-negative STAT3 deletion mutant (STAT3 (1-588)) reduced cyclin D1 transcriptional activity. In addition, CADPE effectively deacetylated histone 4 and prevented STAT3 recruitment to the cyclin D1 promoter, consistent with a role for the CADPE target, STAT3, in the regulation of cyclin D1 transcription. Collectively, these results indicate that CADPE suppresses cyclin D1 expression in HCC cells by blocking both IL-6-mediated STAT3 activation and recruitment of STAT3 to the cyclin D1 promoter.

Topics: Blotting, Western; Caffeic Acids; Carcinoma, Hepatocellular; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Liver Neoplasms; Luciferases; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; STAT3 Transcription Factor; Tumor Cells, Cultured

A common G to A polymorphism (G870A) in the splice donor region of exon 4 of cyclin D1 (CCND1) gene generates two mRNAs (cyclin D1a and D1b) through an alternative splicing at the site of this polymorphism. Cyclin D1a and b proteins differ in their COOH-terminus, a region involved in protein degradation. We examined the association between this CCDN1 genotype and the susceptibility to hepatocellular carcinoma (HCC) in a Turkish population.. The genotype frequency of this polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Hospital-based case-control study was designed consisting of 160 diagnosis subjects with hepatocellular carcinoma and 160 cancer-free control subjects matched on age, gender, smoking and alcohol status.. The allele frequencies of case subjects (A, 0.55; G, 0.45) were significantly different from those of control subjects (A, 0.42; G, 0.58) (p=0.002). The odds ratios (ORs) for the CCND1 870 GA and AA genotypes when compared with the GG genotypes were 1.39 (95% confidence interval [CI] 0.82-2.36, p=0.22) and 2.52 (95% CI 1.38-4.62, p=0.003) respectively. The presence of at least one CCND1 870A allele was associated with increased risk for HCC (OR, 1.73; 95% CI, 1.06-2.82, p=0.03). When combining the GG and GA genotypes as a reference genotype, we found that the OR for the AA genotype was 2.06 (95% CI 1.24-3.44, p=0.006).. Our results suggest that the CCND1 G870A single nucleotide polymorphism is associated with an increased risk of HCC in our Turkish population.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Case-Control Studies; Cyclin D1; Female; Gene Frequency; Genetic Predisposition to Disease; Humans; Liver Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Population; Population Surveillance; Risk Factors; Turkey; Young Adult

The Wnt/beta-catenin signaling pathway regulates various aspects of development and plays important role in human carcinogenesis. Nemo-like kinase (NLK), which is mediator of Wnt/beta-catenin signaling pathway, phosphorylates T-cell factor/lymphoid enhancer factor (TCF/LEF) factor and inhibits interaction of beta-catenin/TCF complex. Although, NLK is known to be a tumor suppressor in Wnt/beta-catenin signaling pathway of colon cancer, the other events occurring downstream of NLK pathways in other types of cancer remain unclear. In the present study, we identified that expression of NLK was significantly up-regulated in the HCCs compared to corresponding normal tissues in five selected tissue samples. Immunohistochemical analysis showed significant over-expression of NLK in the HCCs. Targeted-disruption of NLK suppressed cell growth and arrested cell cycle transition. Suppression of NLK elicited anti-mitogenic properties of the Hep3B cells by simultaneous inhibition of cyclinD1 and CDK2. The results of this study suggest that NLK is aberrantly regulated in HCC, which might contribute to the mitogenic potential of tumor cells during the initiation and progression of hepatocellular carcinoma; this process appears to involve the induction of CDK2 and cyclin D1 and might provide a novel target for therapeutic intervention in patients with liver cancer.

Topics: Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Gene Expression; Gene Expression Regulation; Gene Silencing; Humans; Immunohistochemistry; Liver Neoplasms; Membrane Transport Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factor TFIIIA

Recently, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. However, the functions of these miRNAs in HCC remain largely undefined.. The expression profiles of miR-193b were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was also be used to screen the potential target genes of miR-193b. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-193b in hepatoma cells was examined further.. miR-193b was significantly down-regulated in most of the HCC tissues compared to the matching non-tumoural liver tissues. Furthermore, ectopic expression of miR-193b dramatically suppressed the ability of hepatoma cells to form colonies in vitro and to develop tumours in nude mice. CCND1 and ETS1 were revealed to be regulated by miR-193b directly. By regulating the expressions of these oncogenes, miR-193b induced cell cycle arrest and inhibited the invasion and migration of hepatoma cells.. miR-193b may function as a tumour suppressor in the development of HCC by acting on multiple tumourigenic pathways.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Cycle; Cell Movement; Cell Transformation, Neoplastic; Cyclin D1; Down-Regulation; Female; Humans; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Neoplastic Stem Cells; Proto-Oncogene Protein c-ets-1

The F-box protein family member FBXO31 has rarely been studied in human hepatocellular carcinoma (HCC). This study was designed to investigate the expression profile of FBXO31 in HCC and the possibility that FBXO31 might function as a tumor suppressor in HCC cell lines. We report that FBXO31 is strongly down-regulated in HCC cell lines and specimens. Ectopic expression of FBXO31 inhibited cell proliferation and colony formation in HepG2 and Hep3B cells. The endogenous expression of FBXO31 was fluctuated through cell cycle in the HepG2 cells with maximal expression from late G2 to early G1 phase. Ectopic expression of FBXO31 in HepG2 resulted in the accumulation of cells at the G1 phase of the cell cycle. Possible mechanism might be cyclin D1 degradation mediate by FBXO31 through ubiquitin ligase pathway. Ectopic overexpression of FBXO31 resulted in down-regulation of cyclin D1 which leads to the accumulation of cells at the G1 phase of the cell cycle. Cytoplasmic location of FBXO31 was consistent with cyclin D1 degradation in cytoplasm. Together, our findings suggested that down-regulation of FBXO31 might function as a tumor suppressor in HCC.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cyclin D1; Down-Regulation; F-Box Proteins; Hep G2 Cells; Humans; Liver Neoplasms; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Time Factors; Transfection; Tumor Suppressor Proteins; Ubiquitination

Dysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.. Salirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.. Our results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Farnesol; Female; Hep G2 Cells; Humans; Inhibitor of Apoptosis Proteins; Insulin-Like Growth Factor II; Liver Neoplasms; Mice; Mice, Nude; Microtubule-Associated Proteins; ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; Salicylates; Survivin; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Sulfatase 2 (SULF2), an extracellular heparan sulphate 6-O-endosulphatase, has an oncogenic effect in hepatocellular carcinoma (HCC) that is partially mediated through glypican 3, which promotes heparin-binding growth factor signalling and HCC cell growth. SULF2 also increases phosphorylation of the anti-apoptotic Akt kinase substrate GSK3β and SULF2 expression is associated with a decreased apoptotic index in human HCCs.. We investigated the functional and mechanistic effects of SULF2 on drug-induced apoptosis of HCC cells using immunohistochemistry, Western immunoblotting, gene transfection, real-time quantitative polymerase chain reaction, MTT and apoptosis assays and immunocytochemistry.. The increased expression of SULF2 in human HCCs was confirmed by immunohistochemistry and immunoblotting. Treatment with inhibitors of MEK, JNK and PI3 kinases decreased the viability of SULF2-negative Hep3B HCC cells and induced apoptotic caspase 3 and 7 activity, which was most strongly induced by the PI3K inhibitor LY294002. Forced expression of SULF2 in Hep3B cells significantly decreased activity of the apoptotic caspases 3 and 7 and induced resistance to LY294002-induced apoptosis. As expected, LY294002 inhibited activation of Akt kinase by PI3K. Conversely, knockdown of SULF2 using an shRNA construct targeting the SULF2 mRNA induced profound cell growth arrest and sensitized the endogenously SULF2-expressing HCC cell lines Huh7 and SNU182 to drug-induced apoptosis. The effects of knockdown of SULF2 on HCC cells were mediated by decreased Akt phosphorylation, downregulation of cyclin D1 and the anti-apoptotic molecule Bcl-2, and upregulation of the pro-apoptotic molecule BAD.. The prosurvival, anti-apoptotic effect of SULF2 in HCC is mediated through activation of the PI3K/Akt pathway.

Topics: Apoptosis; bcl-Associated Death Protein; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Survival; Chromones; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sulfatases; Sulfotransferases; Transfection

The present study investigated the effect of mammalian target of rapamycin (mTOR) inhibition on HCC cells in vitro and in vivo, either alone or in combination with cytotoxic agents. In vitro, HCC cell lines were exposed to RAD001, an mTOR inhibitor, either alone or in combination with cisplatin. Alone, RAD001 suppressed cell proliferation in all cell lines tested, but did not induce apoptosis. RAD001 in combination with cisplatin induced a significant increase in the number of apoptotic cells, downregulated the expression of pro-survival molecules, Bcl-2, survivin and cyclinD1, and increased the cleavage of PARP, compared to RAD001 or cisplatin alone. Transfection of p53 into the Hep3B cell line increased the sensitivity of tumor cells to cisplatin. The suppression of HCC tumor growth in vivo was enhanced by RAD001 combined with cisplatin, accompanied by a significant increase in the number of apoptotic cells in tumor tissues. This study demonstrates that inhibition of mTOR suppresses tumor growth and sensitizes tumor cells to chemocytotoxic agents.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Genes, p53; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Male; Mice; Mice, Nude; Microtubule-Associated Proteins; Neoplasm Proteins; Neoplasm Transplantation; Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Survivin; TOR Serine-Threonine Kinases

Mice lacking hepatocyte IKKbeta (Ikkbeta(Delta hep)) are defective in TNFalpha-activation of hepatocellular transcription factor NF-kappaB, and highly susceptible to hepatotoxicity. Following diethylnitrosamine (DEN) exposure, Ikkbeta(Delta hep) mice develop more hepatocellular carcinoma (HCC) than control mice due partly to enhanced DEN-induced hepatocyte death. Here we show that Ikkbeta(Delta hep) hepatocytes display growth advantages over normal hepatocytes consisting of precocious PCNA and cyclin D1 expression during liver regeneration (shortened hepatocyte G(0)-->G(1) transitions), and enhanced recovery efficiency, cyclin D1 expression and cell proliferation after plating. Ex vivo deletion of Ikkbeta also accelerates hepatocyte growth. Ikkbeta(Delta hep) hepatocyte proliferative responses show heightened sensitivity to TGFalpha and TNFalpha, and heightened expression of fibronectin, collagens I/III, nidogen, beta-actin and integrin beta1 mRNAs. These findings suggest that altered mitogen signaling and expression of extracellular matrix and its associated components underlie growth advantages. Increased HCC development in Ikkbeta(Delta hep) mice may also be caused by growth advantages of surviving Ikkbeta-deleted hepatocytes.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; G1 Phase; Gene Deletion; Gene Targeting; Hepatocytes; I-kappa B Kinase; Liver Neoplasms; Liver Regeneration; Mice; Mice, Knockout; Proliferating Cell Nuclear Antigen; Resting Phase, Cell Cycle; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

There are around 350 million of hepatitis B surface antigen (HBsAg) carriers worldwide, and among them, high risk of developing hepatocellular carcinoma (HCC) has been identified by epidemiological studies. To date, the molecular role of HBsAg in HCC development has not been fully studied. We have previously reported that in cell cultures, HBsAg up-regulated the expression of lymphoid enhancer-binding factor 1 (LEF-1), a key component of the Wnt pathway. In this study we aimed to study this effect of HBsAg on LEF-1 in the development of HCC.. Expression of HBsAg, LEF-1 and its downstream effector genes were compared among 30 HCCs, their peritumor tissue counterparts and 9 normal control liver tissues by quantitative real-time PCR. In addition, immunohistochemical staining studies on HBsAg and LEF-1 expression were conducted among these samples.. The expression of LEF-1 was compared between 13 HBsAg positive HCC tissues and 17 HBsAg negative HCC tissues. Simultaneous detection of LEF-1 and HBsAg was observed in HBsAg positive HCC tissues and, additionally, the simultaneous detection of HBsAg and LEF-1 was more pronounced in peritumor tissues, compared to that in the tumor tissues. The distribution of cellular LEF-1 in peritumor tissues was predominantly in the cytoplasm; while LEF-1 in the tumor tissues was located either exclusively in the nucleus or both in the nucleus and cytoplasm. By real-time PCR, the expression levels of LEF-1 downstream effector genes cyclin D1 and c-myc were higher in peritumor cells compared to that of the tumor cells. However, a 38 kDa truncated isoform of LEF-1, rather than the 55 kDa wild-type LEF-1, was significantly elevated in the HBsAg positive tumor cells.. Data indicate that deregulation of the Wnt pathway by HBsAg occurred in HBV-associated HCCs, but was more pronounced in the peritumor cells. It is speculated that HBsAg could stimulate proliferation and functional modification of hepatocytes via LEF-1 through the Wnt pathway at the pre-malignant stage.

Topics: Carcinoma, Hepatocellular; Cyclin D1; Hepatitis B Surface Antigens; Humans; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Protein Isoforms; Proto-Oncogene Proteins c-myc; Signal Transduction; Up-Regulation; Wnt Proteins

Many tumors are resistant to drug-induced cell-cycle arrest and apoptosis. We have reported that apoptosis can be restored in human multidrug-resistant (MDR) hepatocellular carcinoma cell lines by celecoxib. Here we show that P-glycoprotein (P-gp) mediates cell-cycle arrest and autophagy induced by celecoxib in human MDR overexpressing hepatocellular carcinoma cell line by down-regulation of the HGF/MET autocrine loop and Bcl-2 expression. Exposure of cells to a low concentration of celecoxib down-regulated the expression of mTOR and caused G1 arrest and autophagy, while higher concentration triggered apoptosis. Cell growth inhibition and autophagy were associated with up-regulation of the expression of TGFbeta1, p16(INK4b), p21(Cip1) and p27(Kip1) and down-regulation of cyclin D1, cyclin E, pRb and E2F. The role of P-glycoprotein expression in resistance of MDR cell clone to cell-cycle arrest, autophagy and apoptosis was shown in cells transfected with MDR1 small interfering RNA. These findings demonstrate that the constitutive expression of P-gp is involved in the HGF/MET autocrine loop that leads to increased expression of Bcl-2 and mTor, inhibition of eIF2alpha expression, resistance to autophagy/apoptosis and progression in the cell-cycle. Since mTor inhibitors have been proposed in treatment of "drug resistant" cancer, these data may help explain the reversing effect of mTor inhibitors.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Celecoxib; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclooxygenase Inhibitors; Drug Resistance, Multiple; G1 Phase; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); Hepatocyte Growth Factor; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; RNA, Neoplasm; RNA, Small Interfering; Sulfonamides

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; Epiregulin; G1 Phase; Gene Knockdown Techniques; Gene Silencing; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Resting Phase, Cell Cycle; Retinoblastoma Protein; RNA, Small Interfering

To investigate the influence of DNA methyltransferase (DNMT) 3b on the expression of cyclin D1 gene and methylation of its promoters and to investigate the function of DNMT3b.. Human hepatocellular carcinoma cells of the line SMMC7721 were cultured and randomly divided into 3 groups: experimental group transfected with siRNA to silence the DNMT3b, control group transfected with control siRNA, and normal group without transfection. The transfection rate of siRNA was detected by fluorescence microscopy. MTT method was used to measure the survival rate of the SMMC-7721 cells. Western blotting and cell proliferation assay were performed to evaluate the expression of cyclin D1 and cell growth. Methylation specific PCR (MSP) was performed to investigate whether the promoter of cyclin D1 was methylated.. Fluorescence microscopy showed that the transfection rate of siRNA was over 90%. MTT method showed that 24 h and 36 h after transfection the A value and survival rate of the SMMC7721 cells of the experimental group were both significantly higher than those of the control d normal groups (all P < 0.05). Western blotting showed that the expression levels of DNMT3b and cyclin D1 of the experimental group decreased significantly compared with the control and the normal groups. MSP showed no obvious change of the state of methylation among the 3 groups.. DNMT3b may regulate the expression and the function of cyclin D1 gene in the human hepatocellular carcinoma cells, but does not change its methylation state. DNMT3b may play their role as a signal transduction element rather than as a DNA methyltransferase.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Promoter Regions, Genetic; RNA, Small Interfering; Transfection

Frizzled (Fz), a receptor of Wnt ligands, plays key roles in liver carcinogenesis. Its expression was analyzed as part of a search for a target of molecular therapy for hepatocellular carcinoma (HCC) and hepatoblastoma (HB). Fz genes were analyzed by RT-PCR in HCC cell lines HLE, HLF, PLC/PRF/5, Huh-7 and Hep3B, HB cell lines Huh-6 and HepG2, HeLa cells, human normal fetal and adult liver. We transfected PLC/PRF/5, HLE, Huh-6, and HeLa cells with Fz9-small interfering RNA (Fz9-siRNA). Five days after transfection, cell proliferation was analyzed by MTS assay and cell motility by wound assay with H&E staining. Subsequently, the expressions of cyclin D1 and caspase-3 were analyzed by Western blot analysis. Fz9-siRNA decreased the expression of Fz9 gene in all cell lines. MTS assay showed that Fz9-siRNA significantly suppressed cell proliferation and cell motility in all cell lines. The expression of cyclin D1 was also suppressed by Western blotting. Cleaved caspase-3 did not appear and apoptosis was not observed in any of the cell lines tested. We demonstrated that Fz9 plays an essential role in carcinogenesis of HB and HCC, concluding that Fz9-siRNA could represent a useful therapeutic target for HB and HCC.

Topics: Adult; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Frizzled Receptors; HeLa Cells; Humans; Liver Neoplasms; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Time Factors; Transfection

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. The number of cases of HCC has continued to increase in recent decades. Previous studies have suggested that S100P, a member of the S100P calcium-binding protein family, is aberrantly regulated in several malignant neoplasms. However, the underlying molecular mechanisms of the dysregulation of S100P remain to be elucidated. To investigate biological effects of S100P on hepatocarcinogenesis, aberrant expression of S100P was investigated by immunohistochemistry (IHC), Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) in HCC tissues and cell lines. Endogenous expression of S100P was disrupted by the RNA interference-mediated protein knockdown method in the human Hep3B liver cancer cell line. Then, cell growth and cellular apoptosis were compared with control siRNA transfectants. The effects of S100P-silencing on the major components of cell cycle regulation were assessed by Western blot analysis. As results, elevated levels of S100P were observed in the HCC tissues compared to the corresponding normal tissues. Targeted disruption of S100P suppressed cell growth and augmented cellular apoptosis. In addition, inhibition of S100P resulted in the down-regulation of cyclinD1 and CDK2. In conclusion, this study showed over-expression of S100P in HCC. The aberrant regulation of S100P in HCC might activate cyclin D1 and CDK expression and contribute to the mitogenic potential of tumor cells during HCC carcinogenesis. These findings provide information that suggests new therapeutic strategies for the treatment of liver cancer.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver Neoplasms; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference

The mammalian target of rapamycin (mTOR) inhibitors play a key role in regulating signal transduction by blocking the mTOR pathway and combining anticancer and immunosuppressive properties. This study was undertaken to determine the prevalence and clinicopathological relevance of phospho-p70S6 (p-p70S6) kinase in hepatocellular carcinoma (HCC) and to investigate the effects of rapamycin on HCC in vitro.. A total of 196 patients with HCCs were treated either with surgical resection (n=106) or liver transplantation (n=90). Tumour tissue was investigated for p-p70S6, phospho-AKT, Ki-67, Cyclin-D1 and apoptosis, and staining results were correlated with clinicopathologically relevant parameters.. Overall, p-p70S6 was detected in 24.5% (48/196) of HCCs. In the resection group, 26.4% (28/106) of HCC were positive and 22.2% (20/90) in the transplant group. p-p70S6 was significantly associated with elevated Cyclin-D1 immunoexpression and was correlated with decreased overall survival (P=0.011) in patients resected with a clear margin. In multivariate COX regression analysis, p-p70S6 was identified as an independent prognostic parameter in patients resected with a clear margin. Rapamycin induced apoptosis and growth inhibition by G0/G1 cell cycle arrest in vitro. However, in HCC patients p-p70S6 kinase was not associated with proliferation or apoptosis.. Activation of p70S6 kinase indicates aggressive tumour behaviour in patients with clear margin-resected HCC. Identification of p-p70S6 kinase in HCC selects high-risk patients who may benefit from drugs targeting the mTOR pathway.

Topics: Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cyclin D1; Enzyme Activation; Female; Humans; Immunohistochemistry; Immunosuppressive Agents; In Situ Nick-End Labeling; Male; Microarray Analysis; Phosphorylation; Prognosis; Regression Analysis; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Survival Analysis

Topics: Carcinoma, Hepatocellular; Cyclin D1; Hepatitis B virus; Humans; Liver; Liver Cirrhosis; NF-kappa B; Trans-Activators; Viral Regulatory and Accessory Proteins

The small heterodimer partner (SHP; NROB2), a member of the nuclear receptor superfamily, contributes to the biological regulation of several major functions of the liver. However, the role of SHP in cellular proliferation and tumorigenesis has not been investigated before. Here we report that SHP negatively regulates tumorigenesis both in vivo and in vitro. SHP-/- mice aged 12 to 15 months old developed spontaneous hepatocellular carcinoma, which was found to be strongly associated with enhanced hepatocyte proliferation and increased cyclin D1 expression. In contrast, overexpressing SHP in hepatocytes of SHP-transgenic mice reversed this effect. Embryonic fibroblasts lacking SHP showed enhanced proliferation and produced increased cyclin D1 messenger RNA and protein, and SHP was shown to be a direct negative regulator of cyclin D1 gene transcription. The immortal SHP-/- fibroblasts displayed characteristics of malignant transformed cells and formed tumors in nude mice.. These results provide first evidence that SHP plays tumor suppressor function by negatively regulating cellular growth.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cells, Cultured; Cyclin D1; Embryo, Mammalian; Fibroblasts; Hepatocytes; Liver Neoplasms; Mice; Mice, Knockout; Mice, Nude; Mice, Transgenic; Neoplasms; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transcription, Genetic; Tumor Suppressor Proteins; Up-Regulation

To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2beta gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells.. We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-x(L) and bcl-x(S) were detected with western blot.. We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved in down-regulating bcl-x(L) and up- regulating bcl-x(S).. LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.

Topics: Apoptosis; bcl-X Protein; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lentivirus; Liver Neoplasms; Methionine Adenosyltransferase; RNA Interference; S-Adenosylmethionine; Transfection

The potential molecular mechanisms of the antitumor role of Ras association domain family 1 A (RASSF1A) has not been well understood. This study was to explore the molecular mechanisms of proliferation-suppressing ability of wild type RASSF1A in hepatocellular carcinoma (HCC) QGY-7703 cells.. Vectors containing wild type or mutant RASSF1A were transfected into QGY-7703 cells. Cell cycle was determined by flow cytometry (FCM). Western blot was used to examine the protein levels of Cyclin D1, Cyclin E and P21. Luciferase activity assay was performed to study the effect of wild type RASSFIA expression on cyclin D1 promoter activity in QGY-7703 cells. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the mRNA level of cyclin D1.. Wild type RASSF1A expression resulted in G1/S arrest in QGY-7703 cells, decreased the protein level of Cyclin D1, but had no effect on the protein levels of Cyclin E and P21, the promoter activity and mRNA level of cyclin D1. The exogenous expression of cyclin D1 rescued the G1 phase arrest induced by wild type RASSF1A.. Wild type RASSF1A expression could induce G1 phase arrest in QGY-7703 cells, which is accompanied by a down-regulation of Cyclin D1 protein expression through a post-transcriptional mechanism.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Liver Neoplasms; Plasmids; RNA, Messenger; Transfection; Tumor Suppressor Proteins

The canonical Wnt signalling pathway acts by slowing the rate of ubiquitin-mediated beta-catenin degradation. This results in the accumulation and subsequent nuclear translocation of beta-catenin, which induces the expression of a number of genes involved in growth, differentiation and metabolism. The mechanisms regulating the Wnt signalling pathway in the physiological context is still not fully understood. In the present study we provide evidence that changes in glucose levels within the physiological range can acutely regulate the levels of beta-catenin in two macrophage cell lines (J774.2 and RAW264.7 cells). In particular we find that glucose induces these effects by promoting an autocrine activation of Wnt signalling that is mediated by the hexosamine pathway and changes in N-linked glycosylation of proteins. These studies reveal that the Wnt/beta-catenin system is a glucose-responsive signalling system and as such is likely to play a role in pathways involved in sensing changes in metabolic status.

Topics: Animals; Axin Protein; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Cytoskeletal Proteins; DNA Primers; Gene Expression Regulation, Neoplastic; Glucose; Homeostasis; Humans; Liver Neoplasms; Macrophages; Mice; RNA; Wnt Proteins

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.

Topics: Carbazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA, Antisense; Gene Expression Regulation, Neoplastic; Humans; Indoles; Isoenzymes; Neoplasm Invasiveness; Protein Kinase C-alpha; Protein Kinase Inhibitors; RNA, Small Interfering; Tumor Suppressor Protein p53

The tumor-suppressing role of Ras-association domain family 1A (RASSF1A) has been described in several systems. In this study, we tested its tumor-suppressing ability and the potential molecular mechanisms in hepatocellular carcinoma (HCC) from Qidong County.. Reverse transcription polymerase chain reaction and Northern blotting were employed to detect the expression of RASSF1A in HCC. After establishing stable RASSF1A (wild type or mutant) expressing 'qi dong gan ai yan jiu suo' ([Qidong Institute of Liver Cancer] QGY)-7703 cell lines, we tested the effects of RASSF1A expression on cell growth by cell proliferation rate, cell colony formation, and cell cycle progression. We also tested the effects of RASSF1A expression on tumorigenesis in nude mice and on cellular sensitivity to mitomycin treatment.. The RASSF1A transcript was not found in 75% (three of four) of HCC cell lines and 67% (32/48) of HCC primary biopsies. The stepwise regression analyses indicated that the loss of RASSF1A expression was more frequent in patients who were hepatitis B virus surface antigen positive (HBsAg+) compared to those who were HBsAg(-), both in tumor and corresponding non-cancerous tissues. The wild-type (wt)-RASSF1A expression in the QGY-7703 cell line resulted in fewer and smaller clones, decreased xenograft tumor volume and weight, and G(1)/S arrest in vitro and in vivo. The wt-RASSF1A expression also decreased the cyclin D1 protein expression, which appeared to be at the level of post-transcriptional control. In addition, the wt-RASSF1A expression increased cell growth inhibition and the percentage of cells with sub-G(1) DNA content when the cells were treated with mitomycin.. RASSF1A is a tumor suppressor in HCC. The loss of RASSF1A expression may be related to HBsAg+ in hepatocarcinogenesis. Its inactivation may play an important role in the development of HCC.

Topics: Adult; Animals; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; China; Cyclin D1; DNA Replication; Female; Gene Expression Regulation, Neoplastic; Hepatitis B; Hepatitis B Surface Antigens; Humans; Liver Neoplasms; Logistic Models; Male; Mice; Mice, Nude; Mitomycin; Mutation; Odds Ratio; Risk Assessment; Risk Factors; Time Factors; Transfection; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

The initiation of endoplasmic reticulum (ER) stress has been suggested to play potential roles in hepatocarcinogenesis. However, many obstacles remain as to whether ER stress plays a role in carcinogenesis or tumoricide. This study sought to identify the signals that can serve as anticancer effectors in cells in response to ER stress. Tunicamycin (an N-glycosylation inhibitor) inhibited cell proliferation with IC(50) values of 0.19 and 0.62 microg/ml in hepatoma (Hep) 3B and HepG2 cells, respectively. It induced G1 arrest of the cell cycle in both cell lines. The anticancer mechanism of tunicamycin was investigated in Hep3B cells. Tunicamycin induced a rapid decline of cyclin D1 and cyclin A expression and an early increase of glucose-related protein (GRP) 78 and growth arrest and DNA damage-inducible transcription factor (GADD) 153 levels. Cyclin A was the most sensitive regulator to tunicamycin-triggered degradation mechanism. The association of p27(Kip1) with cyclin D1/cyclin-dependent kinase (Cdk) 4 was also increased by tunicamycin. The inhibition of GADD153 expression by transfection of GADD153 antisense did not modify tunicamycin-induced G1 arrest and cyclin/Cdk expressions. The knockdown of GRP78 expression by the siRNA transfection technique moderately increased tunicamycin-induced apoptosis but not the antiproliferative effect by sulforhodamine B assay. We suggest that tunicamycin induces G1 arrest through down-regulation of cyclins and Cdks, in which cyclin A is more susceptible to ER stress-triggered degradation mechanism in Hep3B cells. The increased association of p27(Kip1) with cyclin D1/Cdk4 may also contribute to tunicamycin-induced cell-cycle arrest. GADD153 and GRP78 play a minor role in tunicamycin-mediated antiproliferative effect, although GRP78 moderately inhibits apoptosis in Hep3B cells. These data provide evidence that cell-cycle regulators are susceptible factors in hepatocellular carcinoma (HCC) responsive to ER stress.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Molecular Chaperones; Transcription Factor CHOP; Tunicamycin

Fucoxanthin, a major carotenoid in brown sea algae, has recently been demonstrated by us to inhibit the proliferation of colon cancer cells, and this effect was associated with growth arrest. These results, taken together with previous studies with fucoxanthin, suggest that it may be useful in chemoprevention of other human malignancies. The present study was designed to evaluate the molecular mechanisms of fucoxanthin against hepatic cancer using the human hepatocarcinoma HepG2 cell line (HepG2). Fucoxanthin reduced the viability of HepG2 cells accompanied with the induction of cell cycle arrest during the G0/G1 phase at 25 microM. This concentration of fucoxanthin inhibited the phosphorylation of the retinoblastoma protein (Rb) at Serine 780 (Ser780) position 18 h after treatment. The kinase activity of cyclin D and cdk4 complex, responsible for the phosphorylation of Rb Ser780 site, was down-regulated 18 h after the treatment. Western blotting analysis revealed that the expression of cyclin D-type protein was suppressed by treatment of fucoxanthin. This reduction was partially blocked by concurrent treatment with the proteasome inhibitor MG132, indicating the involvement of the proteasome-mediated degradation. In addition, RT-PCR analysis revealed that fucoxanthin also appeared to repress cyclin D mRNA. Thus, both the protein degradation and transcriptional repression seem to be responsible for suppressed cyclin D level in fucoxanthin-treated HepG2 cells which may be related to the antitumorgenic activity.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D; Cyclin D1; Cyclin D3; Cyclins; Dose-Response Relationship, Drug; Down-Regulation; G1 Phase; Gene Expression; Humans; Hydrolysis; Liver Neoplasms; Molecular Structure; Phosphorylation; Resting Phase, Cell Cycle; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Xanthophylls

Hepatocellular carcinoma (HCC) is a multi-factorial and multi-step process. However, the molecular mechanisms, which play a pivotal role during progressive development of HCC, are not known. Accordingly Sprague-Dawley rats were administered diethylnitrosamine (DEN) for one to three months in order to understand the molecular alterations during progressive development of liver tumor. In this study involvement of G1/S regulatory proteins, MAP kinases and cell survival factors were analyzed using RT-PCR, western blotting and in vitro kinase assays. The data showed overexpression of cyclin D1 and increased expression and activation of ERK1/2, p38 kinase and JNK1/2 with progression of tumor suggesting that MAP kinases play an important role during tumorigenesis. These molecular alterations were supported by Akt upregulation and increase in the levels of inactive GSK3beta with progression of liver tumor. Further, p21-actived kinase1 (Pak1) was found to be upregulated with tumor progression, which is a novel observation during progressive liver carcinogenesis. These results indicate that elevated levels of all the three MAP kinases (ERK1/2, p38 and JNK1/2), Akt/GSK3beta and Pak1 are associated with cyclin D1 upregulation, which helps in the disruption of the G1/S regulatory point of the cell cycle and leads to abnormal cell proliferation during progressive hepatocarcinogenesis.

Topics: Animals; Carcinogens; Carcinoma, Hepatocellular; Cyclin D1; Diethylnitrosamine; Disease Progression; Gene Expression Regulation; Liver Neoplasms; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; p21-Activated Kinases; p38 Mitogen-Activated Protein Kinases; Proliferating Cell Nuclear Antigen; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley

To study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma.. Paraffin-embedded tissue samples of 25 patients (18 males and 7 females) with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method (avidin-biotin-peroxidase) for detection of p53, cyclinD1, RB1, c-fos and N-ras proteins.. Six (24%), 5 (20%), 12 (48%) and 2 samples (8%) were positive for p53, cyclinD1, C-fos and N-ras expression, respectively. Twenty-two (88%) samples had alterations in the G1 cell-cycle checkpoint protein expression (RB1 or cyclinD1). P53 positive samples showed a higher (9 times) risk of being positive for RB1 protein than p53 negative samples. Loss of expression of RB1 in association with p53 over-expression was observed in 4 (66.7%) of 6 samples. Loss of expression of RB1 was seen in all cyclinD1 positive, 20 (90.9%) N-ras negative, and 11 (50%) C-fos positive samples, respectively. CyclinD1 positive samples showed a higher (2.85 and 4.75 times) risk of being positive for c-fos and N-ras expression than cyclinD1 negative samples.. The expression of p53, RB1 and c-fos genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cyclin D1; Female; Genes, ras; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Proto-Oncogene Proteins c-fos; Retinoblastoma Protein; Tumor Suppressor Protein p53

Hepatocellular carcinoma (HCC) is a major public health concern because of the absence of early diagnosis and effective treatments. Efficient diagnosis modalities and therapies to treat HCC are needed. Kruppel-like factor (KLF) family members, such as KLF6, are involved in cell proliferation and differentiation. KLF6 is inactivated in solid tumors, which may contribute to pathogenesis. However, KLF6 status in HCC is controversial. Thus, we undertook the characterization of KLF6 expression and function in HCC and HCC-derived cell lines. We found that HCC, HepG2 and HuH7 cells expressed KLF6 messenger ribonucleic acid and protein. Next, using RNA interference, we demonstrated that inhibiting KLF6 expression in vitro strongly impaired cell proliferation-induced G1-phase arrest, inhibited cyclin-dependent kinase 4 and cyclin D1 expression, and subsequent retinoblastoma phosphorylation. Finally, KLF6 silencing caused p53 upregulation and inhibited Bcl-xL expression, to induce cell death by apoptosis. Taken together, these data demonstrated that KLF6 is essential for HCC-derived cells to evade apoptosis.

Topics: Apoptosis; Base Sequence; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Liver Neoplasms; Molecular Sequence Data; Mutation; Phosphorylation; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Sequence Homology, Nucleic Acid

To investigate the effect of ceramide on the cell cycle in human hepatocarcinoma Bel7402 cells. Possible molecular mechanisms were explored.. [3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, plasmid transfection, reporter assay, FACS and Western blotting analyses were employed to investigate the effect and the related molecular mechanisms of C2-ceramide on the cell cycle of Bel7402 cells.. C2-ceramide was found to inhibit the growth of Bel7402 cells by inducing cell cycle arrest. During the process, the expression of p21 protein increased, while that of cyclinD1, phospho-ERK1/2 and c-myc decreased. Furthermore, the level of CDK7 was downregulated, while the transcriptional activity of PPARgamma was upregulated. Addition of GW9662, which is a PPARgamma specific antagonist, could reserve the modulation action on CDK7.. Our results support the hypothesis that cell cycle arrest induced by C2-ceramide may be mediated via accumulation of p21 and reduction of cyclinD1 and CDK7, at least partly, through PPARgamma activation. The ERK signaling pathway was involved in this process.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Humans; Liver Neoplasms; PPAR gamma; Signal Transduction; Sphingosine

Menatetrenone, a vitamin K2 analogue, plays an important role in the production of blood coagulation factors. Menatetrenone has also bee shown to have antineoplastic effects against several cancer cell lines including hepatocellular carcinoma (HCC) cells. However, the mechanisms by which vitamin K2 inhibits HCC cell growth have not bee fully clarified, and we therefore investigated the molecular basis of vitamin K2-induced growth inhibition of HCC cells.. HCC cells were treated with vitamin K2 and the expression of several growth-related genes including cyclin-dependent kinase inhibitors and cyclin D1 was examined at the mRNA and protein levels. A reporter gene assay of the cyclin D1 promoter was done under vitamin K2 treatment. The regulation of nuclear factor kappaB (NF-kappaB) activation was investigated by a NF-kappaB reporter gene assay, an electrophoretic mobility shift assay, a Western blot for phosphorylated IkappaB, and an in vitro kinase assay for IkappaB kinase (IKK). We also examined the effect of vitamin K2 on the growth of HCC cells transfected with p65 or cyclin D1.. Vitamin K2 inhibited cyclin D1 mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-kappaB binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)-, TNF-alpha-, and interleukin (IL)-1-induced activation of NF-kappaB binding and transactivation. Concomitant with the suppression of NF-kappaB activation, vitamin K2 also inhibited the phosphorylation and degradation of IkappaBalpha and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1 and p65 became resistant to vitamin K2 treatment.. Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1 expression through the IKK/IkappaB/NF-kappaB pathway and might therefore be useful for treatment of HCC.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Separation; Cyclin D1; Electrophoretic Mobility Shift Assay; Enzyme Activation; Flow Cytometry; Humans; I-kappa B Kinase; Liver Neoplasms; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transfection; Vitamin K 2

Signal regulatory protein alpha1 (Sirpalpha1) is a member of Sirps families containing four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) domains in the cytoplasm of and an activated substrate of receptor tyrosine kinase (RTK), that negatively regulates the RTK-dependent cell proliferating signal transduction pathway. Previously we found that Sirpalpha1 was closely associated with the occurrence and development of hepatocellular carcinoma (HCC) as well as liver regeneration. Since it is unclear about the regulatory mechanisms, we established the cell line transfected Sirpalpha1 gene and preliminarily clarified the mechanisms by which Sirpalpha1 negatively regulates the carcinogenesis and development of HCC.. Liver cancer Sk-Hep1 cell was respectively transfected with plasmids of pLXSN, pLXSN-Sirpalpha1 and pLXSN-Sirpalpha1delta4Y2, screened with the drug of G418 (1200 microg/ml), and various transfected Sk-Hep1 cell lines were obtained. The protein expressions of P65, P50, IkappaBalpha, cyclin D1 and Fas in various Sk-Hep1 cell lines were determined by Western blotting, and P65 and P50 were localized by the immunofluorescence technique.. Sirpalpha1 could significantly upregulate the protein expression of IkappaBalpha (vs. other cell lines, P<0.05) in the Sk-Hep1 cell, and downregulate the protein expressions of P65, P50 and cyclin D1 (vs. other cell lines, P<0.05) in the Sk-Hep1 cell. P65 protein expression was mainly localized in the cytoplasm in the pLXSN Sk-Hep1 cell, and in the nucleus of the Sk-Hep1 cell with mutant Sirpalpha1delta4Y2, but in nucleus of the Sk-Hep1 cell with wild Sirpalpha1. P50 protein expression was localized in the cytoplasm and nucleus of the pLXSN Sk-Hep1 cell, but in the nucleus of the Sk-Hep1 cell with wild Sirpalpha1 and mutant Sirpalpha1delta4Y2 plasmid.. Sirpalpha1 might negatively regulate and control the abnormal proliferation of liver cancer cells by influencing the protein content and localization of nuclear factor-kappa B, then influence the expression of cyclins such as cyclin D1 in the signal transduction pathway. It may be one of the important mechanisms by which Sirpalpha1 negatively regulates the carcinogenesis and development of HCC.

Topics: Antigens, Differentiation; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; I-kappa B Proteins; Liver Neoplasms; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Receptors, Immunologic; S Phase; Signal Transduction; Transcription Factor RelA

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated and regulates cell growth and survival of various cancer cells. We investigated the anti-tumor effect of AG490, a Janus kinase 2 specific inhibitor, inhuman hepatoma cells.. Effects of AG490 on STAT3 activation, on cell-growth and survival, and on the expression of cell-cycle- and apoptosis-related proteins were evaluated in Huh-1, Huh-7, HepG2 and Hep3B cells. Next, whether AG490 renders hepatoma cells susceptible to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was examined in vitro and in vivo.. Constitutively activated STAT3 through tyrosine phosphorylation was detected in all hepatoma cells. AG490 inhibited the phosphorylation of STAT3 and its activity. AG490 induced cell cycle arrest in Huh-1, Huh-7 and HepG2 through cyclin D1 downregulation, and induced marked apoptosis in Hep3B. AG490 downregulated at least one of the anti-apoptotic proteins, Bcl-xL, survivin or XIAP in all hepatoma cells. AG490 sensitized Huh-1, Huh-7 and HepG2 to TRAIL-induced apoptosis in vitro. Intraperitoneal injection of AG490, the combination of AG490 and TRAIL more greatly, repressed the growth of subcutaneous Huh-7 tumors in athymic mice.. Abrogation of constitutive activation of STAT3 by AG490 enhances the anti-tumor activity of TRAIL against human hepatoma cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Humans; Janus Kinase 2; Liver Neoplasms; Mice; Mice, Nude; Phosphorylation; STAT3 Transcription Factor; TNF-Related Apoptosis-Inducing Ligand; Tyrphostins; Xenograft Model Antitumor Assays

Obesity serves as an important risk factor for incidences of both cirrhotic and non-cirrhotic hepatocellular carcinoma (HCC), which is the third leading cause of cancer death worldwide. Leptin, the obesity biomarker molecule secreted systemically by body fat mass and locally by activated hepatic stellate cells, is proposed to play a certain role in HCC growth. Here, we show both proliferative and anti-apoptotic effects of leptin in HCC cells. Leptin stimulated cyclin D1 promoter activity to increase cyclin D1 protein expression, which accelerated the cell cycle progression. The reduced ratio between anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) Bcl-2 family proteins by transforming growth factor (TGF)-beta 1 caused HCC cells degradation of poly(ADP-ribose) polymerase and consequential apoptosis; whereas, leptin protected cells from apoptosis by reversing TGF-beta 1-reduced Bcl-2/Bax ratio as a result of down-regulating Bax. Any inhibitor specific for Janus kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) blocked these leptin functions. When intrahepatocytic JAK2 was activated by leptin, the active JAK2 afterward triggered a signaling cascade involving activations of PI3K/Akt and MEK/ERK1/2 in order of occurrence. As yet, in most cases, the crosstalks among signaling pathways primarily studied in diverse cancer cell types for mediating somatotropic effect of leptin are not well clarified and seem to be cell-type dependent. For the first time, our results demonstrate the direct effects of leptin on HCC growth and define its signal pathway with a crosstalking JAK2-PI3K/Akt-MEK/ERK1/2 connection. The identified hierarchy of intrahepatocytic leptin signaling pathway provides a clear basis potentially beneficial to make accurate and effectual strategies for facing both cirrhotic and non-cirrhotic liver carcinogenesis.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cyclin D1; Down-Regulation; Humans; Janus Kinase 2; Leptin; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, Leptin; Transforming Growth Factor beta1; Up-Regulation

The identification of the specific molecular targets, which underlie liver carcinogenesis is essential for the establishment of an effective strategy for the prevention and/or treatment of hepatocellular carcinomas (HCCs). We previously found that a malfunction of RXRalpha due to its aberrant phosphorylation was associated with the development of HCCs. However, it has remained unclear whether the abnormalities in the expression of RXRalpha or the other retinoid receptors play a role in the early stage of liver carcinogenesis. The present study was designed to determine whether alterations in the expression of RXRalpha and the other retinoid receptors RARalpha and RARbeta are involved in hepatocarcinogenesis using a 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-induced rat liver carcinogenesis model. We found that immunohistochemical expression of RXRalpha was decreased in liver cell tumors (HCCs and adenoma) and glutathione S-transferase placental form (GST-P)-positive foci, which is a precancerous lesion of HCC, when compared with the non-cancerous tissues. Western blot and RT-PCR analyses revealed a progressive decrease in the expression levels of RXRalpha, RARalpha, and RARbeta proteins and their mRNAs in 3'-MeDAB-induced HCCs and their surrounding tissues, when compared with the normal liver tissues from the control group. Moreover, the expression level of beta-catenin, the heterodimeric partner for both RXRalpha and RARalpha, was immunohistochemically observed in the cytoplasm and, in some cases, in the nucleus of HCC cells. The nuclear expression of cyclin D1, the downstream target molecule of beta-catenin, was also increased in HCC cells when compared with their adjacent normal appearing tissues. Our findings suggest that loss of retinoid receptors, especially RXRalpha, plays a critical role in the chemically-induced rat liver carcinogenesis and this might be associated with the activation of beta-catenin-related signaling pathway.

Topics: Adenoma; Animals; beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cell Nucleus; Cyclin D1; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Immunoenzyme Techniques; Liver Neoplasms, Experimental; Male; Methyldimethylaminoazobenzene; Rats; Rats, Inbred F344; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-knockout (Mdr2-KO) mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling showed that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC data sets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time reverse transcription-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We show that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in most dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA, level. These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of beta-catenin activation. In conclusion, the Mdr2-KO mouse may serve as a model for beta-catenin-negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; beta Catenin; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Gene Expression Profiling; Genes, Tumor Suppressor; Hepatocytes; Liver Neoplasms; Mice; Mice, Knockout

beta-Catenin, the chief oncogenic component of the canonical Wnt pathway, is known to be involved in a variety of cancers, including hepatocellular carcinoma (HCC). Although the mechanism of beta-catenin activation in HCC is multifactorial, it is indisputably implicated at various stages of hepatocarcinogenesis, making it an attractive therapeutic target. Here we investigate the effect of small interfering RNA-mediated beta-catenin knockdown on the growth and survival of human hepatoma cell lines with (HepG2) and without (Hep3B) beta-catenin mutations. Transfection of HepG2 and Hep3B cells with human beta-catenin (CTNNB1) small interfering RNA resulted in a significant beta-catenin decrease, as confirmed by Western blot analyses and immunofluorescence, also leading to decreased expression of known target genes such as cyclin D1 and glutamine synthetase. The decrease in beta-catenin activity was confirmed by TOPflash reporter luciferase assay. The functional impact of diminished beta-catenin was exhibited as temporal decrease in tumor cell viability by the MTT assay. A concomitant decrease in tumor cell proliferation was also evident with [(3)H]thymidine incorporation and verified with soft agar assays. Thus, beta-catenin is essential for the survival and growth of hepatoma cells independent of mutations in the beta-catenin gene and provide a proof of principle for the significance of the therapeutic inhibition of beta-catenin in HCC.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Fluorescent Antibody Technique; Glutamate-Ammonia Ligase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Liver Neoplasms; Mutation; RNA, Small Interfering

To explore the possible relationship between the expressions of macrophage migration inhibitor factor (MIF), cyclin D1, cyclin-dependent kinase 4 (CDK4), phosphorylated-retinoblastoma susceptibility gene product Rb protein (phospho-Rb) and the development of hepatocellular carcinoma (HCC).. 93 HCC tissues and 5 normal liver tissues were used to investigate the expressions of MIF, cyclin D1, CDK4 and phospho-Rb by tissue microarray and immunohistochemistry methods.. The expression rates of MIF, cyclin D1, CDK4 and phospho-Rb in the HCC tissues were 71%, 41%, 82% and 14% respectively, and in the normal liver tissues, they were 0%, 0%, 80% and 20% respectively. The expression rates of MIF and cyclin D1 were significantly different between the tumor and the normal liver tissues and the expression rates of CDK4 and phospho-Rb were not significantly different between the tumor and the normal liver tissues. The rate difference (69% versus 48%) of MIF expression between the larger tumors (> 3.5 cm) and the smaller tumors (< 3.5 cm) was of statistical significance (P < 0.01). The expression rate (62%) of cyclin D1 in the tumors with metastasis was significantly higher than the expression rate (35%) in the tumors without metastasis (P < 0.05). MIF expression was positively correlated with cyclin D1 expression in the tumor tissues (P < 0.01). CDK4 and phospho-Rb expressions were not significantly associated with the tumor sizes and metastasis status.. Our results indicate that MIF and cyclin D1 might be related to the growth and metastasis of HCC.

Topics: Adolescent; Adult; Aged; Carcinoma, Hepatocellular; Child; Cyclin D1; Female; Humans; Liver Neoplasms; Macrophage Migration-Inhibitory Factors; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Young Adult

Hepatoblastoma (HBL), a major childhood malignant neoplasm, represents the most frequent malignant liver tumor in childhood. Recent reports have shown the CTNNB1 coding beta-catenin protein to be frequently mutated or deleted at hot-spot regions involving exon 3 in HBL. We investigated the genetic alterations of the CTNNB1 coding beta-catenin protein and expression of several genes downstream of Wnt signals in 4 benign and 17 malignant pediatric liver tumors (PLTs) consisting of 15 HBL, 1 hepatocellular carcinoma, and 1 hepatic immature sarcoma. Of 17 malignant PLTs, 10 (56%) revealed pathogenic alterations of the CTNNB1 gene, including 4 with missense mutations at codons 28, 32, 34 or 44, and 6 with interstitial deletions that partially or totally affected exon 3. All 6 deletions were in-frame deletions without a frame shift. The high frequency without any correlation to histological type indicates that the CTNNB1 gene alteration is a crucial event in the tumorigenesis of malignant PLTs. The immunohistochemical studies in 17 malignant PLTs demonstrated the nuclear/cytoplasmic accumulation of beta-catenin to be positive in all tumor specimens except for one hepatic sarcoma. A histological examination revealed all HBL cases involving tumors without detectable CTNNB1 gene alterations to show high expression of beta-catenin, thus indicating the accumulation of beta-catenin to be a common event in malignant PLTs, including HBL and hepatocellular carcinoma. Among the Wnt signal genes downstream of beta-catenin, E-cadherin is expressed in all malignant PLTs, while cyclin D1 expression was significantly detected in malignant PLTs with an advanced stage of disease. An immunohistological examination of nuclear accumulation of beta-catenin may thus be useful for diagnosing malignant PLTs. On the other hand, the expression of cyclin D1, a gene downstream of beta-catenin, might play a role in tumor progression.

Topics: beta Catenin; Cadherins; Carcinoma, Hepatocellular; Child; Child, Preschool; Cyclin D1; DNA Mutational Analysis; DNA, Neoplasm; Female; Hepatoblastoma; Humans; Immunohistochemistry; Infant; Liver Neoplasms; Male; Mutation; Prognosis

To understand the role of P120ctn in E-cadherin-mediated cell-cell adhesion and signaling as well as in hepatoma cell biological function.. We stably overexpressed p120ctn isoform 3A in BEL-7404 human hepatoma cells and studied the effect of p120ctn on beta-catenin and E-cadherin binding as well as p120ctn and beta-catenin subcellular localization using immunoprecipitation, Western blotting and confocal microscopy. We also investigated the inhibitory effect of p120ctn transfection on the expression of apoptotic protein survivin survivin and cell cycle regulator cyclin D1 in the cells.. Western blotting indicated that p120ctn expression increased after cells were transfected with p120ctn isoform 3A. The protein was located mainly at membrane under immunofluorescent microscope. Beta-catenin nuclear expression was reduced after overexpression of p120ctn isoform 3A. The p120ctn-E-cadherin binding increased after transfection of p120ctn isoform 3A. Furthermore, overexpression of p120ctn down regulated the expression of apoptotic protein survivin and cell cycle regulator cyclin D1. These effects led to reduction of cell proliferation.. Our results indicate that p120ctn plays an important role in regulating the formation of E-cadherin and -catenin complex, cell apoptosis, cell cycle and cancer cell biological function.

Topics: Apoptosis; beta Catenin; Blotting, Western; Cadherins; Carcinoma, Hepatocellular; Catenins; Cell Adhesion Molecules; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Delta Catenin; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Microscopy, Confocal; Microtubule-Associated Proteins; Neoplasm Proteins; Phosphoproteins; Protein Binding; Signal Transduction; Survivin; Transfection

TNFSF14/LIGHT is a member of the tumor necrosis factor superfamily that binds to lymphotoxin-beta receptor (LTbetaR) to induce cell death via caspase-dependent and caspase-independent pathways. It has been shown that cellular inhibitor of apoptosis protein-1 inhibits cell death by binding to LTbetaR-TRAF2/TRAF3 complexes and caspases. In this study, we found that both Kaposi's sarcoma-associated herpesvirus K7 (KSHV-K7), a viral inhibitor of apoptosis protein, and the structurally related protein survivin-DeltaEx3 could inhibit LTbetaR-mediated caspase-3 activation. However, only survivin-DeltaEx3 could protect cells from LTbetaR-mediated cell death. The differential protective effects of survivin-DeltaEx3 and KSHV-K7 can be attributed to the fact that survivin-DeltaEx3, but not KSHV-K7, is able to maintain mitochondrial membrane potential and inhibit second mitochondria-derived activator of caspase/DIABLO release. Moreover, survivin-DeltaEx3 is able to inhibit production of reactive oxygen species and can translocate from nucleus to cytosol to associate with apoptosis signal-regulating kinase 1 after activation of LTbetaR. Furthermore, survivin-DeltaEx3 protects LTbetaR-mediated cell death in caspase-3-deficient MCF-7 cells. Thus, survivin-DeltaEx3 is able to regulate both caspase-dependent and caspase-independent pathways, whereas inhibition of caspase-independent pathway is both sufficient and necessary for its protective effect on LTbetaR-mediated cell death.

Topics: Antibodies, Monoclonal; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Cyclin D1; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Interferon-gamma; Intracellular Signaling Peptides and Proteins; Lymphotoxin beta Receptor; MAP Kinase Kinase Kinase 5; Membrane Proteins; Microtubule-Associated Proteins; Mitochondrial Proteins; Neoplasm Proteins; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Signal Transduction; Survivin; Transfection; Tumor Necrosis Factor Ligand Superfamily Member 14; Tumor Necrosis Factor-alpha; Viral Proteins

To investigate the role of Wnt/beta-catenin signaling transduction pathway in rat hepatocarcinogenesis.. The mRNAs of Wnt1, beta-catenin, APC, cyclin D1 and c-myc genes were amplified by using of semiquantitative reverse transcription polymerase chain reaction (RT-PCR) from normal rat livers, atypical hyperplasia livers and hepatoma tissues, respectively. Then the proteins expression of beta-catenin, APC and cyclin D1 was examined by immunohistochemical staining.. In normal rat livers, the mRNAs of Wnt1, cyclin D1 and c-myc genes were not detected and only beta-catenin protein was observed to have low expression at cellular membrane. However, 14 weeks after cancer induction in atypical hyperplasia livers, beta-catenin protein and APC protein were accumulated in cytoplasm. Meanwhile, cyclin D1 protein was detected in cytoplasm and/or nucleus in some cells. 16 weeks after cancer induction in hepatoma tissues, the mRNAs and protein expression of beta-catenin, APC, cyclin D1 and c-myc genes were detected except Wnt1.. The activation of Wnt/beta-catenin signaling transduction pathway might be one of the reasons for rat hepatocarcinogenesis.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cyclin D1; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Genes, myc; Hyperplasia; Immunohistochemistry; Liver; Liver Neoplasms, Experimental; Male; Rats; RNA, Messenger; Signal Transduction; Wnt Proteins

The phospho-Ser/Thr-Pro specific prolyl-isomerase PIN1 is over-expressed in more than 50% of hepatocellular carcinomas (HCCs). To investigate its potential oncogenicity, we over-expressed PIN1 in a non-transformed human liver cell line MIHA. This resulted in up-regulation of beta-catenin and cyclin D1, leading to anchorage-independent growth in soft agar and tumorigenicity in nude mice. To further validate the role of PIN1 in hepatocarcinogenesis, PIN was suppressed by RNA interference (siRNA) in the HCC cell line PLC/PRF/5. siRNA-PIN1 transfection of PLC/PRF/5 cells led to repression of PIN1 expression, resulting in decreased levels of beta-catenin and cyclin D1. siRNA-PIN1 transfectants showed lower cell proliferation rates, reduced colony formation, and retarded cell cycle progression, with an increase in cells residing in G0/G1. Furthermore, soft agar colony formation was depressed, and tumorigenicity in nude mice was abrogated. These findings implicate PIN1 expression as an important step in hepatic carcinogenesis.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; RNA Interference; RNA, Neoplasm; Transfection; Up-Regulation

C75, a well-known fatty acid synthase (FAS) inhibitor, has been shown to possess potent anti-cancer activity in vitro and in vivo. In this study, we reveal that C75 is a cell cycle arrest inducer and explore the potential mechanisms for this effect in hepatocellular carcinoma (HCC) cell lines with abundant FAS expression: HepG2 and SMMC7721 cells with wt-p53, and Hep3B cells with null p53. The results showed FAS protein expression and basal activity levels were higher in HepG2 cells than in the other two HCC cell lines. Treatment with C75 inhibited FAS activity within 30 min of administration and induced G(2) phase arrest accompanied by p53 overexpression in HepG2 and SMMC7721 cells. By contrast, C75 triggered G(1) phase arrest in Hep3B cells, and RNA interference targeting p53 did not attenuate C75-induced G(2) arrest in HepG2 cells. Similarly, p53 overexpression via p53 plasmid transfection did not affect C75-induced G(1) phase arrest in Hep3B cells. However, we observed a clear correlation between p38 MAPK activation triggered by C75 and the induction of cell cycle arrest in all three HCC cells. Furthermore, treatment with the p38 MAPK inhibitor SB203580 reduced p38 MAPK activity and cell cycle arrest, and also partially restored cyclin A, cyclin B1, cyclin D1 and p21 protein levels. Collectively, it was p38 MAPK but not p53 involved in C75-mediated tumor cell growth arrest in HCC cells.

Topics: 4-Butyrolactone; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin A; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; fas Receptor; Fatty Acid Synthases; Flow Cytometry; G2 Phase; Humans; Immunoprecipitation; Liver Neoplasms; p38 Mitogen-Activated Protein Kinases; Plasmids; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Cyclin D1 is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis Bx (HBx) protein is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Here we show that HBx up-regulates cyclin D1 and that this process is mediated by the NF-kappaB2(p52)/BCL-3 complex. Our experiments indicate that HBx up-regulates BCL-3 in the mRNA level, which subsequently results in the up-regulation of the NF-kappaB2(p52)/BCL-3 complex in the nucleus. Moreover, impaired HBx-mediated BCL-3 up-regulation by small interfering RNA for BCL-3 reduced HBx-mediated cyclin D1 up-regulation. Down-regulation of the HBx protein level by p53 also reduced HBx-mediated cyclin D1 up-regulation. From these results, we conclude that the up-regulation of cyclin D1 by HBx is mediated by the up-regulation of NF-kappaB2(p52)/BCL-3 in the nucleus. This HBx-mediated-cyclin D1 up-regulation might play an important role in the HBx-mediated HCC development and progression.

Topics: Animals; B-Cell Lymphoma 3 Protein; Binding Sites; Carcinoma, Hepatocellular; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Neoplasms; NF-kappa B p52 Subunit; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins; Trans-Activators; Transcription Factors; Up-Regulation; Viral Regulatory and Accessory Proteins

The outcome of surgical treatment of hepatocellular carcinoma (HCC) could be improved by applying patient selection criteria based on tumoral aggressiveness. Here we analyzed the prognostic role of the expression of several genes involved in cell-cycle regulation in a group of patients with HCC.. We retrospectively studied 93 patients (67 transplanted and 26 resections) treated between 1996 and 2000. In micro-thick sections from paraffin-embedded tumoral tissues, the expression of p53, pRb, p16, and cyclin D1 was analyzed. A logistic regression model was used to detect factors related to vascular invasion. A Cox regression model was applied to identify pathologic and molecular factors with the capacity to predict the recurrence of HCC.. Only tumor size>3 cm (odds ratio [OR]: 3.4; 95% CI: 1.2-9.9; P=0.019) and pRb expression (OR: 4.1; 95% CI: 1.02-17; P=0.053) were associated with an increased risk of vascular invasion. The regression model applied to the group of transplanted patients showed three factors that were independently related to recurrence: vascular invasion (OR 7.5; 95% CI: 1.1-51.8; P=0.039); pRb expression (OR: 11; 95% CI: 1.2-96.9; P=0.03); and p16 expression (OR: 69.7; 95% CI: 5.1-9448; P=0.001). In the group of resected patients, pRb expression was associated with higher risk of recurrence only in the univariate analysis (P=0.037). The multivariate analysis showed tumor size>3 cm (OR: 57.5; 95% CI: 1.1-51.8; P=0.039) and vascular invasion (OR: 6.1; 95% CI: 1.05-35.3; P=0.044) to be significantly associated with recurrence.. Of the molecular factors studied, only pRb expression was useful as a predictive factor of vascular invasion in patients with HCC, and also of recurrence in transplanted patients with this carcinoma. pRb expression may be relevant to consider when selecting patients for resection and when identifying transplanted patients with a high risk of recurrence.

Topics: Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Cycle; Cyclin D1; Female; Genes, p16; Humans; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Recurrence; Retinoblastoma Protein; Retrospective Studies; Treatment Outcome; Tumor Suppressor Protein p53

Cyclin D1 overexpression is a frequent change in hepatocellular carcinomas (HCCs). Our present study demonstrated that cyclin D1 overexpression with abundant cyclin E, cdk4, cdk2, and p27Kip1 (p27) occurred in neoplastic hepatocytes from the early stage of mouse hepatocarcinogenesis. While cyclin D1 expression was mainly found in the cytoplasm of the tumor cells, it shifted to the nucleus in association with cell proliferation after the animals were subjected to a partial hepatectomy (PH), and then returned once more to the cytoplasm when the cells became quiescent. Inhibition of PI3 kinase (PI3K) by Ly294002 in mouse HCC cells in vitro suppressed the nuclear shift of cyclin D1 as well as cell proliferation, while PI3K activation by PTEN suppression failed to induce nuclear shift of cyclin D1, suggesting that PI3K activation is essential but not sufficient for the cyclin D1 nuclear shift. While MEK-ERK1/2 inhibition by PD98059 and mTOR inhibition by rapamycin affected the cyclin D1 nuclear shift and cell proliferation to a lesser extent, both these inhibitors reduced cyclin D1 levels. Finally, although p27, cdk4 and calmodulin (CaM) were detected in the cyclin D1 immunoprecipitates from both quiescent and proliferating HCC cells, Hsc70 and SSeCKS were detected only in the immunoprecipitate from quiescent cells, and p21Waf1/Cip1 (p21) was detected only in that from proliferating cells, suggesting that the cyclin D1 complex is different in quiescent and proliferating cells. These observations indicate that the nuclear/cytoplasmic localization of cyclin D1 plays an important role in the proliferation/quiescence of neoplastic hepatocytes.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cytoplasm; Extracellular Signal-Regulated MAP Kinases; Hepatocytes; Liver Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; TOR Serine-Threonine Kinases

Cyclins overexpress in various tumors, but its expression in hepatocellular carcinoma (HCC) and its correlation to cell proliferation and apoptosis are unclear. This study was to determine the expression of Cyclins, proliferating cell nuclear antigen (PCNA), and cell apoptosis in HCC, and to analyze their interrelations.. Tissue microarray technology and SP immunohistochemistry were used to detect expressions of Cyclins A, B1, D1, E, and PCNA in 122 specimens of HCC. In situ terminal deoxyribonucleotide transferase labeling was used to detect cell apoptosis.. In the 122 HCC specimens, positive rate of Cyclin A was 50.0%, of Cyclin B1 was 47.5%, of Cyclin D1 was 42.6%, of Cyclin E was 35.2%. Cyclins levels were significantly higher in HCC tissues of grade II, III, and IV than in HCC tissues of grade I(P 0.05). Densities of apoptotic cells were significantly lower in HCC tissues of grade II, III, and IV than in HCC tissues of grade I(P 0.05); PCNA scores were significantly higher in HCC tissues of grade II, III, and IV than in HCC tissues of grade I(P 0.01). The poorer differentiation, the lower density of apoptotic cells in HCC, the higher PCNA score in HCC. Cyclins levels were negatively related to density of apoptotic cells (r=-0.686, P < 0.01), and positively related to PCNA score (r=0.599, P < 0.01); density of apoptotic cells was negatively related to PCNA score (r=-0.701, P < 0.01).. Cyclins overexpress in HCC, which may shorten tumor cell cycle phase, accelerate cell proliferation and decrease apoptosis, and result in increased malignant phenotypes.

Topics: Adult; Aged; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cyclin A; Cyclin B; Cyclin B1; Cyclin D1; Cyclin E; Cyclins; Female; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Proliferating Cell Nuclear Antigen

While the Wnt pathway has been widely implicated in hepatocarcinogenesis, the role of cyclin D1 as a direct downstream target gene of beta-catenin-lymphoid enhancer factor-1 (LEF-1)/T-cell factor (TCF) signaling is controversely discussed.. By immunohistochemical analyses we studied the subcellular localization of LEF-1/TCF and cyclin D1 in 162 hepatocellular carcinoma (HCC). Single- and double-label imaging by brightfield and confocal laser scanning microscopy was quantitated and correlated with beta-catenin, the Ki67(+) proliferation fraction (PF), tumor size, grade, the Okuda stage and patient survival.. The frequency of nuclear cyclin D1 expression was 28% and closely correlated with LEF-1/TCF (P<0.0001) and the Ki67(+) PF (P=0.03). Nuclear LEF-1/TCF expression was observed in 52% of all cases, but was also present in 42% of cyclin D1(-) cases. Nuclear beta-catenin was identified in 37% of all HCCs and correlated with LEF-1/TCF (P=0.04). The expression of cyclin D1, LEF-1/TCF or beta-catenin did not correlate with other clinico-pathological data.. A large proportion of HCCs does not appear to be linked to a deregulation of cyclin D1. However, the coordinated expression of cyclin D1 and LEF-1/TCF in some cases suggests the role of cyclin D1 as a Wnt target gene in a subset of HCCs.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; beta Catenin; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Nucleus; Child; Cyclin D1; Cytoskeletal Proteins; DNA-Binding Proteins; Female; Fluorescent Antibody Technique; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Male; Microscopy, Confocal; Middle Aged; Survival Rate; Trans-Activators; Transcription Factors

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; L-Lactate Dehydrogenase; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vitamin K; Vitamin K 2; Vitamin K 3; Xenograft Model Antitumor Assays

The aim of this study was to evaluate hepatocellular carcinoma (HCC) using a combination of extracellular signal regulated kinase (ERK) and other markers related to hepatocyte growth factor (HGF) in tumor tissue.. Using specimens from 30 hepatocellular carcinoma patients operated on in our department from 2002 to 2003, we evaluated expression levels of HGF, c-Met, ERK, and cyclin D1 by Western blot.. Expression levels of ERK and cyclin D1 proteins were significantly higher in patients with less well-differentiated type tumors by histologic examination or the presence of intrahepatic metastasis (IM). ERK expression in tumor tissue significantly correlated with both tumor size (p=0.0017, R(2)=0.355) and serum levels of HGF (p=0.0247, R(2)=0.218). Nontumor tissue level of cyclin D1 was significantly higher in patients with poor liver function (p=0.047). In patients with higher expression of ERK in tumor tissue compared with nontumor tissue, the histologic finding was more progressed; but a similar tendency was not observed for cyclin D1. In patients with overexpression of HGF and c-Met, the expression level of ERK was significantly higher, but cyclin D1 expression was not. The detected level of cyclin D1 was significantly higher in patients with overexpressed ERK in tumor tissue. Values of ERK and c-Met were correlated, and IM presence was detected more frequently in patients with high expression of ERK and c-Met protein. Even after complete removal of visible IM tumor, recurrence tumors were detected within 6 months in 7 patients with high expressions of both ERK and c-Met protein.. Combination study of tumor expression of ERK might be useful to estimate the properties of hepatocellular carcinoma, especially for the presence of IM.

Topics: Aged; Blotting, Western; Carcinoma, Hepatocellular; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Male; Proto-Oncogene Proteins c-met

To investigate the effect of antisense cDNA of cyclin D1 on the cyclin D1 gene expression and cell proliferation of human hepatocarcinoma HepG2 cells in vitro.. Plasmids containing cyclin D1 antisense cDNA were constructed and transfected into HepG2 cells. Their effects on cell proliferation were examined by MTT method, RT-PCR, immunohistochemical means, and flow cytometry.. Cyclin D1 antisense cDNA significantly inhibited the growth of HepG2 cells. The inhibition peaked at 48 hour after transfection by MTT method. RT-PCR analysis showed that cyclin D1 antisense cDNA down-regulated cyclin D1 at the mRNA levels. Expression level of cyclin D1 protein was also decreased as shown by immunohistochemical studies. Cell-cycle analysis by flow cytometry showed that transfected HepG2 cells were arrested at the G1 phase of the cell cycle.. Our data suggest that cyclin D1 antisense cDNA could specifically inhibit the expression of cyclin D1 mRNA and protein and regulate cell cycle and cell proliferation of HepG2 cells. Cyclin D1 antisense cDNA may serve as a potential antitumor strategy in regulating cell-cyclin treating advanced HCCs.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Genes, bcl-1; Humans; Liver Neoplasms; Oligodeoxyribonucleotides, Antisense

Hepatocellular carcinoma (HCC) is one of the most common recurrent malignancies, for which, currently, there is no effective therapy. Considering the antihepatotoxic activity of silibinin, a widely used drug and supplement for various liver disorders, together with its strong preventive and anticancer efficacy against various epithelial cancers, we investigated the efficacy of silibin against human HCC cells.. Silibinin effects were examined on growth, cytotoxicity, apoptosis, and cell cycle progression in two different HCC cell lines, HepG2 (hepatitis B virus negative; p53 intact) and Hep3B (hepatitis B virus positive; p53 mutated). At molecular level, cell cycle effects of silibinin were assessed by immunoblotting and in-bead kinase assays.. Silibinin strongly inhibited growth of both HepG2 and Hep3B cells with a relatively stronger cytotoxicity in Hep3B cells, which was associated with apoptosis induction. Silibinin also caused G1 arrest in HepG2 and both G1 and G2-M arrests in Hep3B cells. Mechanistic studies revealed that silibinin induces Kip1/p27 but decreases cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)-2, and CDK4 levels in both cell lines. In Hep3B cells, silibinin also reduced the protein levels of G2-M regulators. Furthermore, silibinin strongly inhibited CDK2, CDK4, and CDC2 kinase activity in these HCC cells.. Together, these results for the first time identify the biological efficacy of silibinin against HCC cells, suggesting the importance of conducting further investigations in preclinical HCC models, especially on in vivo efficacy, to support the clinical usefulness of silibinin against hepatocellular carcinoma in addition to its known clinical efficacy as an antihepatotoxic agent.

Topics: Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin D3; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Flow Cytometry; Humans; Liver Neoplasms; Silybin; Silybum marianum; Silymarin; Tumor Cells, Cultured

The human hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The mechanisms of liver cell oncogenic transformation are still unknown. The beta-catenin mutations are identified in up to 30% of HCC and 80% of hepatoblastoma, suggesting a potential role of beta-catenin in the pathogenesis of liver cancers. To define the biological role of the stabilized beta-catenin in liver cell growth and transformation, we examined the effect of mutant beta-catenin on an immortalized murine hepatocyte cell line, AML12. A cell line that stably expresses mutant beta-catenin was established. The cell proliferation, apoptosis, and cell transformation of this cell line were characterized. Our data indicate that the stabilized beta-catenin enhances hepatocyte proliferation, suppresses TNFalpha/Act D-induced cell apoptosis, and causes weak anchorage-independent cell growth. The stabilized beta-catenin-containing cells did not develop tumor in immune-deficient mice. The target genes, c-myc and cyclin D1, were activated by beta-catenin in the hepatocytes. Our study suggests that mutant beta-catenin can promote cell proliferation and cell survival ability, but the stabilized beta-catenin alone is insufficient for completely oncogenic transformation.

Topics: Animals; Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Cytoskeletal Proteins; Drug Stability; Gene Expression Regulation; Genes, myc; Hepatocytes; Humans; Liver Neoplasms; Mice; Mice, SCID; Mutation; Trans-Activators; Transfection; Tumor Necrosis Factor-alpha

To investigate gene amplification of CCND1 and expression of cyclin D1 in hepatocellular carcinoma (HCC) and to explore the possible relationship between CCND1 gene status and carcinogenesis of HCC.. Differential PCR, RT-PCR and immunohistochemistry were used to detect gene amplification, mRNA and protein expression of cyclin D1 in 20 HCC cases respectively. The relationship between the gene amplification rate and the expression level of cyclin D1 and the histological grades of HCC was analyzed.. CCND1 gene amplification was detected in 30% of the cases HCC. An overexpression of cyclin D1 mRNA and protein could be demonstrated in 45% and 70% cases respectively. The expression of cyclin D1 mRNA correlated with its gene amplification status (P < 0.05) and was responsible for the protein expression level (P < 0.05). There was a close relationship between the expression level of cyclin D1 protein and HCC histological grades (P < 0.05).. CCND1 gene amplification is a common phenomenon in HCC and may be directly responsible for the cyclin D1 mRNA and protein overexpression. Cyclin D1 protein expression level is directly related to HCC histological grades. Therefore, CCND1 amplification and cyclin D1 overexpression may play an important role in development and differentiation of HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cyclin D1; Female; Gene Amplification; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Polymerase Chain Reaction; RNA, Messenger

Wnt signaling pathway is essential for development and tumorigenesis, however, this signaling pathway in the progress of hepatocellular carcinoma (HCC) remains unclear. In this paper, we studied the function of human T-cell transcription factor-4 (TCF4), a key factor of Wnt signaling pathway, on the proliferation of HCC cell line. We showed that the expression of TCF4 mRNA in HCC cell line BEL-7402 was higher than that in immortalized normal liver cell line L02. Blockage of Wnt pathway by Delta-NTCF4, a dominant negative TCF4, could suppress BEL-7402 cells growth and decrease the expression of cyclin D1 and c-myc, two of target genes of Wnt pathway. On the other hand, stimulating Wnt pathway by introducing a degradation-resistant -catenin S37A could increase BEL-7402 cells proliferation. But all the treatments had no effect on L02 cells. Our data indicated that TCF4 might be another key factor in Wnt pathway involved in HCC cells proliferation and TCF4 could be an effective therapeutic target for suppressing the growth of hepatocellular cancers.

Topics: beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cell Line, Tumor; Cyclin D1; Cytoplasm; Cytoskeletal Proteins; DNA-Binding Proteins; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Humans; Immunohistochemistry; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Microscopy, Fluorescence; Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spectrometry, Fluorescence; TCF Transcription Factors; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors; Transfection; Wnt Proteins

To study the relation between the expression of p16 protein and Rb (retinoblastoma) protein and the transcription activity of nuclear factor-kappaB (NF-kappaB) and their roles in hepatocarcinogenesis.. p16 protein and Rb protein in 35 hepatocellular carcinoma (HCC) tissues and the peritumoral areas (more than 3 cm away from the tumor) were examined by Western blot analysis. The transcription activity of NF-kappaB in these tissues was detected by electrophoretic mobility shift assays (EMSA) and super-shift assays.. The expression rates of p16 protein in the HCC tissues and peritumoral areas were 37% (13/35) and 48% (17/35) respectively (P < 0.01). The expression rates of Rb protein in the HCC tissues and peritumoral areas were 34% (12/35) and 74% (26/35) respectively (P < 0.01). The emergence rates of NF-kappaB with transcription activity in the HCC tissues and peritumoral areas were 77% (27/35) and 85% (30/35) respectively (P > 0.05). Four out of the 10 (40%) HCC tissues expressing p16 protein and Rb protein simultaneously turned up NF-kappaB with transcription activity, while 18 out of the 20 (90%) HCC tissues not expressing both p16 and Rb proteins turned up NF-kappaB with transcription activity (P < 0.05). 13 out of the 17 (76%) peritumoral areas expressing two kinds of proteins simultaneously turned up NF-kappaB with transcription activity and the emergence rate of NF-kappaB with transcription activity in the peritumoral areas not expressing the two kinds of proteins was 89% (8/9) (P > 0.05).. Dysfunction of p16 is the early event of hepatocarcinogenesis and dysfunction of Rb is the later event in this course. There is a positive correlation between p16 protein and Rb protein in HCC tissues. The loss of expression of p16 and Rb proteins plays important roles in the carcinogenesis and progression of HCC by affecting NF-kappaB transcription activity which may prevent hepocyctes from apoptosis, besides disturbing cell cycle.

Topics: Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Liver Neoplasms; Male; Middle Aged; NF-kappa B; Retinoblastoma Protein

The peptidyl-proplyl-isomerase, PIN1, upregulates beta-catenin by inhibiting its interaction with APC. beta-catenin accumulation occurs in about 70% of hepatocellular carcinoma (HCC), of which only 20% are due to beta-catenin mutations. The role of PIN1 in beta-catenin upregulation in HCC was investigated. PIN1 was shown to be overexpressed in more than 50% of HCC. All cases with PIN1 overexpression also showed beta-catenin accumulation, with 68% of cases showing concomitant beta-catenin and cyclin D1 accumulation. PIN1 was shown to contribute to beta-catenin and cyclin D1 overexpression directly by in vitro cell-line transfection experiments. Finally, we showed that PIN1 overexpression and beta-catenin gene mutations appeared to be mutually exclusive events, leading to beta-catenin accumulation in HCC. These results showed that PIN1 overexpression leading to beta-catenin accumulation might be a critical event in hepatocarcinogenesis, and that PIN1 is a potential target for therapeutic intervention in HCC.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cyclin D1; Cytoskeletal Proteins; Humans; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Reverse Transcriptase Polymerase Chain Reaction; Trans-Activators; Transfection

It has been shown that a variety of cell cycle-related proteins play important roles in the process of carcinogenesis including hepatocarcinogenesis. In the present study, we evaluated mRNA and protein expression of G1 phase-related cell cycle molecules in the process of hepatocarcinogenesis, using Long-Evans Cinnamon (LEC) rats, an animal model of hepatocellular carcinoma (HCC). The expression of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6 was measured quantitatively by real-time polymerase chain reaction. Cyclin D1 mRNA expression was increased significantly in chronic hepatitis liver compared with normal liver, and then decreased in HCC and the surrounding precancerous liver of LEC rats. Levels of Cdk4 mRNA were increased significantly in HCC compared to precancerous and chronic hepatitis livers. In contrast, mRNA levels of Cdk6 did not change significantly during hepatocarcinogenesis. We also evaluated the protein levels of these G1 phase-related cell cycle molecules by Western blot analyses and confirmed similar results. Total amounts of retinoblastoma protein (pRb) in the liver did not change significantly in the process of hepatocarcinogenesis in LEC rats. However, levels of phosphorylated pRb were increased markedly in the process of hepatocarcinogenesis, and the highest in HCC compared to precancerous, chronic hepatitis and normal livers. These results indicate that cyclin D1 may be involved in the regeneration of hepatocytes rather than hepatocarcinogenesis, while Cdk4 but not Cdk6 may play an important role in the development of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; G1 Phase; Hepatitis, Animal; Hepatitis, Chronic; Liver Cirrhosis; Liver Neoplasms, Experimental; Phosphorylation; Proto-Oncogene Proteins; Rats; Rats, Inbred LEC; Retinoblastoma Protein; RNA, Messenger

Antiproliferative activities of fractions of Hypsizigus marmoreus were examined using HepG2 cells in vitro. The methanol extract of H. marmoreus markedly induced antiproliferative activity, and an active compound from this mushroom was identified as hypsiziprenol A9. Hypsiziprenol A9 inhibited cell proliferation in a time- and concentration-dependent manner by up to 80% on HepG2 cells by inducing arrest of the G1 phase. Further investigation revealed that hypsiziprenol A9 decreased expression of phosphorylated retinoblastoma protein (ppRb), cyclin D1, and cyclin E in a dose-dependent manner. These results suggest that hypsiziprenol A9 can inhibit the growth of HepG2 cells through inducing G1 phase cell cycle arrest due to the inhibition of pRb phosphorylation.

Topics: Agaricales; Carcinoma, Hepatocellular; Cell Cycle; Cyclin D1; Cyclin E; Dose-Response Relationship, Drug; Fatty Alcohols; Humans; Liver Neoplasms; Phosphorylation; Retinoblastoma Protein; Tumor Cells, Cultured

Our aim was to study the anticancer effect of the novel immunomodulator FTY720 in vitro and in vivo by investigation of cell cycle entry, cell cycle regulation, cell survival and apoptosis pathways. Three hepatoma cell lines with different p53 statuses (HepG2, Huh-7 and Hep3B) and one non-tumorigenic immortalized liver cell line (MIHA) were used for an in vitro study. The in vivo effects of FTY720 were evaluated in a nude mouse tumor model. Cell cycle distribution and cell cycle regulator proteins p27(Kip1) and cyclin D1, together with the PI3-K/Akt pathway, mitogen-activated protein kinases and cleaved caspase-3 and caspase-9, were evaluated. FTY720 selectively induced cell apoptosis in hepatoma cell lines with overexpression of cleaved caspase-3 and caspase-9, but the same phenomena were not found in MIHA cells. FTY720 induced Akt dephosphorylation at Ser473 mediated by phosphoinositide 3-kinase (PI3-K) inhibition. Dephosphorylation led to down-regulation of p42/p44 and dephosphorylation of Forkhead transcription factor and GSK-3beta and, subsequently, up-regulation of p27(Kip1) and down-regulation of cyclin D1. In our in vivo model FTY720 induced apoptosis of tumor cells by down-regulation of the Akt pathway. FTY720 suppressed tumor growth without notable side-effects in normal liver. In conclusion, FTY720 is a novel anticancer agent that induces apoptosis of hepatoma cell lines both in vitro and in vivo through PI3-K-mediated Akt dephosphorylation in a p53-independent manner.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Enzyme Inhibitors; Fingolimod Hydrochloride; G1 Phase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunosuppressive Agents; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Phosphorylation; Propylene Glycols; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sphingosine; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Hepatoma is one of the most frequently occurring cancers worldwide. However, effective chemotherapeutic agents for this disease have not been developed. Acyclic retinoid, a novel synthetic retinoid, can reduce the incidence of postsurgical recurrence of hepatoma and improve the survival rate. OSI-461, a potent derivative of exisulind, can increase intracellular levels of cyclic GMP, which leads to activation of protein kinase G and induction of apoptosis in cancer cells. In the present study, we examined the combined effects of acyclic retinoid plus OSI-461 in the HepG2 human hepatoma cell line. We found that the combination of as little as 1.0 micromol/L acyclic retinoid and 0.01 micromol/L OSI-461 exerted synergistic inhibition of the growth of HepG2 cells. Combined treatment with low concentrations of these two agents also acted synergistically to induce apoptosis in HepG2 cells through induction of Bax and Apaf-1, reduction of Bcl-2 and Bcl-xL, and activation of caspase-3, -8, and -9. OSI-461 enhanced the G0-G1 arrest caused by acyclic retinoid, and the combination of these agents caused a synergistic decrease in the levels of expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, decreased the level of hyperphosphorylated forms of the Rb protein, induced increased cellular levels of the p21(CIP1) protein and mRNA, and stimulated p21(CIP1) promoter activity. Moreover, OSI-461 enhanced the ability of acyclic retinoid to induce increased cellular levels of retinoic acid receptor beta and to stimulate retinoic acid response element-chloramphenicol acetyltransferase activity. A hypothetical model involving concerted effects on p21(CIP1) and retinoic acid receptor beta expression is proposed to explain these synergistic effects. Our results suggest that the combination of acyclic retinoid plus OSI-461 might be an effective regimen for the chemoprevention and chemotherapy of human hepatoma and possibly other malignancies.

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chloramphenicol O-Acetyltransferase; Cyclic GMP; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Drug Synergism; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Intracellular Space; Liver Neoplasms; Microfilament Proteins; Models, Biological; Phosphoproteins; Phosphorylation; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Response Elements; Resting Phase, Cell Cycle; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulindac; Trans-Activators; Transfection; Tretinoin; Tumor Suppressor Protein p53

Introns are present in some human pre-tRNAs. They are spliced out during the maturation processes of pre-tRNAs in a way that is irrelevant to their specific nucleotide sequences. This unique characteristic of tRNA splicing can be used for generation of small antisense RNAs by replacing the intron sequences with corresponding antisense sequences. In this work, the intron sequence of human pre-tRNAtyr gene was replaced with a 20 bp antisense sequence targeted to the 5' coding region of cyclin D1, a molecule that was over-expressed in many malignant proliferating cells. Under the control of U6 SnRNA promoter to further enhance transcription efficiency of the modified pre-tRNAtyr gene and subsequent antisense generation, the antisense RNA exhibited obvious suppression of cyclin D1 expression in H22 hepatoma cells. The growth of H22-transplanted tumors in mice was significantly inhibited when treated with naked plasmid DNA harboring the cyclin D1 antisense RNA generating cassette. Such tumor growth inhibition might be due to apoptosis caused by reduced cyclin D1 expression as revealed by immunohistochemical analysis of tumor samples.

Topics: Alternative Splicing; Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cyclin D1; DNA, Ribosomal; Humans; Liver Neoplasms; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; RNA Precursors; RNA Splicing; RNA, Antisense; RNA, Transfer, Tyr; Transcription, Genetic; Transplantation, Heterologous

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Southeast Asia. Hyperphosphorylation of retinoblastoma (pRB) by cyclin/CDKs in G1/S transition is required for its inactivation and cell cycle progression. In the present study, we report that phosphorylation of pRB at Ser780 and Ser795 was detected in 71% (33 of 46) and 63% (29 of 46) of HCCs examined respectively. pRB protein was undetectable in 13% (6 of 46) of HCCs examined. Phosphorylated pRB was localized in the nuclei of hepatocarcinoma cells. Benign hepatocytes exhibited very weakly or no nuclear staining for phosphorylated pRB. Over-expression of E2F-1, cyclin D1, Cdk-2, Cdk-4 and cyclin A was found in 64% (30 of 46), 43% (26 of 46), 28% (11 of 46), 71% (33 of 46) and 63% (29 of 46) of HCCs examined respectively and this was correlated with elevation of ERK. Treatment of HepG2 cells with MEK1/2 inhibitor U0126 resulted in cell cycle arrest, downregulation of cyclin D1 and Cdk-2 expression and inhibition of pRB phosphorylation at Ser780 and Ser795. Ectopic expression of activated MEK1 in HepG2 cells increased cyclin D1 and Cdk-2 expression, phosphorylation of pRB at Ser780 and Ser795, and percentage of cells in S phase. Our data indicate that activated ERK plays an important role in cyclin D1 and Cdk-2 expression and phosphorylation of pRB at Ser780 and Ser795 in liver cancer cells.

Topics: Carcinoma, Hepatocellular; CDC2-CDC28 Kinases; Cyclin D1; Cyclin-Dependent Kinase 2; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; Phosphorylation; Retinoblastoma Protein; Tumor Cells, Cultured

Recently we found that 20 microg/ml of ginsenoside Rh2 (G-Rh(2)) can inhibit growth, and induce differentiation of hepatocarcinoma cell line SMMC-7721. Re-activation of telomerase may play an important role in carcinogenesis, and cellular immortalization. This study was to explore the mechanism of G-Rh(2)-induced differentiation of SMMC-7721 cells by detecting telomerase activity.. After treated with 20 mug/ml of G-Rh(2), telomerase activity in SMMC-7721 cells was detected by polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) coupled with enzyme-linked immune sorbent assay(ELISA); mRNA levels of human telomerase reverse transcriptase (hTERT), and p21 were measured by reverse transcriptase-polymerase chain reaction (RT-PCR); changes of cell cycle, and expression of Cyclin D1, Cyclin E,P16 protein were detected by flow cytometry (FCM).. G-Rh(2) inhibited telomerase activity of SMMC-7721 in a time- dependent manner: the activity decreased from 1.105 to 0.765 (P< 0.01) after 1-day treatment, while from 1.152 to 0.326 (P< 0.01) after treated for 5-day treatment. The mRNA expression of hTERT was significantly reduced by 20 microg/ml of G-Rh(2). Cell cycle assay showed that 20 microg/ml of G-Rh(2) increased the proportion of SMMC-7721 cells in G1 phase, and decreased those in S, and G2/M phases, the increase of G1 phase after 1-day treatment was the most apparent (from 60.85% to 78.53%,P< 0.05). Furthermore,G-Rh(2) weakened the expressions of positive-regulating factors(Cyclin D1,Cyclin E),and increased the expressions of negative-regulating factors (P16 protein, p21 gene) in SMMC- 7721 cells.. G-Rh(2) may effectively reduce telomerase activity through affecting transcription level of hTERT,and arresting cell cycle progression. The down- regulation of telomerase activity in SMMC-7721 cells may be closely related to G-Rh(2)-induced differentiation.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Drugs, Chinese Herbal; Ginsenosides; Humans; Liver Neoplasms; Panax; Plants, Medicinal; RNA, Messenger; Telomerase

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Helicobacter pylori; Humans; Liver Neoplasms; Proliferating Cell Nuclear Antigen; RNA, Messenger

Increasing evidence has indicated that perturbation of cyclins is one of the major factors leading to cancer. The aim of this study was not only to investigate various cell cycle-related kinase activities in hepatocellular carcinoma (HCC), but also to analyze the difference of cell cycle-related kinase activity levels between hepatitis C virus (HCV)-induced HCC and HCV-induced cirrhosis. The protein levels of cyclins D1, E, A, and H, and of cyclin dependent kinase 1 (Cdk1), Cdk2, Cdk4, Cdk6, and Cdk7 in HCC and in surrounding nontumorous cirrhosis were determined by Western blot. The enzymatic activities of cyclins D1, E, A, Cdk1, Cdk4, Cdk6, Cdk7, and Wee1 were measured using in vitro kinase assays. Protein levels and kinase activities of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 were significantly elevated in HCC compared with surrounding cirrhotic tissues. The enhanced cyclin D1-related kinase activity in HCC was accompanied by the up-regulation of Cdk4 activity, but not Cdk6 activity. The kinase activities of Cdk6, Cdk7, and Cdk1 did not differ between HCC and surrounding cirrhotic tissues. In addition, the protein levels and kinase activities of cyclin D1, Cdk4, and cyclin E were higher in poorly differentiated HCC and advanced HCC. In conclusion, the increases of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 play an important role in the development of HCC from cirrhosis. Cyclin D1, Cdk4, and cyclin E activation may be closely related to the histopathologic grade and progression of HCC.

Topics: Aged; Blotting, Western; Carcinoma, Hepatocellular; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin E; Cyclin H; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Cyclins; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein

Hepatocellular carcinoma (HCC) is a common killer cancer in the world. Recently, abnormal activation of the Wnt pathway has been found to be involved in the carcinogenesis of several human cancers including HCC. The goal of the present study was to investigate the mechanism of inappropriate activation of the Wnt pathway in hepatocarcinogenesis.. We analyzed the alterations of three key components of the Wnt pathway: beta-catenin, glycogen synthase kinase (GSK)-3beta and T-cell factor (Tcf)-4 in 34 HCC and paracancerous normal liver by immunohistochemistry, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP), direct sequencing, and quantitative real-time reverse transcription (RT)-PCR.. We found that 61.8% (21/34) of all HCC examined showed an abnormal beta-catenin protein accumulation in the cytoplasm or nuclei. The RT-PCR-SSCP and direct sequencing showed that beta-catenin exon 3 mutations existed in 44.1% (15/34) of the HCC. No mutations of GSK-3beta or Tcf-4 were detected in HCC. Moreover, messenger RNA of beta-catenin and Tcf-4, but not GSK-3beta, was found to be overexpressed in HCC. On analyzing the relationship between alterations of beta-catenin or Tcf-4 and C-myc or Cyclin D1 expression, we found that mutations of beta-catenin, as well as overexpression of beta-catenin or the Tcf-4 gene were independently correlated with C-myc gene overexpression in HCC.. Our present findings strongly suggest that mutations of beta-catenin, as well as overexpression of beta-catenin and the Tcf-4 gene, independently activate the Wnt pathway in HCC, with the target gene most likely to be C-myc.

Topics: beta Catenin; Biomarkers, Tumor; Carcinoma, Hepatocellular; China; Cyclin D1; Cytoskeletal Proteins; DNA Mutational Analysis; Down-Regulation; Electrophoresis, Agar Gel; Exons; Genes, myc; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunohistochemistry; Liver Neoplasms; Mutation; Polymorphism, Single-Stranded Conformational; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics as Topic; TCF Transcription Factors; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors

Alteration of beta-catenin expression has been implicated in the development of hepatocellular carcinoma (HCC). It has been also reported that beta-catenin can influence the tumor cell proliferation or cyclin D1 expression, one of the target factors of betacatenin. We performed an immunohistochemical analysis of beta-catenin and cyclin D1 in 77 patients with resected HCCs, and examined the relationships between the expressions of beta-catenin and cyclin D1, and other pathologic parameters including the mitotic index. Altered expressions of beta-catenin including nonnuclear overexpression and nuclear expression were detected in 58.4% of HCCs (45/77) and showed significant correlations with large tumor size, poor histologic grade, and high tumor stage. The mean mitotic index of HCCs with nuclear expression (3.2 +/- 3.0) and nonnuclear overexpression (2.7 +/- 2.5) was significantly higher than that of tumors with no overexpression (1.7 +/- 1.4) (p=0.018 and 0.038, respectively), however, no correlation was noted between the expressions of cyclin D1 and beta-catenin. In addition, nonnuclear overexpression out of two altered expression patterns was more frequent (37.7% versus 20.8%) as well as pathologically more significant than nuclear expression. These results indicate that the altered expression of beta-catenin in HCC may play an important role in tumor progression by stimulating tumor cell proliferation, and nonnuclear overexpression may have pathologic significance in HCC.

Topics: beta Catenin; Carcinoma, Hepatocellular; Cell Division; Cyclin D1; Cytoskeletal Proteins; Humans; Immunohistochemistry; Liver Neoplasms; Trans-Activators

In this study we used liver neoplasms induced by several chemical carcinogens to investigate potential nuclear targets associated with beta-catenin/Wnt signaling and potential membrane-associated beta-catenin binding partners. Strong expression of cyclin D1, in a pattern similar to that observed previously for beta-catenin, was observed by Western analysis for all five hepatoblastomas examined regardless of treatment. Increased expression of cyclin D1 was also detected in 12 of 35 (34%) hepatocellular neoplasms. Ten of 15 tumors (67%) that had mutations in the Catnb gene had upregulation of cyclin D1, while only 2 of 20 tumors (10%) without Catnb mutations had increased cyclin D1 expression. Immunohistochemical analysis confirmed strong expression of cyclin D1 in most nuclei of hepatoblastomas and scattered nuclear staining in hepatocellular tumors that had Catnb mutations. Increased c-Jun expression was observed in 19 of 30 (63%) hepatocellular tumors and all hepatoblastomas, although upregulation was not completely correlated with Catnb mutation. C-Myc expression was not increased in the tumors. Reduced expression of E-cadherin, which interacts with beta-catenin at the membrane, was observed in some tumors, but this did not correlate with Catnb mutation. Expression of the epidermal growth factor receptor, which may have a role in beta-catenin tyrosine phosphorylation, was lower in some tumors than in normal tissue depending on chemical treatment. The results provide evidence that increased expression of cyclin D1 and c-Jun may provide an advantage during tumor progression and in the transition from hepatocellular neoplasms to hepatoblastomas. Moreover, it is likely increased cyclin D1 expression results at least in part from Catnb mutation, beta-catenin accumulation, and increased Wnt signaling.

Topics: Adenoma, Liver Cell; Animals; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cyclin D1; Cytoskeletal Proteins; ErbB Receptors; Genes, ras; Hepatoblastoma; Immunoenzyme Techniques; Liver Neoplasms, Experimental; Mice; Mice, Inbred Strains; Mutation; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Trans-Activators; Wnt Proteins; Zebrafish Proteins

p27(Kip1) (p27) might act as an adverse prognostic marker for various types of cancers. However, its clinical usefulness remains uncertain, because it is sometimes overexpressed in aggressive types.. To precisely evaluate the practical significance of p27 in hepatocellular carcinoma (HCC), we immunohistochemically compared the level of p27 expression with Ki-67 labeling in 74 HCCs and focused on tumors in which cell proliferation increased despite a high level of p27 expression. We then analyzed the status of p27 and related cell-cycle regulators using kinase and immunoprecipitation assays, Western blotting, and methylation-specific PCR to understand the rationale for the functional inactivation of p27 in HCC. We also evaluated relationships between the key biological characteristics of HCC and survival.. Immunohistochemical studies showed that 40 (54%) of 74 HCCs expressed high levels of p27 (>50% of the tumor cells). Of these, the Ki-67 labeling index was low (<20%) in 26 (65%) and high (>20%) in 14 (35%). Increased proliferative activities were closely correlated with elevated kinase activities, sequestration of p27 protein, and p16 gene methylation. The association between a loss of p16 and poor prognosis was significant when p27 expression was high (P < 0.01).. The loss of p16 appears to be closely related to the functional inactivation of p27, and assessment of p16 status may be useful for a precise prognostic prediction of individuals with HCC expressing high levels of p27.

Topics: Adult; Aged; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; DNA Methylation; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Polymerase Chain Reaction; Precipitin Tests; Prognosis; Promoter Regions, Genetic; Proto-Oncogene Proteins; Time Factors; Tumor Suppressor Proteins

Characterization of the protein profiles expressed by hepatocellular carcinomas (HCCs) may identify the genes involved in hepatocellular carcinogenesis and offers the possibility of elucidating clinical biomarkers. In an effort to discover such proteins and pathways that are deregulated in hepatocellular carcinogenesis, cellular proteomes of matched normal liver cells and carcinoma were analysed by tissue microdissection and protein microarrays. Using protein microarrays made up of 83 different antibodies, it was possible to monitor alterations of the protein levels in HCC and non-neoplastic liver tissue. Further analysis of altered proteins was performed using western blot analysis and tissue microarrays (TMAs) containing 210 HCC specimens and corresponding liver tissue. The protein microarray approach revealed differential expression between HCC and normal liver of 32 of the 83 proteins examined: 21 of these were up-regulated and 11 down-regulated. IGF (insulin growth factor) II, ADAM (a disintegrin and metalloproteases) 9, STAT (signal transducers and activators of transcription) 3, SOCS (suppressors of cytokine signalling) 3, and cyclin D1 were significantly up-regulated and collagen I, SMAD 4, FHIT (fragile histidine triad), and SOCS1 were down-regulated. The differential expression of these proteins was confirmed using western blot analysis and TMAs. Correlation of differentially regulated proteins with clinico-pathological data showed that cyclin D1 and SOCS1 were associated with tumour prognosis in univariate analysis, but not multivariate analysis. These data indicate that the development of an array-based approach for the determination of protein profiles in HCC may facilitate the identification of new proteins associated with carcinogenesis or prognosis.

Topics: Acid Anhydride Hydrolases; ADAM Proteins; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Collagen Type I; Cyclin D1; Disintegrins; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation; Humans; Insulin-Like Growth Factor II; Intracellular Signaling Peptides and Proteins; Liver; Liver Neoplasms; Membrane Proteins; Metalloendopeptidases; Neoplasm Proteins; Neoplasm Staging; Protein Array Analysis; Proteins; Repressor Proteins; Reproducibility of Results; Smad4 Protein; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Trans-Activators; Transcription Factors

To investigate the expression of five kinds of cyclins in hepatocellular carcinoma (HCC) and their association with degree of tumor differentiation, metastasis and infection of hepatitis B virus (HBV).. The HCC tissue microarrays were composed of those from 273 cases of HCC tissues, 144 surrounding-tumor liver tissues and 10 normal liver tissues obtained from autopsy. The diameter of each specimens on tissue microarrays was 2.0 mm. Immunohistochemistry was used to detect the expression of cyclin A, cyclin B, cyclin D1, cyclin D3 and cyclin E on HCC tissue microarrays. The association of the expression of these cyclins with the infection rate of HBV was also analyzed.. Three paraffin-embedded HCC tissue microarrays were successfully constructed, including 136, 143 and 148 tissue spots, respectively. The positive rates of cyclins in 273 cases of HCC were cyclin A 52.7%, cyclin B 45.4%, cyclin D1 35.9%, cyclin D3 44.3% and cyclin E 23.1%; while the figures in 144 surrounding-tumor tissues were 8.3%, 5.6%, 4.9%, 6.3% and 1.4%, respectively. In 10 normal liver tissues these cyclins exhibited negative staining, with the exception that cyclin D1 was positive in one case of normal liver tissue. The positive rate of cyclins in HCC were significant higher than those in surrounding-tumor liver tissues (P < 0.01), in HCC tissues with histological grade II and III, the cyclins expression were stronger than that in grade I (P < 0.05). The positive rates of cyclins, except cyclin A in HCC with portal vein invasion were higher than those without portal vein invasion (P < 0.01). Infection of HBV did not have significant relationship with the expression of cyclins (P > 0.05).. Cyclins in different cell cycles overexpressed at varied levels in hepatocellular carcinoma, and the increased expression of cyclins may shorten the tumor cell cycle phase, accelerate cell proliferation, and have a close relationship with HCC aggressiveness.

Topics: Carcinoma, Hepatocellular; Cyclin A; Cyclin B; Cyclin D1; Cyclin D3; Cyclin E; Cyclins; Hepatitis B; Humans; Immunohistochemistry; Liver Neoplasms

Acyclic retinoid (ACR), a novel synthetic retinoid, can prevent the recurrence of human hepatoma after surgical resection of primary tumors, but the molecular mechanisms by which ACR exerts antitumor effects are not known. In this study, we found that ACR inhibited the growth of three human hepatoma cell lines. In HepG2 cells, this inhibition was associated with an arrest of the cell cycle in G(0)-G(1), increased cellular levels of p21(CIP1), decreased levels of the hyperphosphorylated form of the retinoblastoma protein, and decreased levels of cyclin D1, but no significant changes were seen in the levels of the p16(INK4a), p27(KIP1), cyclin-dependent kinase 4, cyclin-dependent kinase 6, glycogen synthase kinase 3beta, or beta-catenin proteins. ACR also caused a decrease in the level of cyclin D1 mRNA. Cotreatment of HepG2 human hepatoma cells with the proteasome inhibitor N-acetyl-Leu-Leu-norleu-al did not prevent the ACR-induced decrease in cyclin D1 protein, in contrast to the protective effect of N-acetyl-Leu-Leu-norleu-al on the cyclin D1 protein in cells treated with all-trans-retinoic acid. In transient transfection reporter assays, ACR, but not all-trans-retinoic acid, inhibited transcription from the cyclin D1 promoter. As reported previously in colon carcinoma cells, we found that in hepatoma cells, cyclin D1 promoter activity is markedly stimulated by the beta-catenin/T-cell factor pathway. Nevertheless, even in the presence of excess beta-catenin, ACR markedly inhibited the transcriptional activity of the cyclin D1 promoter. This is the first systematic study of the inhibitory effects of ACR, or any other retinoid compound, on beta-catenin/T-cell factor-stimulated cyclin D1 promoter activity in human tumor cells. These novel effects of ACR provide further evidence that ACR may be a valuable agent in the chemoprevention and therapy of hepatoma and possibly other human malignancies.

Topics: Antineoplastic Agents; beta Catenin; Carcinoma, Hepatocellular; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytoskeletal Proteins; G1 Phase; Growth Inhibitors; Humans; Liver Neoplasms; Phosphorylation; Promoter Regions, Genetic; Retinoblastoma Protein; RNA, Messenger; Trans-Activators; Tretinoin; Tumor Cells, Cultured

D-type cyclins regulate distinct cellular processes such as mitotic cell cycle control, differentiation and transcription. Deregulation of cyclin D1, a component of G1 checkpoint control, can result in enhanced genomic instability, cell transformation, and malignant neoplasia. However, a precise understanding of the molecular and cellular events underlying the regulation of the cyclin D1 gene remains to be elucidated. In this study, we examined the regulation of the cyclin D1 gene during n-nitrosodiethylamine (DEN)-induced sequential liver carcinogenesis. Northern blot studies showed an increase in the level of cyclin D1 mRNA. Southern blot analysis of the DNA restriction fragment showed no alterations and/or amplification in the coding region of the cyclin D1 gene. Bulk chromatin from DEN-treated rat liver is much more sensitive to nuclease digestion than that from normal liver. Increased expression of the cyclin D1 gene is correlated to the upregulation of its transcription, mediated through chromatin decondensation during sequential hepatocarcinogenesis. Thus, the functional inter-relationship between chromatin organization and gene expression appears to be of critical importance for liver tumour development.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Chromatin; Cyclin D1; Diethylnitrosamine; Gene Expression Regulation, Neoplastic; Genes, cdc; Liver Neoplasms, Experimental; Male; Rats; Rats, Inbred Strains; RNA, Messenger; Up-Regulation

To contrast the feature of different periods of p53, p16, and cyclin D1 expression in cells of hepatocarcinoma.. Sixty hepatocarcinoma specimens were collected and divided into three periods in one yearly cycle with each period of 20 cases. Hepatocellular carcinoma was documented in all cases by pathomorphological classification. Edmondson-Steinet grading belonged to II~III stages. The expression of gene proteins of p53, p16, and cyclin D1 was determined by immunohistochemical assay (S-P method). The expressive intensity was analysed by the rank test.. p16 expression showed significant difference of period (H=10.334, P<0. 05). Arpil-July was the high expressive period.. p16 may take an important role in the chronobiologic mechanism of gene control of hepatocyte canceration and hepatocarcinoma cell growth.

Topics: Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Tumor Suppressor Protein p53

Poor prognosis of hepatocellular carcinoma (HCC) is due to its high recurrent rate after operation and early metastasis through portal vein. This study was designed to establish a metastatic HCC cell line and to investigate its molecular and cytogenetic characterization.. Cell culture was performed using the tissue obtained from a metastatic lesion in portal vein of a patient with HCC. After G-banding staining, karyotype, and comparative genomic hybridization (CGH) of the cultured H4M cells were characterized and cyclin D1 gene CCND1 was detected by differential PCR.. The karyotype of metastatic HCC cell strain is a hypertriploid (71-78 chromosomes) with a huge marker chromosome containing a long homogeneously staining region (hsr). The main genetic alterations in H4M cells were a high copy number amplification of 11q13 and loss of 8p. Cyclin D1 gene CCND1 was also amplified significantly in the cells.. The loss of chromosome 8p and high copy number amplification of 11q13 are associated with the metastatic characteristics of H4M cells. The amplification of cyclin D1 gene CCND1 in H4M cells may be the cause of the amplification of chromosome 11q13.

Topics: Aneuploidy; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromosome Deletion; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 8; Cyclin D1; Humans; Karyotyping; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Middle Aged; Portal Vein

In this study, the effects of HMBA on the expression of G0/G1 phase arrest-associated genes in human hepatocarcinoma SMMC-7721 cells were investigated. Immunocytochemistry and in situ hybridization assay revealed that the levels of p21WAF1/CIP1 and p16 proteins and the level of p21WAF1/CIP1 mRNA were increased while the levels of CDK4 and Cyclin D1 proteins and c-myc mRNA were decreased in the cells treated with HMBA. These results showed that HMBA could up-regulate the expression of p21WAF1/CIP1 and p16 genes, down-regulate the activity of Cyclin D1-CDK4 and the transcription of c-myc gene which were necessary for cells entering into S phase, and so arrest the cells in G0/G1 phase, and induce the differentiation of human hepatocarcinoma SMMC-7721 cells.

Topics: Acetamides; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Liver Neoplasms; Neoplasm Proteins; Proto-Oncogene Proteins c-myc

The cyclin D1 gene, CCND1, located within chromosome 11q13, plays an important role in the regulation of cell-cycle progression and has oncogenic properties. Cyclin D1 frequently is overexpressed in a variety of cancers, including hepatocellular carcinoma (HCC), as a result of gene amplification. In a previous study, we showed threefold to 20-fold amplification of CCND1 in four of 30 (13%) HCC tissues from Taiwan but not in any control liver tissues or in two HCC cell lines. A common A870G polymorphism located within the splice donor region of exon 4 of CCND1 has been reported to enhance alternate splicing. Two forms of mRNA are present in subjects with the heterozygous genotype. The relationship between the variant allele and susceptibility to HCC and clinical-pathologic outcome was investigated in 97 Taiwanese HCC patients and 35 control subjects. In this small sample, CCND1 genotype frequencies were similar in cases and controls and were not associated with susceptibility to the development of HCC. All nine patients homozygous for the G allele (GG genotype) had poorly differentiated tumors, but this association was not statistically significant, perhaps owing to the small sample. Overexpression of cyclin D1 protein, through gene amplification, correlates with poor prognosis in several cancers, but its role in HCC is the subject of controversy. Increased expression of cyclin D1 may play an important role in the development of HCC owing to the perturbation of normal control of the cell cycle. The A870G polymorphism in CCND1 may influence differentiation and prognosis in HCC patients but requires further study.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Differentiation; Chromosomes, Human, Pair 11; Cyclin D1; DNA Primers; Genotype; Humans; Liver Neoplasms; Polymorphism, Genetic

Although hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the human liver, the molecular changes and mechanisms that regulate its development and progression remain unclear. In the present study, we investigated the correlation between beta-catenin expression and clinical outcome in 51 patients with relatively small (maximal diameter < 30 mm), solitary HCCs.. The tumors were classified according to histological tumor differentiation (grade I, 11 tumors; grade II, 28 tumors; grade III, 12 tumors). Using immunohistochemical methods to detect nuclear accumulation of beta-catenin, we investigated the correlation between beta-catenin expression and clinical outcome and compared the correlation with cyclin D1, Ki-67, and E-cadherin.. Focal or generalized nuclear beta-catenin expression was observed in 36.4% (4 of 11) of the grade I tumors, 39.3% (11 of 28) of the grade II tumors, and 25% (3 of 12) of the grade III tumors. Nuclear beta-catenin-positive grade III tumors were associated with significantly poorer survival (P = 0.004), whereas none of the patients with nuclear beta-catenin-negative grade I tumors died. With regard to proliferative activity, positive nuclear beta-catenin staining correlated significantly with an increased Ki-67 labeling index in grade I (P < 0.0001) and grade III (P = 0.0045) tumors and with reduced epithelial cadherin expression in the cell membrane (P < 0.001). In contrast, no association with the expression of cyclin D1, one of the target factors of beta-catenin, was detected.. Our present data suggest that beta-catenin plays important roles in promoting tumor progression by stimulating tumor cell proliferation and reducing the activity of cell adhesion systems and is associated with a poor prognosis, especially in patients with poorly differentiated HCCs.

Topics: Aged; beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Adhesion; Cell Differentiation; Cell Division; Cell Nucleus; Cyclin D1; Cytoskeletal Proteins; Disease Progression; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Prognosis; Time Factors; Trans-Activators

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

Preneoplastic and neoplastic hepatocytes undergo c-Myc up-regulation and overgrowth in rats genetically susceptible to hepatocarcinogenesis, but not in resistant rats. Because c-Myc regulates the pRb-E2F pathway, we evaluated cell cycle gene expression in neoplastic nodules and hepatocellular carcinomas (HCCs), induced by initiation/selection (IS) protocols 40 and 70 weeks after diethylnitrosamine treatment, in susceptible Fisher 344 (F344) rats, and resistant Wistar and Brown Norway (BN) rats. No interstrain differences in gene expression occurred in normal liver. Overexpression of c-myc, Cyclins D1, E, and A, and E2F1 genes, at messenger RNA (mRNA) and protein levels, rise in Cyclin D1-CDK4, Cyclin E-CDK2, and E2F1-DP1 complexes, and pRb hyperphosphorylation occurred in nodules and HCCs of F344 rats. Expression of Cdk4, Cdk2, p16(INK4A), and p27(KIP1) did not change. In nodules and/or HCCs of Wistar and BN rats, low or no increases in c-myc, Cyclins D1, E, and A, and E2F1 expression, and Cyclin-CDKs complex formation were associated with p16(INK4A) overexpression and pRb hypophosphorylation. In conclusion, these results suggest deregulation of G1 and S phases in liver lesions of susceptible rats and block of G1-S transition in lesions of resistant strains, which explains their low progression capacity.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; G1 Phase; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Liver; Liver Neoplasms, Experimental; Male; Phosphorylation; Proto-Oncogene Proteins c-myc; Rats; Rats, Inbred BN; Rats, Inbred F344; Rats, Wistar; Retinoblastoma Protein; S Phase; Transcription Factor DP1; Transcription Factors

To elucidate the relationship between expression of p16 and cyclinD1 proteins and evaluate the role of p16 gene exon 2 mutation in hepatocellular carcinogenesis.. Streptavidin-peroxidase conjugated method(SP) was used to detect expressions of p16 and cyclinD1 proteins in 44 cases of hepatocellular carcinoma(HCC), and polymerase chain reaction single-strand conformation polymorphism analysis(PCR-SSCP) to detect p16 gene mutation in exon 2 in 12 cases of HCC and liver tissues adjacent to HCC.. Expression rate and positive signal intensity of p16 protein in HCC were significantly lower(P < 0.01) and those of cyclinD1 protein in HCC were significantly higher (P < 0.05) than those in pericarcinomatous tissues. Of 12 fresh HCC tissues, p16 gene mutation in exon 2 was found in 2 cases, whereas that was not found in pericarcinomatous tissues.. 1. Inactivation or/ and deletion of p16 protein may be one of important reasons which result in proliferation unbalance of cells. 2. p16 gene mutation in exon 2 presents in HCC, but it does not frequently occur in Chinese hepatocarcinogenesis. 3. p16 gene abnormality and cyclinD1 over expression may coact in HCC.

Topics: Adolescent; Adult; Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Exons; Female; Genes, p16; Humans; Liver Neoplasms; Male; Middle Aged; Mutation

Cyclin D1 gene amplification and cyclin D1 protein overexpression have been reported in various human tumors, including hepatocellular carcinoma (HCC). However, their significance is still controversial. In the present study, we examined the expression of cyclin D1 and its relationships to p53 and Ki-67 in HCCs.. The expression and topological distribution of cyclin D1, p53 and Ki-67 in 50 cases of HCC were examined immunohistochemically, and the relationship between the expression of these proteins and their pathologic features was investigated.. Overexpression of cyclin D1 was noted in 58% of the HCC cases, and significantly associated with a well-differentiated histology and a low Ki-67 labeling index (LI). Cyclin D1 overexpression was also observed in all (7 of 7) dysplastic nodules and in non-neoplastic hepatocytes. On the other hand, aberrant p53 expression was detected in 36% of the cases, which showed positive relationships with poor differentiation, portal vein invasion, and KI-67 LI. Only eight of the 50 cases examined (16%) were positive for both cyclin D1 and p53, which showed only a small number of cyclin D1-positive cells. There was no significant relationship between the expressions of cyclin D1 and p53.. Our results suggest that cyclin D1 overexpression may be an early event in hepatocarcinogenesis and that it plays a role in tumor differentiation. In addition, cyclin D1 expression is not correlated with tumor cell proliferation in HCCs.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Nucleus; Cyclin D1; Female; Focal Nodular Hyperplasia; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Tumor Suppressor Protein p53

Deregulation of the cell cycle by overexpression of G1 cyclins, cyclin E and cyclin D1 genes, has been demonstrated to be a prerequisite for the development of human cancer. Recently, cyclin E is proposed to be sufficient for the progression of the G1 cell cycle without cyclin D1. Here we show that the proposed model system was specifically present in human hepatocellular carcinoma (HCC) unlike other human cancers. Of 31 HCC tissues analyzed, 21 (67.7%) exhibited an overexpression of cyclin E protein. In contrast to cyclin E gene expression, cyclin D1 expression was strongly downregulated in 19 (61.2%) HCCs. Interestingly, 65% of HCC tissues with overexpression of the cyclin E gene exhibited downregulation of cyclin D1, suggesting reciprocal deregulation of these cyclins in the G1 progression of the cell cycle. Southern blot analysis proved the amplification of cyclin E gene in HCC with a high level of overexpression. The present findings suggest that the reciprocal deregulation of cyclin E lacking cyclin D1 expression might play a role in G1 progression and the development of HCC.

Topics: Blotting, Southern; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cyclin D1; Cyclin E; Down-Regulation; Humans; Liver Neoplasms

p16, cyclin D1 and retinoblastoma protein (pRB) regulate G1 to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA, Neoplasm; Female; Gene Deletion; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinoblastoma Protein; Sequence Analysis, DNA

Cyclin D1 is a known oncogene and a key regulator of cell cycle progression. Amplification of the cyclin D1 gene and its overexpression have been associated with aggressive forms of human hepatocellular carcinoma (HCC). In this study, two independent lines of transgenic mice have been generated that express cyclin D1 under the control of the rat liver fatty acid binding protein promoter. This transgene specifically directs expression in the liver and the intestines. RNA and protein analysis demonstrated increased expression of the cyclin D1 gene product in the liver and bowel when compared with wild-type siblings. Both transgenic lines developed progressive liver disease. Examination of H&E stained sections of the liver and bowel revealed hyperplastic changes in the liver by 3 months of age. By 6 months of age, transgenic mice had obvious hepatomegaly and histological evidence of dysplasia in the liver. These early changes were significantly more dramatic in male animals when compared with female animals. By 9 months of age adenomas of the liver appeared, progressing to HCC over the ensuing 6-month period. By 15-17 months of age, 87% of male and 69% of female animals had either adenomatous nodules or HCCs. By 17 months of age, 31% of male and female animals had disease that had progressed to HCC. These animals represent a unique and significant new model for the study of human HCC. This study demonstrates that overexpression of cyclin D1 is sufficient to initiate hepatocellular carcinogenesis.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cyclin D1; DNA, Complementary; Female; Gene Expression Regulation, Neoplastic; Hepatomegaly; Intestinal Mucosa; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Rats; Sex Factors; Time Factors; Transgenes

Deranged expression of cell cycle modulators has been reported to contribute to the development and progression of hepatocellular carcinoma (HCC). However, their expression patterns remain poorly understood in hepatitis B virus (HBV)-related HCC, which constitutes about 65-70% of HCC in Korea. The aims of this study were to evaluate the expressions of G1-S modulators in HBV-related HCCs and dysplastic nodules (DNs), and to correlate with the histopathologic features of HCCs. Immunohistochemical expressions of cyclin D1, cyclin E, p53, p27, p21, p16, Rb, and PCNA proteins were investigated in 80 HCCs and 22 DNs. Cyclin D1 overexpression showed positive relationships with advanced tumor stage, poor differentiation, larger tumor size, microvascular invasion, intrahepatic meta-stasis, no tumor capsule formation, infiltrative growth, aberrant p53 expression, and high PCNA labeling index (LI) of HCC (p<0.05). Aberrant p53 expression showed positive relationship with poor differentiation of HCC (p<0.01). Expression of cyclin D1 or p53 was not observed in DNs. The p27 LI and p16 LI were lower in HCCs with intrahepatic metastasis (p<0.05). Cyclin D1 overexpression and aberrant p53 expression could be associated with the progression of HBV-related HCC, and might have a less crucial role in the DN-HCC sequence. In addition, elevated expression of p27 and p16 proteins might have inhibitory action to the intrahepatic metastasis of HBV-related HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; G1 Phase; Hepatitis B; Humans; Immunohistochemistry; Liver Neoplasms; Male; Microfilament Proteins; Middle Aged; Muscle Proteins; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Retinoblastoma Protein; S Phase; Tumor Suppressor Protein p53

Evaluation of the biological character of carcinomas requires understanding of cell cycle regulators. In the present study, we investigated the expression of p57 (Kip2) in 90 hepatocellular carcinomas and 66 noncancerous lesions. The average p57 labeling index in noncancerous lesions was 72.3 +/- 19.7. The labeling index significantly decreased (p < 0.0001) in hepatocellular carcinoma (54.9 +/- 19.7). It was significantly lower in hepatocellular carcinoma cases with high biological aggressiveness such as advanced stage (p = 0.0041), poor differentiation (p < 0.0001), larger size (p = 0.0400), portal invasion (p < 0.0001), satellite tumor (p = 0.0023), high proliferating activity (p = 0.0002) and cyclin D(1) overexpression (p = 0.0416). Furthermore, cases with low p57 expression showed worse outcomes for disease-free survival in univariate analysis (p = 0.0235), although p57 expression could not be recognized as an independent prognostic factor. These findings suggest that p57 contributes to the downregulation of cell proliferation and to the progression of hepatocellular carcinoma.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p57; Disease-Free Survival; Female; Hepatitis, Chronic; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Staging; Nuclear Proteins; Retrospective Studies; Survival Analysis

Mutation in cell cycle genes is the most common genetic change in malignant tumor cells. Telomerase activation, considered as essential in the immortality of cancer cells, is found in most cancers, where there may be an association with an active cell cycle.. In this study study we used the TRAP assay to determine telomerase activity in liver tumor specimens from 25 cases of hepatocellular carcinoma (HCCs) as well as in corresponding non-cancerous liver tissue in each patient. The expression of cyclin D1, cdk2, and cdk4 protein was also examined by Western blot.. Twenty-one of the 25 cases of HCC were found to have increased telomerase activity, whereas only five out of the 25 non-cancerous liver samples were found to have weak telomerase activity. Telomerase activity was not found to be related to tumor size, HBsAg, HBeAg, anti-HCV, transaminase, or alpha-fetoprotein serum titer. Furthermore, three out of the 25 cases of HCC showed cyclin D1 overexpression, whereas 15 of the 23 cases of HCC showed decreased cyclin D1 expression. Down regulation of cyclin D1, cdk2, cdk4 protein correlated with telomerase activity (p<0.004, p<0.013, and p<0.001 respectively).. The results indicate that genetic defects in HCC facilitate the reactivation of telomerase activity, a process which may be dependent on cyclin D1 with its cyclin dependent kinase (cdk) partner defect.

Topics: Adult; Aged; Blotting, Western; Carcinoma, Hepatocellular; CDC2-CDC28 Kinases; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Female; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Neoplasm Proteins; Nucleic Acid Amplification Techniques; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Telomerase

In hepatitis B virus-related hepatocellular carcinoma (HCC), at least 20-40 years of continuous necro-inflammation is necessary for the hepato-carcinogenesis to occur. However, HCC in childhood shows an unusually short latent period and rapid progression. In our previous report, mutation of c-met was found only in childhood HCC, but not in adult HCC. In order to determine the specific biological tumorous features of childhood HCC, a comparison study of childhood and adult HCC was performed.. Eighteen cases of HBV positive HCC (nine children and nine adults aged more than 40 years) were selected. The expression of G1 phase regulatory proteins (cyclin D1, cyclin E and cdk4) was studied using immunohistochemical methods. Loss of heterozygosity (LOH) on chromosomal arms 8p, 13q and 17p was analyzed.. Cyclin D1 expression was significantly lower in childhood HCC than in adult HCC (cases of cyclin D1 expression under 3+: childhood 5/9 vs. adult 1/9, p=0.046). No difference in cyclin E and cdk4 expression was found between childhood and adult HCC. LOH frequency on 13q was relatively higher in childhood than in adult HCC (66.7% vs. 22.2%, p=0.058). LOH frequency on 8p and 17p was 44.4% and 33.3% in childhood HCC and 44.4% and 75% in adult HCC with no statistical significance between the two groups.. Our data suggest that childhood HCC may be less dependent on cyclin D1 protein for tumor growth and progression than adult HCC, and that the LOH on 13q may be an important feature of childhood HCC.

Topics: Adolescent; Adult; Carcinoma, Hepatocellular; Cell Cycle Proteins; Child; Chromosomes, Human; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 8; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA, Neoplasm; Fluorescent Antibody Technique, Indirect; Hepatitis B virus; Humans; Liver Neoplasms; Loss of Heterozygosity; Middle Aged; Polymerase Chain Reaction; Proto-Oncogene Proteins

In spite of the importance of periodic screening for hepatocellular carcinoma (HCC) by ultrasonography (US) in patients with underlying liver disease, the clinicopathological characteristics of hyperechoic nodules have not been clearly evaluated. The aim of this study was to characterize the pathological and proliferating features of small hyperechoic nodules. Tissue specimens of 55 hyperechoic and 107 hypoechoic nodules less than 20 mm in diameter in patients with chronic liver disease were obtained by echo-guided needle biopsy and examined histopathologically. Of these, 42 (76%) hyperechoic and 56 (52%) hypoechoic nodules were diagnosed as HCC, and 82% of hyperechoic HCCs contained fatty change and/or clear cell change. In addition, immunohistochemical staining using cyclin D1, p53, and Ki-67 was examined. A high-level expression of cyclin D1 was found in only 5% of hyperechoic HCCs, in contrast to 38% of hypoechoic HCCs (P <.02). The labeling index of Ki-67 in hyperechoic HCCs was lower than in hypoechoic HCCs (4.2% vs. 8.9%; P <.003). However, there was no difference on p53 staining between them. Retrospective follow-up study revealed that hyperechoic nodules showed slow growth (doubling time, median: 1,403 days) initially, and came to show rapid growth (doubling time, median: 56 days). From these results, small hyperechoic nodules in chronic liver diseases are worth notice as candidates for well-differentiated HCC with low cyclin D1 and Ki-67 expression.

Topics: Adult; Aged; Aged, 80 and over; Biopsy, Needle; Carcinoma, Hepatocellular; Cell Nucleus; Cyclin D1; Female; Humans; Ki-67 Antigen; Liver; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Retrospective Studies; Tumor Suppressor Protein p53; Ultrasonography

The peroxisome proliferators are rodent non-genotoxic hepatocarcinogens that suppress apoptosis and induce DNA replication, cell proliferation and liver tumours. In order to investigate the effect of peroxisome proliferators on cell cycle progression, we arrested the well-differentiated rat hepatoma cell line FaO in the G1 phase of the cell cycle. Under these conditions, CDK2 and CDK4 protein expression remained unchanged compared with proliferating cells, but expression of cyclin D1 and p27(KIP1) was down-regulated and cyclin E accumulated in the inactive form. G1-arrested cells were able to enter the cell cycle on addition of exogenous growth factors such as epidermal growth factor (EGF) or hepatocyte growth factor (HGF) and replicate their DNA within 12 to 24 h of re-stimulation. Upon release from G1 arrest, CDK2 protein expression was down-regulated and, surprisingly, p27(KIP1) expression was restored. Cyclin D1 and phosphorylated cyclin E accumulated at 12 h but were degraded by 24 h after addition of EGF. Importantly, the peroxisome proliferator nafenopin and tumour necrosis factor alpha were able to induce DNA replication. Thus, the profile of expression of cell cycle regulatory proteins upon stimulation with nafenopin is comparable with that induced by growth factors such as EGF.

Topics: Animals; Butyrates; Carcinogens; Carcinoma, Hepatocellular; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; G1 Phase; Microtubule-Associated Proteins; Nafenopin; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rats; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins

Expression of cell-cycle modulators at the G1-S boundary, retinoblastoma gene product (pRb), p21, p16, p27, p53, cyclin D1, and cyclin E was investigated with 104 hepatocellular carcinomas (HCC), as well as 90 of their adjacent noncancerous lesions and 9 normal liver control specimens. The labeling indices (LI) of pRb, p21, p16, and p27 were higher in HCC lesions than in the adjacent noncancerous lesions and normal controls. Especially, p27 LI in noncancerous lesions was significantly higher than that in normal livers (P =.011). Aberrant p53 expression and cyclin D1 and E overexpression were observed exclusively in HCC lesions. pRb was positive in 85.6% of the HCC cases and was not related to any clinicopathological parameters. The p21 LI was generally low (average, 5.5 +/- 9.8). Although a negative regulator, p21 LI was higher in cases with intrahepatic metastasis (P =.0359). The p16 LI was significantly decreased (P =.0121) in cases with advanced stage. p27 LI was significantly decreased in cases with portal invasion (P =.0409), poor differentiation (P <.0001), larger size (P =.0421), and intrahepatic metastasis (P =.0878, borderline significance). On the other hand, aberrant p53 expression showed positive relationships with poor differentiation (P =.0004) and Ki-67 LI (P =. 0047). Cyclin D1 overexpression was found in 32.6% of the cases and occurred more frequently in those with high Ki-67 LI (P =.0032), pRb expression (P =.0202), poor differentiation (P =.0612, borderline significance), and intrahepatic metastasis (P =.0675, borderline significance). Cyclin E was overexpressed in 35.5% and had positive relationships with Ki-67 LI (P =.0269) and stage (P =.0125). In univariate analysis, cases with p27 LI < 50 (P =.0004), cyclin D1 overexpression (P =.0041), and cyclin E overexpression (P =.0572, borderline significance) showed poorer outcomes for disease-free survival (DFS). In multivariate analysis, p27 expression could be recognized as an independent prognostic marker for DFS. These findings suggest that in HCC: 1) p27 is active against HCC progression in early phases and, possibly, hepatocarcinogenesis as a negative regulator and can be a novel prognostic marker for DFS; and 2) cyclin D1 predominantly works for cell-cycle progression at the G1-S boundary.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Female; Follow-Up Studies; G1 Phase; Genes, Retinoblastoma; Humans; Liver Neoplasms; Male; Microtubule-Associated Proteins; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Prognosis; Recurrence; Retinoblastoma Protein; S Phase; Survival Analysis; Time Factors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The amplification and/or rearrangement of the cyclin D1 gene, a positive regulatory element of the G1 to S phase of the cell cycle, has been reported in various human tumors, suggesting an oncogenic role of this gene. In this study, we investigated the expression of cyclin D1 in the formalin-fixed and paraffin-embedded human hepatocellular carcinoma (HCC) tissues of 25 patients, using monoclonal antibody 5D4 raised against cyclin D1. Two distinct patterns of staining were observed in HCC cells, nuclear and cytoplasmic. The nuclear staining pattern of cyclin D1 was detected in the tissues of only 2 of the 25 HCC patients (8%) examined and no particular clinicopathological characteristics were found in these patients. In contrast, the cytoplasmic staining pattern, without nuclear staining, was detected in 8 of the 25 patients with HCC (32%). A significant correlation was found between the expression of cytoplasmic cyclin D1 and patients with tumor thrombus in the portal vein (Vp), as well as those with intrahepatic metastasis (IM). These results indicate that the cytoplasmic cyclin D1 expression appears to be related to the prognosis of HCC. The Ag nucleolar organizer regions (NORs) counts in cyclin D1-positive and -negative patients were not significantly different, suggesting that immunostaining for cyclin D1 has the potential to be a unique prognostic marker in human HCC. Simultaneous immunohistochemical study with p53 antibody in the same series of HCC revealed that 88% of the patients positive for cyclin D1 also expressed p53 and that in 91% of the patients negative for p53, cyclin D1 was not expressed. These results suggest that cyclin D1 is expressed later than the alteration of p53 in the progression of human HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cyclin D1; Cytoplasm; Female; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Nucleolus Organizer Region; Prognosis; Silver Staining; Tumor Suppressor Protein p53

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinases; Cytoplasm; Humans; Liver Neoplasms

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) is a key molecule in intracellular signal transducing pathways that transport extracellular stimuli from cell surface to nuclei. MAPK/ERK has been revealed to be involved in the physiological proliferation of mammalian cells and also to potentiate them to transform. However, its role in the outgrowth of human hepatocellular carcinoma (HCC) has yet to be clarified. Therefore, in this study, we investigated the activation of MAPK/ERK and its associated gene expression in HCC. MAPK/ERK was activated in 15 of 26 cases of HCC we examined (58%), and its activity level was significantly higher in HCC than in the adjacent non-cancerous lesions. Besides, MAPK/ERK activation in HCC was positively correlated with protein expression of transcription factor c-Fos. Furthermore, in 25 of 26 cases of HCC which genomic DNA was available, 22 cases without genomic DNA amplification exhibited positive correlation, not only between protein expression of c-Fos and cyclin D1, but also between MAPK/ERK activation and cyclin D1 expression. Concerning the relationship between MAPK/ERK activation and the clinicohistopathological features of HCC, the tumor (HCC) versus non-tumor (non-cancerous counterpart) ratio (T/N) of MAPK/ERK activity was positively correlated with tumor size, but neither with the stage of HCC nor the degree of differentiation of HCC. In conclusion, these findings suggest that MAPK/ERK activation in human HCC may play an important role in multistep hepatocarcinogenesis, especially in the progression of HCC; at least in part, through cyclin D1 up-regulation primarily induced by MAPK/ERK via c-Fos.

Topics: Aged; Aged, 80 and over; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Hepatocellular; Cyclin D1; Enzyme Activation; Female; Genes, fos; Genes, jun; Humans; Liver Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases

The major regulatory events leading to cell proliferation occur in the G1 phase of cell cycle, and the deranged expression of G1 cyclins is related to oncogenesis. In this study, we analyzed the aberrant expressions of cyclins D1 and E, and their role in hepatocellular carcinoma.. We examined paired hepatocellular carcinoma and liver RNAs taken from 71 patients who had been followed for more than 4 years after tumor resection, using reverse transcription-polymerase chain reaction supplemented with Northern blotting and immunohistochemistry. The genetic alterations of the p53 gene were also studied.. Downregulation of cyclin D1 mRNA was detected in 29 hepatocellular carcinomas (40.8%), while overexpression was detected in only 4 hepatocellular carcinomas (5.6%). Downregulation of cyclin D1 was associated significantly with large hepatocellular carcinomas (p=0.0006) and poorly differentiated (grades III-IV) hepatocellular carcinoma (p=0.057), but not seen in any of 15 minute hepatocellular carcinomas (< or =2.5 cm in size). Cyclin E mRNA was overexpressed in 40 hepatocellular carcinomas, regardless of tumor size. Overexpression of cyclin E was significantly associated with poorly differentiated and invasive hepatocellular carcinoma (p=0.001 and p=0.015, respectively). Downregulation of cyclin D1 and overexpression of cyclin E were significantly associated with the p53 mutation (p=0.023 and p=0.005, respectively). Hepatocellular carcinomas expressing both downregulation of cyclin D1 and overexpression of cyclin E had the worst 4-year survival (p<0.03), and higher frequencies of the p53 mutation (p<0.001), large hepatocellular carcinoma (p<0.001), and invasive tumor (p<0.01).. The deranged expressions of G1 cyclins correlate with the p53 mutation, tumor progression, and tumor biologic behavior of hepatocellular carcinoma. Overexpression of cyclin E occurs early, and downregulation of cyclin D1 late in hepatocellular carcinoma growth.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cyclin D1; Cyclin E; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Liver Neoplasms; Male; Middle Aged; Mutation; Neoplasm Invasiveness; Prognosis; Recurrence; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

A protein kinase inhibitor K252a suppressed the growth of HuH7 hepatoma cells and the hyperphosphorylation of retinoblastoma protein (pRb) at late G1 phase of cell cycle. However, K252a treatment did not alter the levels of cyclin D1, cyclin E, cyclin A and Cdk2 protein bound to cyclin E or cyclin A. Therefore, the K252a inhibition of pRb phosphorylation is considered to be brought about probably by inhibiting the action of Cdk-cyclin complex rather than by changing its cellular level. These results also suggest that K252a is a useful tool for investigating the mechanism of phosphorylation of pRb mediated by Cdk-cyclin.

Topics: Carbazoles; Carcinoma, Hepatocellular; CDC2-CDC28 Kinases; Cell Cycle; Cell Line; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Humans; Indole Alkaloids; Kinetics; Liver Neoplasms; Oncogene Proteins; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Retinoblastoma Protein; Time Factors; Tumor Cells, Cultured

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

We analyzed the genetic alterations of the cyclin D1 and INT-2 genes in hepatocellular carcinomas (HCCs) from 45 patients. Among these, expression of the cyclin D1 mRNA was also analyzed in 18 of them by Northern blotting. The cyclin D1 gene was amplified 3-16 fold in five HCCs (11%); among these, the INT-2 gene was also amplified 2-10 fold in four HCCs. We analyzed the mRNA of cyclin D1 in four HCCs with gene amplifications, and 6-10 fold overexpressions were detected in all of them. Because the cyclin D1 gene was amplified in patients at an advanced stage of HCC with rapid tumor growth, it appeared to be associated with the aggressive behavior of tumors. Studies on loss of heterozygosity on chromosome 13q, where the retinoblastoma (RB) gene is located, indicated that all HCCs with an amplified cyclin D1 gene retained heterozygosity on chromosome 13q. These results suggest that amplification and overexpression of the cyclin D1 gene result in the rapid growth of a subset of HCC, even though the function of the RB gene is retained.

Topics: Carcinoma, Hepatocellular; Chromosome Deletion; Chromosomes, Human, Pair 13; Cyclin D1; Cyclins; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Gene Amplification; Gene Expression; Humans; Liver Neoplasms; Oncogene Proteins; Proto-Oncogene Proteins; RNA, Messenger

Amplification of the chromosome 11q13 region occurs in several types of human cancer including esophageal, breast, lung, bladder and hepatocellular carcinoma (HCC). The gene cyclin D1 maps to this region in close proximity to two proto-oncogenes hst-1 and int-2. We previously demonstrated that cyclin D1 was not only amplified but also overexpressed in about 30% of human esophageal cancers. To investigate the role of cyclin D1 in human hepatocellular carcinoma (HCC), DNA from 30 HCC and 5 control liver tissues from Taiwan and also the HCC cells lines HepG2 and Hep3B, were examined for amplification of the cyclin D1 gene. A 3 to 20-fold amplification was found in 4 of the 30 (13%) HCC samples but not in any of the 5 control tissues or the 2 cell lines. Immunohistochemical analysis of cyclin D1 indicated overexpression of this protein in tumors that displayed gene amplification. Weak or negative staining was observed in the other HCC samples as well as in the control tissues and cell lines. These data suggest that increased expression of cyclin D1 may play an important role in the development of a subset of human HCC, perhaps by perturbing normal control of the cell cycle.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Hepatocellular; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; DNA; DNA, Neoplasm; Gene Amplification; Gene Expression; Humans; Immunohistochemistry; Liver Neoplasms; Oncogene Proteins; RNA, Neoplasm