tretinoin and Carcinoma--Hepatocellular

Processed from natural minerals, arsenic trioxide (ATO) as an ancient Chinese medicine has been used to treat diseases for over 2000 years. And it was applied to treat acute promyelocytic leukemia (APL) since the 1970s in China. Summarizing the clinical evidence of ATO in cancer is conducive to further understanding, development, and promotion of its pharmacological research.. It is the first time to comprehensively assess and summarize the evidence of ATO in cancer treatment via umbrella review.. 8 databases in English or Chinese from their inception to February 21, 2023 were searched by two reviewers separately and suitable meta-analyses (MAs) were included in this umbrella review. Their methodological quality and risk of bias were evaluated and data of outcomes was extracted and pooled again. The evidence certainty of pooled results was classified.. 17 MAs with 27 outcomes and seven comparisons in three cancers were included in this umbrella review. However, their methodological quality was unsatisfactory with 6 MAs as low quality and 12 MAs as critically low quality. Their shortcomings were mainly focused on protocol, literature selecting, bias risk, small sample study bias, and conflicts of interest or funding. And they were all assessed as high risk in bias. It was suggested that ATO had an advantage in enhancing complete remission rate, event-free survival, and recurrence free survival and decreasing recurrence rate, cutaneous toxicity, hyper leukocyte syndrome, tretinoin syndrome, edema and hepatotoxicity in different comparisons of APL with low or moderate certainty. Besides, compared with transcatheter arterial chemoembolization (TACE) alone, ATO plus TACE also could improve objective response rate, disease control rate, survival rate (0.5, 1, 2, and 3-year) and life quality and reduce the level of alpha fetoprotein in primarily hepatocellular carcinoma with low or moderate certainty. However, no significant results were found in MM. Finally, key findings were as followed. ATO has potential broad-spectrum anticancer effects but the clinical transformation is rarely achieved. Route of administration may affect the antitumor effects of ATO. ATO can act synergistically in combination with a variety of antitumor therapies. The safety and drug resistance of ATO should be paid more attention to.. ATO may be a promising drug in anticancer treatment although earlier RCTs have dragged down the level of evidence. However, high-quality clinical trials are expected to explore its broad-spectrum anticancer effects, wide application, appropriate route of administration, and compound dosage form.

Topics: Arsenic Trioxide; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Humans; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Randomized Controlled Trials as Topic; Tretinoin

There is a need for agents that eliminate cancer stem cells, which sustain cancer and are also largely responsible for disease relapse and metastasis. Conventional chemotherapeutics and radiotherapy are often highly effective against the bulk of cancer cells, which are proliferating, but spare cancer stem cells. Therapeutics that target cancer stem cells may also provide a

Topics: Carcinoma, Hepatocellular; Child; Humans; Liver Neoplasms; Male; Necroptosis; Neoplastic Stem Cells; Prostatic Neoplasms; Receptors, Retinoic Acid; Tretinoin

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

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Obesity and its related metabolic disorders are serious health problems worldwide, and lead to various health-related complications, including cancer. Among human cancers, hepatocellular carcinoma (HCC) is one of the most common malignancies affected by obesity. Therefore, obesity and its related disorders might be a key target for the prevention of HCC. Recently, new research indicates that the molecular abnormalities associated with obesity, including insulin resistance/hyperinsulinemia, chronic inflammation, adipokine imbalance, and oxidative stress, are possible molecular mechanisms underlying the pathogenesis of obesity-related hepatocarcinogenesis. Green tea catechins and branched-chain amino acids, both of which are classified as nutraceutical agents, have been reported to prevent obesity-related HCC development by improving metabolic abnormalities. The administration of acyclic retinoid, a pharmaceutical agent, reduced the incidence of HCC in obese and diabetic mice, and was also associated with improvements in insulin resistance and chronic inflammation. In this article, we review the detailed molecular mechanisms that link obesity to the development of HCC in obese individuals. We also summarize recent evidence from experimental and clinical studies using either nutraceutical or pharmaceutical agents, and suggest that nutraceutical and pharmaceutical approaches targeting metabolic abnormalities might be a promising strategy to prevent the development of obesity-related HCC.

Topics: Amino Acids, Branched-Chain; Animals; Carcinoma, Hepatocellular; Catechin; Chemoprevention; Diabetes Mellitus, Experimental; Dietary Supplements; Humans; Liver Neoplasms; Mice; Non-alcoholic Fatty Liver Disease; Obesity; Tea; Tretinoin

The poor prognosis for patients with hepatocellular carcinoma (HCC) is associated with its high rate of recurrence in the cirrhotic liver. Therefore, more effective strategies need to be urgently developed for the chemoprevention of this malignancy. The malfunction of retinoid X receptor α, a retinoid receptor, due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), a synthetic retinoid, can prevent HCC development by inhibiting retinoid X receptor α phosphorylation and improve the prognosis for this malignancy. Supplementation with branched-chain amino acids (BCAA), which are used to improve protein malnutrition in patients with liver cirrhosis, can also reduce the risk of HCC in obese cirrhotic patients. In experimental studies, both ACR and BCAA exert suppressive effects on HCC development and the growth of HCC cells. In particular, combined treatment with ACR and BCAA cooperatively inhibits the growth of HCC cells. Furthermore, ACR and BCAA inhibit liver tumorigenesis associated with obesity and diabetes, both of which are critical risk factors for HCC development. These findings suggest that pharmaceutical and nutraceutical approaches using ACR and BCAA may be promising strategies for preventing HCC and improving the prognosis of this malignancy.

Topics: Amino Acids, Branched-Chain; Carcinoma, Hepatocellular; Chronic Disease; Clinical Trials as Topic; Dietary Supplements; Humans; Liver Diseases; Liver Neoplasms; Phosphorylation; Retinoid X Receptor alpha; Retinoid X Receptors; Retinoids; Tretinoin

Quiescent hepatic stellate cells (HSCs) in healthy liver store 80% of total liver retinols and release them depending on the extracellular retinol status. However, HSCs activated by liver injury lose their retinols and produce a considerable amount of extracellular matrix, subsequently leading to liver fibrosis. Emerging evidence suggests that retinols and their metabolites such as retinoic acids (RAs) contribute to liver regeneration, fibrosis and tumor. However, it is not clear yet why HSCs lose retinol, which enzymes are involved in the retinol metabolism of HSCs and what function of retinol metabolites on HSCs upon liver injury. Recently, our group and collaborators have demonstrated that during activation, HSCs not only lose retinols but also metabolize them into RAs by alcohol dehydrogenases and retinaldehyde dehydrogenases. As transcriptional factors, metabolized RAs induce retinoic acid early inducible-1 and suppressor of cytokine signaling 1 in HSCs, which plays an important role in the interaction between HSCs and natural killer cells. In addition, RAs released from HSCs may induce hepatic cannabinoid receptor 1 expression in alcoholic liver steatosis or regulate immune responses upon liver inflammation. The present review summarizes the role of endogenous metabolized RAs on HSCs themselves and on other liver cells including hepatocytes and immune cells. Moreover, the effects of exogenous retinol and RA treatments on HSCs and liver disease are discussed.

Topics: Animals; Carcinoma, Hepatocellular; Extracellular Matrix; Fatty Liver; Hepatic Stellate Cells; Hepatitis; Humans; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Liver Regeneration; Tretinoin; Vitamin A

The poor prognosis for hepatocellular carcinoma (HCC) is associated with its high rate of recurrence in the cirrhotic liver. Therefore, development of effective strategies for preventing recurrence and secondary tumors will improve the clinical outcome of HCC patients. A malfunction of the retinoid X receptor-α (RXRα) due to phosphorylation by the Ras-MAPK signaling pathway is profoundly associated with liver carcinogenesis, and thus, may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), which inhibits Ras-MAPK activation and RXRα phosphorylation, successfully prevents HCC recurrence, thus improving patient survival. The fundamental concept of HCC chemoprevention by ACR is "clonal deletion," which is defined as the removal of latent malignant clones from the liver before they expand into clinically detectable HCC. "Combination chemoprevention" using ACR as a key drug holds great promise of a new effective strategy for the prevention of HCC because of its synergism. ACR is also expected to prevent the development of HCC in obese people, who are at an increased risk to HCC, because this agent significantly inhibits obesity-related liver tumorigenesis in the rodent model. Here, we review the detailed effects of ACR on preventing HCC development, especially based on the results of our basic and clinical research.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Clonal Deletion; Humans; Liver Neoplasms; Receptors, Retinoic Acid; Retinoid X Receptors; Tretinoin

The prognosis for patients with hepatocellular carcinoma (HCC) is poor and effective prevention strategies are urgently required. Here, we review abnormalities in the expression and function of retinoids and their receptors, and how they play a critical role in the development of HCC. In particular, a malfunction of RXRalpha due to phosphorylation by Ras-MAPK signaling pathway is profoundly associated with liver carcinogenesis and thus may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), a synthetic retinoid, inhibits Ras-MAPK activation and RXRalpha phosphorylation, thereby suppressing growth in HCC-derived cells. In clinical trials, ACR has been shown to improve patient survival by preventing viral HCC development, a possible manifestation of the concept of "clonal deletion" therapy. "Combination chemoprevention" with ACR as the key drug has great potential to become an effective strategy for the prevention of liver carcinogenesis. In summary, both basic and clinical research strongly suggest that ACR plays a critical role in preventing the development of HCC and that "clonal deletion" therapy is one of the most practical approaches for this purpose.

Topics: Carcinoma, Hepatocellular; Chemoprevention; Clinical Trials as Topic; Clonal Deletion; Humans; Liver Neoplasms; Receptors, Retinoic Acid; Retinoid X Receptors; Tretinoin

Hepatocellular carcinoma (HCC) is a major health care problem worldwide. The prognosis of patients with HCC is poor because even in the early stages when surgical treatment might be expected to be curative, the incidence of recurrence in patients with underlying cirrhosis is very high due to multicentric carcinogenesis. Therefore, strategies to prevent recurrence and second primary HCC are required to improve the prognosis. One of the most practical approaches to prevent the multicentric development of HCC is 'clonal deletion' therapy, which is defined as the removal of latent (i.e. invisible) (pre)malignant clones from the liver in a hypercarcinogenic state. Retinoids, a group of structural and functional analogs of vitamin A, exert their biological function primarily through two distinct nuclear receptors, retinoic acid receptors and retinoid X receptors (RXR), and abnormalities in the expression and function of these receptors are highly associated with the development of various cancers, including HCC. In particular, a malfunction of RXRalpha due to phosphorylation by the Ras-mitogen-activated protein kinase signaling pathway is profoundly associated with the development of HCC and thus may be a critical target for HCC chemoprevention. Acyclic retinoid, which has been clinically shown to reduce the incidence of a post-therapeutic recurrence of HCC, can inhibit Ras activity and phosphorylation of the extracellular signal-regulated kinase and RXRalpha proteins. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its physiological function as a master regulator for nuclear receptors may be a potentially effective strategy for HCC chemoprevention and clonal deletion. Acyclic retinoid, which targets phosphorylated RXRalpha, may thus play a critical role in preventing the development of multicentric HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Chemoprevention; Humans; Liver Neoplasms; Phosphorylation; Retinoid X Receptor alpha; Tretinoin

Topics: Amino Acids, Branched-Chain; Angiotensin-Converting Enzyme Inhibitors; Antineoplastic Agents; Carcinoma, Hepatocellular; Diabetes Mellitus; Humans; Immunotherapy, Adoptive; Interferons; Lamivudine; Liver Neoplasms; Neoadjuvant Therapy; Neoplasm Recurrence, Local; Obesity; Randomized Controlled Trials as Topic; Risk Factors; Tretinoin; Vitamin K

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Receptors, Retinoic Acid; Retinoid X Receptors; Tretinoin

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer deaths. Surgical resection, with or without transplantation, can result in long-term survival. However, surgery can only be performed in about 15% of patients with HCC and the 5-year survival rate is only approximately 33%-50% after potentially curative resection. Percutaneous ethanol injection, radiofrequency ablation, and transarterial chemoembolization are invasive techniques that have shown efficacy in reducing tumor bulk. Similarly, systemic chemotherapy may induce tumor responses, but a survival benefit has not been clearly demonstrated. In addition, the lack of efficacy of antiandrogens, tamoxifen, and single-agent interferon has now been confirmed.Sorafenib is a multikinase inhibitor with antiangiogenic, proapoptotic, and Raf kinase inhibitory activity. In a large, multicenter, randomized, phase 3 trial there was a significant improvement in both time to disease progression and overall survival with sorafenib compared with placebo. Sorafenib is the first agent to demonstrate a consistent improvement in overall survival for patients with advanced HCC. Further studies are required to determine the role of other molecular-targeted therapies, either alone or in combination with sorafenib in patients with advanced HCC. Further studies are also required to determine the role of sorafenib in combination with locoregional therapies (eg, transarterial chemoembolization), and the role of sorafenib as adjuvant therapy following surgery.

Topics: Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Catheter Ablation; Chemoembolization, Therapeutic; Estrogens; Humans; Injections, Intravenous; Interferons; Liver Neoplasms; Niacinamide; Palliative Care; Phenylurea Compounds; Pyridines; Risk Factors; Sorafenib; Tretinoin

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Chemoprevention; Fibroblast Growth Factors; Humans; Liver Neoplasms; Signal Transduction; Tretinoin

More than 18 million adults in the United States abuse alcohol, a prevalence 5 times higher than that of hepatitis C. Chronic alcohol use of greater than 80 g/day for more than 10 years increases the risk for hepatocellular carcinoma (HCC) approximately 5-fold; alcohol use of less than 80 g/day is associated with a nonsignificant increased risk for HCC. The risk for HCC in decompensated alcohol induced cirrhosis approaches 1% per year. The risk does not decrease with abstinence, and HCC can occur in a noncirrhotic liver. Alcohol use in chronic hepatitis C doubles the risk for HCC as compared with the risk in hepatitis C alone. Furthermore, there may be synergism between alcohol and hepatitis C in the development of HCC, and in these patients HCC may occur at an earlier age and the HCC may be histologically more advanced. Studies in the United States and Italy suggest that alcohol is the most common cause of HCC (accounting for 32%-45% of HCC). The mechanisms by which alcohol causes HCC are incompletely understood, but may include chromosomal loss, oxidative stress, a decreased retinoic acid level in the liver, altered DNA methylation, and genetic susceptibility. Alcohol use is increasing in many countries, suggesting that alcohol will continue to be a common cause of HCC throughout the world.

Topics: Alcohol Drinking; Carcinoma, Hepatocellular; Chromosome Aberrations; DNA Methylation; Hepatitis C, Chronic; Humans; Incidence; Liver Cirrhosis, Alcoholic; Liver Neoplasms; Oxidative Stress; Risk Factors; Tretinoin; United States

Hepatocellular carcinoma (HCC) develops frequently in the liver of patients with chronic hepatitis and cirrhosis caused by persistent infection with hepatitis B virus or hepatitis C virus in Asia. Several studies including ours have revealed that the risk of HCC increases in parallel with the progression of hepatic fibrosis associated with chronic hepatitis and liver cirrhosis. It may be delineated reasonably, therefore, that suppression of fibrosis by chemical (or biological) agents would be able to decrease the risk of HCC. We have demonstrated that antifibrotic agents, such as HOE 077, TJ-9 and interferon, can reduce the risk of HCC. Accordingly, antifibrotic agents are promising candidates for chemoprevention of HCC. The results of our study are discussed within the scope of the current state of the art as regards chemopreventing HCC.

Topics: Animals; Antineoplastic Agents; Antiviral Agents; Carcinoma, Hepatocellular; Chemoprevention; Drugs, Chinese Herbal; Forecasting; Hepatitis B; Hepatitis C; Humans; Interferons; Liver Cirrhosis, Experimental; Liver Neoplasms; Pyridines; Tretinoin

Topics: Anticarcinogenic Agents; Carcinoma, Hepatocellular; Chemoprevention; Humans; Liver Neoplasms; Tretinoin

Hepatocellular carcinoma (HCC) often develops in patients with chronic liver diseases associated with hepatitis B (HBV) and hepatitis C (HCV) virus infections with high incidences. Particularly, post-therapeutic recurrence encountered after the curative treatment of the preceding HCC may limit the prognosis. Thus, prevention of HCC is of great significance. In the present review, immunopreventions with alpha-interferon and glycyrrhizin, as well as chemoprevention with acyclic retinoid, are discussed. alpha-Interferon prevents the development of HCC not only in patients with a long-term elimination of HCV (sustained virological responders), but in ones with normalized serum aminotransferases (sustained biochemical responders). Glycyrrhizin also suppresses serum aminotransferases and thereby prevents the tumor development, even though the compound does not have antiviral activity for HBV or HCV by itself. Therefore, suppression of hepatic necroinflammation by these drugs may serve to prevent hepatocarcinogenesis. In contrast, acyclic retinoid suppresses the post-therapeutic recurrence in cirrhotic patients who underwent curative treatment of preceding tumors. The retinoid induces the disappearance of serum lectin-reactive alpha-fetoprotein (AFP-L3), a tumor marker indicating the presence of unrecognizable tumors in the remnant liver, suggesting a deletion of such minute (pre)malignant clones (clonal deletion). As a molecular mechanism of the clonal deletion, a novel mechanism of apoptosis induction by the retinoid via tissue transglutaminase is implicated. In future, a combination of immunopreventive and chemopreventive therapies may give a clue to the further advances of cancer prevention, and thereby to the improvement of the prognosis of cirrhotic patients.

Topics: alpha-Fetoproteins; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Carcinoma, Hepatocellular; Clonal Deletion; Glycyrrhizic Acid; Humans; Interferon-alpha; Liver Neoplasms; Prognosis; Tretinoin

Pre- and/or postoperative adjuvant therapy for hepatocellular carcinoma (HCC) is discussed. There is a high recurrence rate of HCC of up to 50% or more within three years after hepatectomy. More than 80% of those recurrences are in the form of intrahepatic metastases. Therefore it is extremely important to administer successful adjuvant therapy to prevent intrahepatic recurrence. There are two types of intrahepatic recurrence: simple dissemination from the primary focus of HCC; and newly developed HCC in the remnant liver. TAE is one option for preoperative adjuvant therapy to prevent intrahepatic recurrence. Postoperative adjuvant chemotherapy via the hepatic artery has occasionally been administered, but it is not yet established as an effective adjuvant therapy. However, a report by Muto et al showed that retinoid administration can prevent intrahepatic recurrence of newly developed HCC after hepatectomy. On the other hand, adjuvant therapy must not be tooaggressive, because: 1) HCC develops mainly in cirrhotic liver (with poor liver function); and 2) locoregional therapy for intrahepatic recurrence results in good survival rats even after detection of an established recurrence.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Combined Modality Therapy; Disease-Free Survival; Embolization, Therapeutic; Humans; Immunotherapy; Infusions, Intra-Arterial; Liver Neoplasms; Neoplasm Recurrence, Local; Postoperative Care; Preoperative Care; Tretinoin

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Neoplasms, Second Primary; Tretinoin

Structurally and functionally altered retinoic acid receptors have been associated with rare human neoplasms: acute promyelocytic leukemia and hepatocellular carcinoma. Whereas the retinoic acid receptor beta (RARbeta) rearrangement in hepatocellular carcinoma is unique, in acute promyelocytic leukemia (APL), RARalpha fusion to the promyelocytic leukemia (PML) gene by the t(15;17) translocation is a general feature of the disease. APL is an important model in cancer biology because retinoic acid induces complete remissions in this malignancy, providing the first example of differentiation therapy and of an antineoplastic drug directly targeted at the underlying genetic lesion. The molecular basis of PML/RARalpha fusion leukemogenesis is discussed with respect to dominant negative inhibition of nuclear receptor and PML functions.

Topics: Animals; Carcinoma, Hepatocellular; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Humans; Leukemia, Myeloid; Liver Neoplasms; Mice; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Carrier Proteins; Chick Embryo; DNA; Gene Expression Regulation, Neoplastic; Leukemia, Promyelocytic, Acute; Mice; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Proteins; Nuclear Proteins; Receptors, Cell Surface; Receptors, Retinoic Acid; Retinoid X Receptors; Retinol-Binding Proteins; Transcription Factors; Transcription, Genetic; Tretinoin

Chronic hepatitis B virus (HBV) infection is etiologically related to human hepatocellular carcinoma (HCC). Most HCCs contain integrated HBV DNA in the liver cellular DNA, suggesting that the integration may be involved in carcinogenesis. From a comparison of a single HBV integration site present in a hepatoma with the corresponding unoccupied site in the non-tumourous tissue of the same liver, we have shown that HBV DNA inserted in a putative cellular exon with striking similarity to the DNA-binding domain of the thyroid/steroid hormone receptors. The corresponding cDNA has been isolated (hap gene) and shown to encode the retinoic acid receptor. In the original patient, integration took place so that the first codons of the viral surface protein gene became fused in frame with most of the hap gene. Because retinoic acid is known to regulate the transcription of target genes crucial for cellular growth and differentiation, it is most probable that consequent to the HBV insertion, hap, usually transcribed at a very low level in normal hepatocytes, became inappropriately expressed as an altered chimaeric retinoic acid receptor, thus contributing to the cell transformation. These results strongly support the possibility that HBV may play a direct role in liver carcinogenesis by insertional mutagenesis.

Topics: Amino Acid Sequence; Base Sequence; Carcinoma, Hepatocellular; Carrier Proteins; Cell Transformation, Neoplastic; DNA; DNA, Neoplasm; DNA, Viral; Exons; Gene Expression Regulation, Neoplastic; Genes, Viral; Hepatitis B; Hepatitis B virus; Humans; Liver Neoplasms; Molecular Sequence Data; Mutation; Organ Specificity; Receptors, Retinoic Acid; Recombination, Genetic; Tretinoin

The majority of hepatocellular carcinoma (HCC) cases are diagnosed at an advanced stage. Currently, there are only a few therapeutic methods available for patients with advanced HCC and extrahepatic metastasis (EHM). Systemic chemotherapy, such as FOLFOX4 (infusions of fluorouracil, leucovorin, and oxaliplatin), has been reported for treating advanced HCC with EHM, but its effectiveness is very poor. In this randomized, double-blind, placebo-controlled study, we aimed to assess the efficacy and safety of FOLFOX4 with all-trans-retinoic acid (ATRA) as a palliative treatment for HCC patients with EHM, compared to FOLFOX4 with a placebo. The primary endpoint was overall survival (OS), and subsequently, an exploratory model was developed based on bioinformatics to predict the efficacy of FOLFOX4-ATRA treatment. A total of 108 patients were randomly assigned in a 1:1 ratio to receive either FOLFOX4-ATRA or FOLFOX4-placebo. The intention-to-treat (ITT) population showed a median OS of 16.2 months for the FOLFOX4-ATRA group, compared with 10.7 months for the FOLFOX4-placebo group (HR 0.56, 95% CI 0.33-0.93; p = 0.025). The median progression-free survival (PFS) was 7.1 months for the FOLFOX4-ATRA group and 4.2 months for the FOLFOX4-placebo group (HR 0.62, 95% CI 0.41-0.94; p = 0.024). A panel of proteins with unique upregulation during complete response (CR) (SOD3, TTR, SSC5D, GP5, IGKV1D-33) and partial response (PR) (TGFB1, GSS, IGHV5-10-1) effectively predicted CR and PR in patients treated with FOLFOX4-ATRA, as compared to FOLFOX4-placebo. The results suggest that FOLFOX4-ATRA is a safe and effective treatment for patients with advanced HCC and EHM in eastern China.

Topics: Carcinoma, Hepatocellular; Fluorouracil; Humans; Liver Neoplasms; Treatment Outcome; Tretinoin

Among patients with hepatocellular carcinoma (HCC), 85% of patients have an advanced disease stage at diagnosis and curative therapies cannot be performed. Prognosis has been quite poor as until recently there was no proven effective chemotherapy. Our group found that all-trans-retinoic acid (ATRA) could improve the efficacy of platinum in HCC in vivo and in vitro, thus we wish to validate the efficiency of ATRA in clinical practice.. This is a double-blinded, 1:1 randomized, controlled, multicenter clinical trial. Three hundred and sixty-eight patients with HCC and extrahepatic metastases will receive palliative chemotherapy at the Eastern Hepatobiliary Surgery Hospital, First Hospital of Jilin University and Fujian Provincial Cancer Hospital. Subjects will be randomly assigned to one of the two arms, either ATRA + oxaliplatin + 5-fluorouracil/leucovorin (FOLFOX4) or FOLFOX4 alone. ATRA 20 mg will be given orally three times/day for 3 days prior to the initiation of FOLFOX4. ATRA will be discontinued at the end of FOLFOX4.. Overall survival rate is the primary endpoint. Secondary endpoints are time to progression according to the modified response evaluation criteria in solid tumors (mRECIST) criteria, acute and chronic adverse events, and quality of life.. Chinese Clinical Trial Registry, ChiCTR-IIR-17012916 . Registered on 9 October 2017.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; China; Disease Progression; Double-Blind Method; Female; Fluorouracil; Humans; Leucovorin; Liver Neoplasms; Male; Multicenter Studies as Topic; Organoplatinum Compounds; Palliative Care; Progression-Free Survival; Randomized Controlled Trials as Topic; Time Factors; Tretinoin

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; 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Oral administration with acyclic retinoid, a synthetic vitamin A analog, for a limited period of 12 months (48 weeks) prevented the development of second primary hepatocellular carcinoma (HCC) and also improved the survival of patients who underwent curative treatments of the initial tumor. Following that randomized controlled study reported in 1996 and 1999, we have continued to follow up the patients by medical imaging and blood chemical analyses, and found that the preventive effect of acyclic retinoid lasted up to 199 weeks after randomization (or 151 weeks after completion of retinoid administration). The retinoid's effect was not mediated by reduction in hepatic necro-inflammation since no significant decrease in serum aminotransferase activity was seen in the retinoid group. Such observation seems quite distinct from the cancer-preventive mechanism of interferon, a potent immunopreventive agent for HCC. We have also shown here the reduction by the retinoid in serum levels of lectin-reactive alpha-fetoprotein (AFP-L3) and protein induced by vitamin K absence or antagonist-II (PIVKA-II), both of which indicate the presence of latent HCC cells. These results suggest that acyclic retinoid may delete such malignant clones before they expand to clinically detectable tumors and thereby inhibited second primary HCC. Once such latent clones are eradicated, it may well take at least several years for the next cancer clone to arise clinically. This may possibly explain a reason for the long-term effect of the retinoid even after the limited period of administration.

Topics: alpha-Fetoproteins; Antineoplastic Agents; Aspartate Aminotransferases; Biomarkers; Carcinoma, Hepatocellular; Humans; Japan; Liver Neoplasms; Neoplasms, Second Primary; Protein Precursors; Prothrombin; Survival Analysis; Tretinoin

Twenty-nine chemotherapy-naive patients with primary hepatocellular carcinoma were treated with oral beta-all trans-retinoic acid (retinoic acid, TRA 50 mg/m2 t.i.d.) on a 3-week on/one week off schedule until progression or grade 3 or 4 toxicity. Eligibility requirements allowed abnormal liver function tests as long as the creatinine and bilirubin levels were normal. No responses were seen and the median survival was four months. Grade 3 side effects occurred in II patients and grade 4 in four and included a wide range of toxicities. The results indicate that oral TRA is ineffective against primary hepatocellular carcinoma and suggest that dose-modification of this retinoid may be required in patients with significant malignant hepatic involvement.

Topics: Administration, Oral; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Survival Rate; Tretinoin

A goal of cancer chemoprevention is the deletion of latent premalignant or malignant clones before they expand to a clinically detectable tumor. However, such clonal deletion has not been demonstrated in clinical studies. We have evaluated serum levels of lectin-reactive alpha-fetoprotein (AFP-L3), which suggests the presence of latent hepatoma cells, in a randomized controlled trial that used acyclic retinoid to prevent second primary hepatomas in patients who had received treatments that cured initial hepatomas. The trial involved 21 patients in each acyclic retinoid (600 mg daily) and placebo group and consisted of a 12-month period of drug administration and a subsequent follow-up period. Serum AFP-L3 was determined at entry and at the end of the 12-month treatment period using lectin-affinity electrophoresis and antibody-affinity blotting. Although neither treatment affected serum levels of total AFP, acyclic retinoid significantly reduced AFP-L3 levels after a 12-month administration (P < 0.01). Acyclic retinoid not only deleted AFP-L3 from patients who had been positive for AFP-L3 at entry but also prevented the appearance of AFP-L3 in patients who had been negative at entry (P < 0.01). In contrast, placebo significantly raised the incidence of AFP-L3-positive patients after a 12-month administration from that at entry (P < 0.05). Patients positive for AFP-L3 after a 12-month treatment had a significantly higher risk of second primary hepatomas in the subsequent follow-up period (P = 0.03). Acyclic retinoid may have deleted a clone of latent hepatoma cells producing AFP-L3 and thereby inhibited second primary hepatomas. Serum AFP-L3 may be a useful intermediate biomarker in the chemoprevention of second primary hepatomas by acyclic retinoid.

Topics: alpha-Fetoproteins; Antineoplastic Agents; Carcinoma, Hepatocellular; Disease-Free Survival; Female; Follow-Up Studies; Humans; Lectins; Liver Neoplasms; Male; Middle Aged; Neoplasms, Second Primary; Placebos; Time Factors; Tretinoin

In patients with hepatocellular carcinoma (hepatoma), the rate of recurrent and second primary hepatomas is high despite surgical resection and percutaneous ethanol-injection therapy. We developed an acyclic retinoid, polyprenoic acid, that inhibits hepatocarcinogenesis in the laboratory and induces differentiation and apoptosis in cell lines derived from human hepatoma. In a randomized, controlled study, we tested whether the compound reduced the incidence of recurrent and second primary hepatomas after curative treatment.. We prospectively studied 89 patients who were free of disease after surgical resection of a primary hepatoma or the percutaneous injection of ethanol. We randomly assigned the patients to receive either polyprenoic acid (600 mg daily) or placebo for 12 months. We studied the remnant liver by ultrasonography every three months after randomization. The primary end point of the study was the appearance of a histologically confirmed recurrent or new hepatoma.. Treatment with polyprenoic acid significantly reduced the incidence of recurrent or new hepatomas. After a median follow-up of 38 months, 12 patients in the polyprenoic acid group (27 percent) had recurrent or new hepatomas as compared with 22 patients in the placebo group (49 percent, P = 0.04). The most striking difference was in the groups that had second primary hepatomas--7 in the group receiving polyprenoic acid as compared with 20 in the placebo group (P = 0.04 by the log-rank test). Cox proportional-hazards analysis demonstrated that as an independent factor, polyprenoic acid reduced the occurrence of second primary hepatomas (adjusted relative risk, 0.31; 95 percent confidence interval, 0.12 to 0.78).. Oral polyprenoic acid prevents second primary hepatomas after surgical resection of the original tumor or the percutaneous injection of ethanol.

Topics: Aged; Carcinoma, Hepatocellular; Embolization, Therapeutic; Ethanol; Female; Follow-Up Studies; Humans; Injections, Intralesional; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasms, Second Primary; Proportional Hazards Models; Prospective Studies; Survival Analysis; Treatment Outcome; Tretinoin

A surfeit of mitochondrial reactive oxygen species (ROS) and inflammation serve as obligatory mediators of lipid-associated hepatocellular maladies. While retinoid homeostasis is essential in restoring systemic energy balance, its role in hepatic mitochondrial function remains elusive. The role of lecithin-retinol acyltransferase (LRAT) in maintenance of retinoid homeostasis is appreciated earlier; however, its role in modulating retinoic acid (RA) bioavailability upon lipid-imposition is unexplored. We identified LRAT overexpression in high-fat diet (HFD)-fed rats and palmitate-treated hepatoma cells. Elevation in LRAT expression depletes RA production and deregulates RA signaling. This altered RA metabolism enhances fat accumulation, accompanied by inflammation that leads to impaired mitochondrial function through enhanced ROS generation. Hence, LRAT inhibition could be a novel approach preventing lipid-induced mitochondrial dysfunction in hepatoma cells.

Topics: Animals; Carcinoma, Hepatocellular; Inflammation; Lipids; Liver Neoplasms; Mitochondria; Rats; Reactive Oxygen Species; Retinoids; Tretinoin; Vitamin A

A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Liver Neoplasms; Mice; Neoplastic Cells, Circulating; Tretinoin

Hepatocellular carcinoma is characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of maintaining the immunosuppressive tumor microenvironment. Therefore, targeting MDSCs will improve immunotherapies for cancers. It has been shown that all-trans retinoic acid (ATRA) can differentiate MDSCs into mature myeloid cells. However, whether ATRA suppression of MDSCs function could inhibit the growth of liver cancer remains unknown. Here we found that ATRA significantly inhibited hepatocellular carcinoma promotion, tumor cell proliferation, and angiogenesis markers. Moreover, ATRA decreased the number of mononuclear myeloid-derived suppressor cells (M-MDSCs), granulocytic myeloid-derived suppressor cells (G-MDSCs) and tumor-associated macrophages (TAMs) in spleens. In addition, ATRA significantly reduced the intratumoral infiltrating G-MDSCs and the expression of protumor immunosuppressive molecules (arginase 1, iNOS, IDO and S100A8 + A9), which was accompanied by increased cytotoxic T cell infiltration. Our study demonstrates that ATRA not only has direct intrinsic inhibitory effect on tumor angiogenesis and fibrosis, but also reeducates the tumor microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces ATRA as a potential druggable target for treatment of hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Myeloid Cells; Myeloid-Derived Suppressor Cells; Tretinoin; Tumor Microenvironment

All‑trans retinoic acid (ATRA) has been implicated in the differentiation of hepatic stellate cells (HSCs). In the present study, the liver‑targeting hyaluronic acid micelles (ADHG) were prepared for co‑delivery of ATRA and doxorubicin (DOX) to block the HSC‑hepatoma interrelation. To simulate the tumor microenvironment, an

Topics: Animals; Carcinoma, Hepatocellular; Doxorubicin; Hepatic Stellate Cells; Hyaluronic Acid; Hydrogen-Ion Concentration; Mice; Tretinoin; Tumor Microenvironment

Sorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms.. CCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues.. We observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC. Our findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients. Video Abstract.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; p21-Activated Kinases; Sorafenib; Tretinoin

Chemotherapy is a common treatment for advanced hepatocellular carcinoma, but the effect is not satisfactory. The study aimed to retrospectively evaluate the effects of adding all-trans-retinoic acid (ATRA) to infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) for advanced hepatocellular carcinoma (HCC).. We extracted the data of patients with advanced HCC who underwent systemic chemotherapy using FOLFOX4 or ATRA plus FOLFOX4 at the Eastern Hepatobiliary Surgery Hospital, First Hospital of Jilin University, and Zhejiang Sian International Hospital and retrospectively compared for overall survival. The Cox proportional hazards model was used to calculate the hazard ratios for overall survival and disease progression after controlling for age, sex, and disease stage.. From July 2013 to July 2018, 111 patients with HCC were included in this study. The median survival duration was 14.8 months in the ATRA plus FOLFOX4 group and 8.2 months in the FOLFOX4 only group ( P  < 0.001). The ATRA plus FOLFOX4 group had a significantly longer median time to progression compared with the FOLFOX4 group (3.6 months vs. 1.8 months, P  < 0.001). Hazard ratios for overall survival and disease progression were 0.465 (95% confidence interval: 0.298-0.726; P  = 0.001) and 0.474 (0.314-0.717; P  < 0.001) after adjusting for potential confounders, respectively.. ATRA plus FOLFOX4 significantly improves the overall survival and time to disease progression in patients with advanced HCC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Disease Progression; Fluorouracil; Humans; Leucovorin; Liver Neoplasms; Oxaliplatin; Retrospective Studies; Tretinoin

Retinoic acid and retinoid acid receptor (RA-RAR) signaling exhibits suppressive functions in the progression of hepatocellular carcinoma (HCC) through multiple mechanisms. However, whether RA-RAR signaling induces autophagy that contributes its anti-tumor activity in HCC remains elusive. In the current study, the effects of RA-RAR pathway on autophagy were investigated in two HCC cell lines: alpha-fetoprotein (AFP) positive PLC/PRF/5 and AFP negative HLE cells. Cell autophagy was analyzed with western blot for detection of LC3 conversion and p62/SQSTM1 degradation while autophagy flux was assayed using the mRFP-GFP-LC3 reporter. Cell apoptosis and viability were analyzed by caspase-3 activity, TdT-mediated dUTP nick end labeling (TUNEL) assay, and Cell Counting Kit (CCK)-8, respectively. Chromatin immunoprecipitation (ChIP) was employed to detect the binding of RAR onto the promoter of autophagy-relevant 7 (ATG7), and co-immunoprecipitation (CoIP) was used to analyze the interaction of AFP and RAR. The results showed that ATRA dosage and time-dependently induced high levels of cell autophagy in both the PLC/PRF/5 and HLE cells, which was accompanied with up-regulation of ATG7. ChIP assay showed that RAR was able to bind to its responsive elements on ATG7 promoter. Impairment of ATG7 induction or blockade of autophagy with chloroquine aggravated ATRA induced apoptosis of HCC cells. Furthermore, intracellular AFP was able to complex with RAR in PLC/PRF/5 cells. Knockdown of AFP in PLC/PRF/5 cells augmented the up-regulation of ATG7 by ATRA while overexpression of AFP in HLE cells attenuated ATRA induced ATG7 expression and autophagy. Thus, ATRA induced ATG7 and autophagy participated in its cytotoxicity on HCC cells and AFP interfere with the induction of ATG7 and autophagy through forming complex with RAR.

Topics: alpha-Fetoproteins; Autophagy; Autophagy-Related Protein 7; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Intracellular Space; Liver Neoplasms; Protein Binding; Receptors, Retinoic Acid; Signal Transduction; Transcription, Genetic; Tretinoin

Low molecular weight branched polyethylenimines (LMW bPEIs) are almost nontoxic but display poor transfection efficiency due to lack of adequate complexation ability with nucleic acids followed by transportation across the cell membrane. Here, a series of amphiphilic retinoyl-bPEI conjugates (RP-1, RP-2 and RP-3) has been synthesized by allowing the reaction between bPEI (1.8 kDa) and a bioactive and hydrophobic vitamin A metabolite, all-trans-retinoic acid (ATRA), in varying amounts. In aqueous medium, these conjugates self-assembled into core-shell RP nanocomposites with size ranging from ~113-178 nm and zeta potential from ~ +15-35 mV. Evaluation of pDNA complexes of RP nanocomposites revealed that all the complexes exhibited significantly enhanced transfection efficiency without compromising on the cytocompatibility. RP-3/pDNA complex, with the highest content of retinoic acid, exhibited the best transfection efficiency. Further, due to anticancer properties of ATRA, these nanocomposites significantly reduced the viability of cancer cells (HepG2 and MCF-7 cells) without affecting the viability of non-cancerous cells (HEK 293 cells) demonstrating the cell-selective nature of the formulated nanocomposites. The intracellular trafficking and co-localization studies involving RP-3 nanocomposites also showed their higher uptake with intracellular and nuclear accumulation properties. Altogether, the results demonstrate the promising potential of the RP conjugates that can be used in future hepatocellular carcinoma targeted gene delivery applications.

Topics: Carcinoma, Hepatocellular; Gene Transfer Techniques; HEK293 Cells; Humans; Liver Neoplasms; Nanocomposites; Plasmids; Polyethyleneimine; Transfection; Transgenes; Tretinoin

A chronic, local inflammatory milieu can cause tissue fibrosis that results in epithelial-to-mesenchymal transition (EMT), endothelial-to-mesenchymal transition (EndMT), increased abundance of fibroblasts, and further acceleration of fibrosis. In this study, we aimed to identify potential mechanisms and inhibitors of fibrosis using 3D model-based phenotypic screening. We established liver fibrosis models using multicellular tumor spheroids (MCTSs) composed of hepatocellular carcinoma (HCC) and stromal cells such as fibroblasts (WI38), hepatic stellate cells (LX2), and endothelial cells (HUVEC) seeded at constant ratios. Through high-throughput screening of FDA-approved drugs, we identified retinoic acid and forskolin as candidates to attenuate the compactness of MCTSs as well as inhibit the expression of ECM-related proteins. Additionally, retinoic acid and forskolin induced reprogramming of fibroblast and cancer stem cells in the HCC microenvironment. Of interest, retinoic acid and forskolin had anti-fibrosis effects by decreasing expression of α-SMA and F-actin in LX2 cells and HUVEC cells. Moreover, when sorafenib was added along with retinoic acid and forskolin, apoptosis was increased, suggesting that anti-fibrosis drugs may improve tissue penetration to support the efficacy of anti-cancer drugs. Collectively, these findings support the potential utility of morphometric analyses of hepatic multicellular spheroid models in the development of new drugs with novel mechanisms for the treatment of hepatic fibrosis and HCCs.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Colforsin; Drug Synergism; Epithelial-Mesenchymal Transition; Fibroblasts; Hep G2 Cells; Hepatic Stellate Cells; Human Umbilical Vein Endothelial Cells; Humans; Liver Cirrhosis; Liver Neoplasms; Small Molecule Libraries; Sorafenib; Spheroids, Cellular; Tretinoin; Tumor Microenvironment

A 69-year-old man was referred to the hematology department for the evaluation of pancytopenia. He had been treated with radiation and epirubicin for hepatocellular carcinoma, and with docetaxel and pembrolizumab for lung adenocarcinoma. Bone marrow smears exhibited markedly increased promyelocytes, and polymerase chain reaction (PCR) study demonstrated chimeric fusion genes of

Topics: Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Carcinoma, Hepatocellular; Docetaxel; Epirubicin; Humans; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Lung Neoplasms; Male; Neoplasms, Multiple Primary; Treatment Outcome; Tretinoin

Liver cancer is a serious liver disease in which hepatoma cells and activated hepatic stellate cells (HSCs) overproduce extracellular matrix (ECM), which involves the Golgi apparatus. Here chondroitin-modified lipid nanoparticles (CSNs) were prepared and loaded with doxorubicin (DOX) and retinoic acid (RA) using a thin-film hydration-high pressure homogenization method. The resulting DOX + RA-CSNs were efficiently taken up by SMMC-7721 hepatoma cells and HSCs in culture, where they accumulated in the Golgi apparatus and destroyed it, inhibiting ECM production. Injecting DOX + RA-CSNs into mice with primary liver cancer or H22 allografts led to significantly higher tumor penetration by DOX and RA, greater antitumor efficacy, and lower DOX-related toxicity than injecting a solution of the two drugs. Immunofluorescence and immunohistochemistry of liver tissues showed that DOX + RA-CSNs dramatically reduced expression of the ECM components. These results suggest that CSNs show potential for targeting drugs to the Golgi apparatus of liver cancer cells and potentially other types of tumors.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Chondroitin; Doxorubicin; Drug Delivery Systems; Extracellular Matrix; Golgi Apparatus; Humans; Lipids; Liver Neoplasms; Male; Mice; Nanoparticles; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Previous studies have found that alpha-fetoprotein (AFP) can promote the proliferation of hepatoma cells and accelerate the progression of hepatocellular carcinoma (HCC). However, the exact mechanism of action remains unclear. Recent bioinformatics studies have predicted the possible interaction between AFP and retinoic acid receptors (RARs). Thus, the purpose of this study was to investigate the molecular mechanism through which AFP promotes tumour cell proliferation by interfering with the RA-RAR signal pathway. Our data indicated that AFP could significantly promote the proliferation and weaken ATRA-induced apoptosis of hepatoma cells. Besides, cytoplasmic AFP interacts with RAR, disrupting its entrance into the nucleus, which in turn affects the expression of the Bcl-2 gene. In addition, knockdown of AFP in HepG2 cells was synchronously associated with an incremental increase of RAR binding to DNA, as well as down-regulation of Bcl-2; the opposite effect was observed in AFP gene-transfected HLE cells. Moreover, a similar effect of AFP was detected in tumour tissues with high serum AFP, but not in adjacent non-cancerous liver tissues, or HCC tissues with low serum AFP levels. These results indicate that AFP acts as signalling molecule and prevents RAR from entering into the nucleus by interacting with RAR, thereby promoting the expression of Bcl-2. Our data reveal a novel mechanism through which AFP regulates Bcl-2 expression and further suggest that AFP may be used as a novel target for treating HCC.

Topics: alpha-Fetoproteins; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Retinoic Acid; Signal Transduction; Tretinoin

Activated hepatic stellate cells promote hepatocellular carcinoma (HCC) progression. Hepatic stellate cells play a key role in retinoid metabolism, and activation of stellate cells increases retinoic acid (RA) in the liver. However, the role of RA in HCC proliferation remains unclear. We aimed to analyse the mechanism of RA in HCC proliferation. Thirty-eight patients who had undergone hepatic resection for HCCs were recruited. Paired non-tumour tissues, adjacent and distal to HCCs, were collected, and the RA levels in the tissues were analysed. The mechanisms of RA and HCC proliferation were assessed in liver cancer cell lines by protein and gene expression analyses. Early recurrence of HCC was significantly higher in patients with a higher RA concentration than in those with a lower RA concentration in tissues adjacent to HCCs (61.1% vs. 20%, p = .010). RA promoted HCC cell proliferation and activated the expression of Amphiregulin, a growth factor in hepatocarcinogenesis. The promoter of Amphiregulin contained the binding sites of the RA receptor, RXRα. Wnt signalling also activated the expression of Amphiregulin, and the RA and Wnt pathways acted synergistically to increase the expression of Amphiregulin. Furthermore, RXRα interacted with β-catenin and then translocated to the nucleus to activate Amphiregulin. An increased RA concentration in the tissues adjacent to the tumour was associated with an early recurrence of HCC. RA activated the expression of Amphiregulin, and then promoted HCC proliferation, which might partly contribute to early recurrence of HCC after hepatic resection.

Topics: Amphiregulin; Antineoplastic Agents; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Retinoid X Receptor alpha; Tretinoin; Up-Regulation; Wnt Proteins

Hepatic stellate cells (HSCs) are essential perisinusoidal cells in both healthy and diseased liver. HSCs modulate extracellular matrix (ECM) homeostasis when quiescent, but in liver fibrosis, HSCs become activated and promote excess deposition of ECM molecules and tissue stiffening via force generation and mechanosensing. In hepatocellular carcinoma (HCC), activated HSCs infiltrate the stroma and migrate to the tumor core to facilitate paracrine signaling with cancer cells. Because the function of HSCs is known to be modulated by retinoids, we investigated the expression profile of retinoic acid receptor beta (RAR-β) in patients with cirrhosis and HCC, as well as the effects of RAR-β activation in HSCs. We found that RAR-β expression is significantly reduced in cirrhotic and HCC tissues. Using a comprehensive set of biophysical methods combined with cellular and molecular biology, we have elucidated the biomechanical mechanism by which all trans-retinoic acid promotes HSC deactivation via RAR-β-dependent transcriptional downregulation of myosin light chain 2 expression. Furthermore, this also abrogated mechanically driven migration toward stiffer substrates. Conclusion: Targeting mechanotransduction in HSCs at the transcriptional level may offer therapeutic options for a range of liver diseases.

Topics: Animals; Carcinoma, Hepatocellular; Cardiac Myosins; Case-Control Studies; Cell Movement; Cellular Microenvironment; Extracellular Matrix Proteins; Hepatic Stellate Cells; Humans; Liver Cirrhosis, Experimental; Liver Neoplasms; Mechanotransduction, Cellular; Mice; Myosin Light Chains; Primary Cell Culture; Receptors, Retinoic Acid; Tretinoin

Topics: Camptothecin; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Hep G2 Cells; HT29 Cells; Humans; Irinotecan; Lactic Acid; Liver Neoplasms; Micelles; Nanoparticles; Particle Size; Polyethylene Glycols; Polyglactin 910; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Tretinoin

Hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths worldwide largely due to lack of effective targeted drugs to simultaneously block multiple cancer-driving pathways. The identification of all-trans retinoic acid (ATRA) as a potent Pin1 inhibitor provides a promising candidate for HCC targeted therapy because Pin1 is overexpressed in most HCC and activates numerous cancer-driving pathways. However, the efficacy of ATRA against solid tumors is limited due to its short half-life of 45min in humans. A slow-releasing ATRA formulation inhibits solid tumors such as HCC, but can be used only in animals. Here, we developed a one-step, cost-effective route to produce a novel biocompatible, biodegradable, and non-toxic controlled release formulation of ATRA for effective HCC therapy. We used supercritical carbon dioxide process to encapsulate ATRA in largely uniform poly L-lactic acid (PLLA) microparticles, with the efficiency of 91.4% and yield of 68.3%, and ~4-fold higher C

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line; Delayed-Action Preparations; Drug Carriers; Drug Liberation; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; NIMA-Interacting Peptidylprolyl Isomerase; Polyesters; Tretinoin

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/β-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Epithelial Cell Adhesion Molecule; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; N-Myc Proto-Oncogene Protein; Neoplasm Metastasis; Neoplastic Stem Cells; Prognosis; Tretinoin; Wnt Signaling Pathway

Hepatocellular carcinoma (HCC) is one of the most prevalent and malignant cancers with high inter- and intra-tumor heterogeneity. A central common signaling mechanism in cancer is proline-directed phosphorylation, which is further regulated by the unique proline isomerase Pin1. Pin1 is prevalently overexpressed in human cancers including ~70% of HCC, and promotes tumorigenesis by activating multiple cancer-driving pathways. However, it was challenging to evaluate the significance of targeting Pin1 in cancer treatment until the recent identification of all-trans retinoic acid (ATRA) as a Pin1 inhibitor. Here we systematically investigate functions of Pin1 and its inhibitor ATRA in the development and treatment of HCC. Pin1 knockdown potently inhibited HCC cell proliferation and tumor growth in mice. ATRA-induced Pin1 degradation inhibited the growth of HCC cells, although at a higher IC50 as compared with breast cancer cells, likely due to more active ATRA metabolism in liver cells. Indeed, inhibition of ATRA metabolism enhanced the sensitivity of HCC cells to ATRA. Moreover, slow-releasing ATRA potently and dose-dependently inhibited HCC growth in mice. Finally, chemical or genetic Pin1 ablation blocked multiple cancer-driving pathways simultaneously in HCC cells. Thus, targeting Pin1 offers a promising therapeutic approach to simultaneously stop multiple cancer-driving pathways in HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Enzyme Inhibitors; Gene Knockdown Techniques; Humans; Liver Neoplasms; Mice; NIMA-Interacting Peptidylprolyl Isomerase; Proteolysis; Signal Transduction; Tretinoin; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) has the characristics of tumor invasiveness, frequent intrahepatic spread and extra hepatic metastases, which affects the therapy efficiency and prognosis. Epithelial-mesenchymal transition (EMT) is now recognized as a key process in tumor invasion, metastasis and the generation of cancer initiating cells. All-trans retinoic acid (ATRA) is currently used as a potential chemo-therapeutic or chemo-preventive agent because of its anti-proliferative, pro-apoptotic and antioxidant properties. This study investigated the effects of ATRA at different concentrations on the proliferation, migration, invasion, differentiation and functions of the mouse hepa1-6 hepatocarcinoma cell line and explored whether ATRA regulates EMT in the antitumor process. Trypan blue staining and colony formation assay were used to detect cell proliferation. Wound-healing assay and Transwell Matrigel assay were performed to examine migration. Invasion was assessed by using Transwell invasion assay. In the present study, ATRA significantly inhibited the cell growth, colony formation, migration, and invasion capability of hepa1-6 cells in a dose-dependent manner. Furthermore, ATRA at low concentration (0.1 µmol/l) could generate these influences. After treated in the ATRA medium, the expression of mature hepatic markers ALB (albumin), CK18 (cytokeratin 18), TAT (tyrosine aminotransferase), ApoB (apolipoprotein B) decreased and that of hepatocarcinoma marker AFP (α fetoprotein) increased. At day 7 after ATRA induction, hepa1-6 cells showed comparable indocyanine green (ICG) uptake and glycogen storage function to the blank control. The mRNA expression of mesenchymal markers N-cadherin, vimentin, snail and twist decreased, while expression of epithelial marker E-cadherin increased in hepa1-6 cells after treated with ATRA. Therefore, this study demonstrates that ATRA remarkably suppressed the proliferation, migration, invasion of hepa1-6 hepatocarcinoma cell line and effectively induced its differentiation and liver functions in vitro through the reversal of EMT. HCC may be more sensitive to ATRA than other cancers, suggesting the prospective usefulness of ATRA in the treatment of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Mice; Neoplasm Invasiveness; Tretinoin; Xenograft Model Antitumor Assays

Acyclic retinoid (ACR) is a promising drug under clinical trials for preventing recurrence of hepatocellular carcinoma. The objective of this study was to gain insights into molecular basis of the antitumorigenic action of ACR from a metabolic point of view. To achieve this, comprehensive cationic and lipophilic liver metabolic profiling was performed in mouse diethylnitrosamine (DEN)-induced hepatic tumorigenesis model using both capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography time-of-flight mass spectrometry. ACR significantly counteracted against acceleration of lipogenesis but not glucose metabolism in DEN-treated mice liver, suggesting an important role of lipid metabolic reprogramming in the initiation step of hepatic tumorigenesis. Knowledge-based pathway analysis suggested that inhibition of linoleic acid metabolites such as arachidonic acid, a proinflammatory precursor, played a crucial role in the prevention by ACR of DEN-induced chronic inflammation-mediated tumorigenesis of the liver. As a molecular mechanism of the ACR's effect to prevent the aberrant lipogenesis, microarray analysis identified that a key transcription regulator of both embryogenesis and tumorigenesis, COUP transcription factor 2, also known as NR2F2, was associated with the metabolic effect of ACR in human hepatocellular carcinoma cells. Our study provided potential therapeutic targets for the chemoprevention of hepatocellular carcinoma as well as new insights into the mechanisms underlying prevention of hepatic tumorigenesis.

Topics: Alkylating Agents; Animals; Antineoplastic Agents; Blotting, Western; Carcinogenesis; Carcinoma, Hepatocellular; Chromatography, Liquid; Diethylnitrosamine; Lipogenesis; Liver Neoplasms, Experimental; Male; Metabolome; Metabolomics; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Obese; Real-Time Polymerase Chain Reaction; Receptors, Leptin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tretinoin; Tumor Cells, Cultured

Normal hepatocytes express connexin32 (Cx32), which forms gap junctions at cell‑cell contact areas. The aim of the present study was to investigate whether Cx32 mediates the cell death‑inducing effects of ultrasound microbubbles carrying the herpes simplex virus thymidine kinase (HSV‑TK) suicide gene against hepatocellular carcinoma cells in vitro and in vivo. HepG2 cells were exposed to different concentrations of trans‑retinoic acid (ATRA) in culture, to evaluate the intrinsic antitumor effect of ATRA. Detailed in‑vitro and in‑vivo investigations on the antitumor effects of ATRA via Cx32 mediation were performed, and the possible underlying mechanisms of action of the compound were then examined. The gene expression of HSV‑TK transfected by ultrasound wave irradiation in the HepG2 cells was quantified using reverse transcription‑quantitative polymerase chain reaction analysis. The effects on cell death were assessed using an MTT assay. The protein expression levels of Cx32 in ATRA‑untreated or ATRA‑treated tissues were quantified by immunohistochemical analysis and Western blot assays. The HSV‑TK gene was successfully transfected into the HepG2 cell using ultrasound wave irradiation, and was stably expressed. Compared with the other groups, the HSV‑TK gene group treated with ATRA exhibited an increased number of apoptotic cells (P<0.05) and improved tumor suppression (P<0.05). ATRA significantly increased the expression of Cx32 in the hepatoma tissues (P<0.01). The present study demonstrated that ATRA elevated the protein expression of Cx32 and enhanced the bystander effect of the HSV‑TK/GCV suicide gene therapy system, which may provide a potential strategy for hepatocellular carcinoma treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Bystander Effect; Carcinoma, Hepatocellular; Connexins; Ganciclovir; Gap Junction beta-1 Protein; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Simplexvirus; Thymidine Kinase; Transfection; Transplantation, Heterologous; Tretinoin

4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was reported to function as a tumor inhibitor in various types of cancer cells in vitro. However, little is known concerning its antitumor effect on human hepatocellular carcinoma (HCC) HepG2 cells. The aims of the present study were to investigate the effects of ATPR on the proliferation of HepG2 cells and to explore the probable mechanisms. A series of experiments were performed following the treatment of HepG2 cells with ATRA and ATPR. MTT and plate colony formation assays were used to measure the cell viability. To confirm the influence on proliferation, flow cytometry was used to detect the distribution of the cell cycle. Apoptosis was observed by Hoechst staining and flow cytometry. In addition, to characterize the underlying molecular mechanisms, immunofluorescence was applied to observe the distribution of p53. The transcription and translation levels of p53 were analyzed by real-time quantitative RT-PCR (qRT-PCR) and western blotting. The expression levels of murine double minute 2 (MDM2), apoptosis stimulating proteins of p53 (ASPP), cell cycle- and apoptosis-associated proteins were detected by western blotting. After HepG2 cells were incubated with ATRA and ATPR, the viability of the HepG2 cells was inhibited in a dose- and time-dependent manner. As well, ATPR significantly suppressed HepG2 cell colony formation and arrested cells at the G0/G1 phase, while ATRA had no obvious effects. Both Hoechst staining and flow cytometry unveiled the apoptosis of HepG2 cells. Moreover, the fluorescent density of p53 was higher in the nuclei after exposure to ATPR than that in the ATRA group. HepG2 cells treated with ATPR showed elevated mRNA and protein levels of p53 when compared with these levels in the ATRA-treated cells. Western blotting showed that ATPR increased ASPP1, p21 and Bax expression and decreased MDM2, iASPP, cyclin D and E, cyclin-dependent kinase 6 (CDK6) and Bcl-2 expression, while CDK4 and ASPP2 expression were scarcely altered. Consequently, ATPR exerted a better inhibitory effect on the proliferation of HepG2 cells than ATRA through increased expression of p53 and ASPP1 and downregulation of iASPP, thereby resulting in G0/G1 cell cycle arrest and apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Down-Regulation; G1 Phase; Hep G2 Cells; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Proto-Oncogene Proteins c-mdm2; Repressor Proteins; Resting Phase, Cell Cycle; Retinoids; RNA, Messenger; Signal Transduction; Transcription, Genetic; Tretinoin; Tumor Suppressor Protein p53; Up-Regulation

Objective To investigate the effect of interferon-β (IFN-β) combined with all-trans retinoic acid (ATRA) on the proliferation and apoptosis of HepG2 human hepatocarcinoma cells and the role of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal pathway in the process. Methods HepG2 cells were randomly divided intro three groups and treated with 1000 U/mL IFN-β, 10 μmol/L ATRA and 1000 U/mL IFN-β combined with 10 μmol/L ATRA, respectively for 24 hours. Cell viability was measured by MTT assay and apoptosis rate was detected by flow cytometry. Western blotting was applied to detect the protein levels of p-JAK2, p-STAT3, gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), Bcl-2, Bcl-xl and Bax. Results IFN-β or ATRA inhibited the proliferation and induced the apoptosis of HepG2 cells. The effect was enhanced when IFN-β was combined with ATRA. The expressions of p-JAK2 and p-STAT3 were down-regulated while the expressions of GRIM-19 and Bax were up-regulated after treated with IFN-β or ATRA on HepG2 cells, especially the combination of IFN-β and ATRA. Conclusion Combination of IFN-β and ATRA could suppress the proliferation and induced the apoptosis of HepG2 hepatocarcinoma cells by inhibiting JAK2/STAT3 signal pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Drug Synergism; Flow Cytometry; Hep G2 Cells; Humans; Interferon-beta; Janus Kinase 2; Liver Neoplasms; Microscopy, Fluorescence; NADH, NADPH Oxidoreductases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Tretinoin

To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all-trans retinoic acid (ATRA), NIK-333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 μM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK-333 and Am80. We found that ATRA induced AMP-activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT-1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase-3, was observed. Suppression of AMP-activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells.

Topics: Adenosine Triphosphate; AMP-Activated Protein Kinases; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Benzoates; Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 1; Humans; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Retinoids; Sorafenib; Tetrahydronaphthalenes; Tretinoin

Hepatocellular carcinoma (HCC) is a fatal cancer with no effective therapy. Astrocyte elevated gene-1 (AEG-1) plays a pivotal role in hepatocarcinogenesis and inhibits retinoic acid-induced gene expression and cell death. The combination of a lentivirus expressing AEG-1 shRNA and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We have now developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (Gal) (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). The polymer conjugate was characterized by (1)H-NMR, MALDI, and mass spectrometry; and optimal nanoplex formulations were characterized for surface charge, size, and morphology. Orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) were established in the livers of athymic nude mice and tumor development was monitored by bioluminescence imaging (BLI). Tumor-bearing mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si, and PAMAM-AEG-1si+ATRA. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth, and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. A marked decrease in AEG-1 level was observed in tumor samples treated with PAMAM-AEG-1si. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation, and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anticancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Proliferation; Combined Modality Therapy; Genetic Therapy; Humans; Liver; Liver Neoplasms; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Nude; Nanoparticles; RNA-Binding Proteins; RNA, Small Interfering; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Nuclear accumulation of transglutaminase 2 (TG2) is an important step in TG2-dependent cell death. However, the underlying molecular mechanisms for nuclear translocation of TG2 are still poorly understood. In this study, we demonstrated that acyclic retinoid (ACR) induced nuclear accumulation of TG2 in JHH-7 cells, a hepatocellular carcinoma (HCC) leading to their apoptosis. We further demonstrated molecular mechanism in nuclear-cytoplasmic trafficking of TG2 and an effect of ACR on it. We identified a novel 14-amino acid nuclear localization signal (NLS) (466)AEKEETGMAMRIRV(479) in the 'C' domain and a leucine-rich nuclear export signal (NES) (657)LHMGLHKL(664) in the 'D' domain that allowed TG2 to shuttle between the nuclear and cytosolic milieu. Increased nuclear import of GAPDH myc-HIS fused with the identified NLS was observed, confirming its nuclear import ability. Leptomycin B, an inhibitor of exportin-1 as well as point mutation of all leucine residues to glutamine residues in the NES of TG2 demolished its nuclear export. TG2 formed a trimeric complex with importin-α and importin-β independently from transamidase activity which strongly suggested the involvement of a NLS-based translocation of TG2 to the nucleus. ACR accelerated the formation of the trimeric complex and that may be at least in part responsible for enhanced nuclear localization of TG2 in HCC cells treated with ACR.

Topics: Amino Acid Sequence; Apoptosis; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; GTP-Binding Proteins; Humans; Liver Neoplasms; Molecular Sequence Data; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Tretinoin

Vitamin A status regulates obesity development, hyperlipidemia, and hepatic lipogenic gene expression in Zucker fatty (ZF) rats. The development of hyperlipidemia in acne patients treated with retinoic acid (RA) has been attributed to the induction of apolipoprotein C-III expression. To understand the role of retinoids in the development of hyperlipidemia in ZF rats, the expression levels of several selected RA-responsive genes in the liver and isolated hepatocytes from Zucker lean (ZL) and ZF rats were compared using real-time PCR. The Rarb and Srebp-1c mRNA levels are higher in the liver and isolated hepatocytes from ZF than ZL rats. The Apoc3 mRNA level is only higher in the isolated hepatocytes from ZF than ZL rats. To determine whether dynamic RA production acutely regulates Apoc3 expression, its mRNA levels in response to retinoid treatments or adenovirus-mediated overexpression of hepatocyte nuclear factor 4 alpha (HNF4α) and chicken ovalbumin upstream-transcription factor II (COUP-TFII) were analyzed. Retinoid treatments for 2-6 h did not induce the expression of Apoc3 mRNA. The overexpression of HNF4α or COUP-TFII induced or inhibited Apoc3 expression, respectively. We conclude that short-term retinoid treatments could not induce Apoc3 mRNA expression, which is regulated by HNF4α and COUP-TFII in hepatocytes.

Topics: Animals; Apolipoprotein C-III; Carcinoma, Hepatocellular; Cell Line, Tumor; COUP Transcription Factor II; Gene Expression Regulation; HEK293 Cells; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver Neoplasms; Male; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Rats, Zucker; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Tretinoin

Increasing gap junction activity in tumor cells provides a target by which to enhance antineoplastic therapies. Previously, several naturally occurring agents, including all-trans retinoic acid (ATRA) have been demonstrated to increase gap junctional intercellular communication (GJIC) in a number of types of cancer cells. In the present study, we investigated in vitro whether ATRA modulates the response of human hepatocellular carcinoma (HCC) cells to sorafenib, the only proven oral drug for advanced HCC, and the underlying mechanisms. HepG2 and SMMC-7721 cells were treated with sorafenib and/or ATRA, and cell proliferation and apoptosis were analyzed; the role of GJIC was also explored. We found that ATRA, at non-toxic concentrations, enhanced sorafenib-induced growth inhibition in both HCC cell lines, and this effect was abolished by two GJIC inhibitors, 18-α-GA and oleamide. Whereas lower concentrations of sorafenib (5 µM) or ATRA (0.1 or 10 µM) alone modestly induced GJIC activity, the combination of sorafenib plus ATRA resulted in a strong enhancement of GJIC. However, the action paradigm differed in the HepG2 and SMMC-7721 cells, with the dominant effect of GJIC dependent on the cell-specific connexin increase in protein amounts and relocalization. RT-PCR assay further revealed a transcriptional modification of the key structural connexin in the two cell lines. Thus, a connexin-dependent gap junction enhancement may play a central role in ATRA plus sorafenib synergy in inhibiting HCC cell growth. Since both agents are available for human use, the combination treatment represents a future profitable strategy for the treatment of advanced HCC.

Topics: Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Communication; Cell Line, Tumor; Cell Proliferation; Connexins; Drug Synergism; Gap Junctions; Glycyrrhetinic Acid; Hep G2 Cells; Humans; Liver Neoplasms; Niacinamide; Oleic Acids; Phenylurea Compounds; Protein Kinase Inhibitors; Sorafenib; Tretinoin

We investigated the effects of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), alone and in combination, on apoptosis and intracellular calcium concentration in hepatocellular carcinoma (HepG2) cells. We used HepG2 cells to test the effects of ATRA and ATO, individually and in combination, on cell proliferation, apoptosis, and intracellular-free calcium concentration. The results indicate that each drug decreased cell proliferation, increased apoptosis, and increased intracellular-free calcium in a time- and dose-dependent manner. We also calculated the coefficients of drug interaction for sub-threshold administration of both drugs in combination (1 μmol/L each). ATRA and ATO acted synergistically in inhibition of cell proliferation and additively in the promotion of apoptosis. All-trans retinoic acid and ATO interacted synergistically to reduce cell proliferation in HepG2 cells.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Calcium; Carcinoma, Hepatocellular; Cell Proliferation; Hep G2 Cells; Humans; Liver Neoplasms; Oxides; Tretinoin

To explore the effect of alpha-fetoprotein (AFP) on transduction of the PI3K/ AKT signal in hepatocellular carcinoma cells and the role played by AFP in resistance to cytotoxicity of all-trans retinoic acid (ATRA).. The effects of ATRA of human liver cancer cells was assessed using the BEL-7402 cell line with the MTT assay (to evaluate proliferation), microscopy (to evaluate morphology), flow cytometry (to evaluate apoptosis), laser confocal microscopy and coimmunoprecipitation (co-IP; to evaluate co-localization and interaction of AFP with PTEN), Western blotting (to evaluate expression of phosphorylated-protein kinase B (pAKT) and Src, and RNA interference (RNAi)-mediated knockdown of AFP. Finally, application of the PI3K-specific inhibitor Ly294002 was used to monitor the influence of AFP in transduction of the PI3K signal pathway.. The human hepatoma cell line BEL-7402 were resistant to ATRA cytotoxicity. PTEN and AFP co-localized in the cytoplasm, and co-IP indicated that AFP interacts with PTEN in BEL-7402 cells.RNAi knockdown of AFP expression led to reduced growth of BEL-7402 cells.BEL-7402 cells transfected with AFP-short interfering (si)RNA vectors showed enhanced sensitivity to ATRA and reduced expression of pAKT(Ser473) and Src; Ly294002 reduced the role of AFP in stimulating expression of pAKT(Ser473) and Src.. AFP can activate transduction of the PI3K/AKT signal, and expression of AFP in hepatoma cells is a pivotal event for resisting ATRA-induced apoptosis.

Topics: alpha-Fetoproteins; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytoplasm; Humans; Immunoprecipitation; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA Interference; RNA, Small Interfering; Signal Transduction; Transfection; Tretinoin

The epidermal growth factor receptor (EGFR) is an important surface receptor with N-glycans in its extracellular domain, whose glycosylation is essential for its function, especially in tumor cells. Here, we demonstrated that polylactosamine is markedly increased in H7721 hepatocellular carcinoma cells after treatment with EGF, while it apparently declined after exposure to all-trans retinoic acid (ATRA). In the study of the enzymatic mechanism of this phenomenon, we explored changes in the expression of poly-N-acetyllactosamine (PLN) branching glycosyltransferases using RT-PCR. Among the four glycosyltransferases with altered expression, GnT-V was most elevated by EGF, while GnT-V and B3GNT2 were most declined by ATRA. Next, we conducted co-immunoprecipitation experiments to test whether B3GNT2 and EGFR associate with each other. We observed that EGFR is a B3GNT2-targeting protein in H7721 cells. Taken together, these findings indicated that the altered expression of B3GNT2 will remodel the PLN stucture of EGFR in H7721 cells, which may modify downstream signal transduction.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Glycosylation; Humans; Immunoprecipitation; Liver Neoplasms; N-Acetylglucosaminyltransferases; Phosphorylation; Polysaccharides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The prognosis of patients with hepatocellular carcinoma (HCC) is poor and the development of effective treatments for this malignancy, including combination chemotherapy, is required. This study examined the possible combined inhibitory effects of bevacizumab, an anti-vascular endothelial growth factor monoclonal antibody, and acyclic retinoid (ACR), which can prevent the development of HCC, on the growth of Huh7 human HCC cells. Xenograft tumors were produced by subcutaneously injecting Huh7 cells into nude mice. Starting 1 wk after the tumor cell injection, the mice were treated with bevacizumab alone (5 mg/kg body weight, subcutaneous injection, twice a week), ACR alone (given in a diet containing 0.03%), or their combination for 6 wk, and the effects of these regimens on xenograft growth were examined. Combined treatment with bevacizumab plus ACR significantly suppressed the growth of Huh7 xenografts. The combination of these agents significantly inhibited the phosphorylation of the Akt protein in tumor tissues. With combination therapy, the population of Ki-67-positive cells in xenografts decreased, while that of TUNEL-positive cells increased. The combination of bevacizumab and ACR exerts growth-suppressing effects on HCC cells by inhibiting cell proliferation and inducing apoptosis. This combination might be an effective regimen for the treatment of HCC.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bevacizumab; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Mice; Mice, Nude; Neoplasm Transplantation; Phosphorylation; Proto-Oncogene Proteins c-akt; Tretinoin; Vascular Endothelial Growth Factor A

Aberrant promoter methylation of tumor suppressor genes including retinoic acid receptor-β2 (RAR-β2) is frequently detected in hepatitis C virus (HCV)-associated hepatocellular carcinoma; however, the mechanism and its significance are relatively unknown. Here, we showed that HCV Core induced promoter hypermethylation of RAR-β2 to inhibit its expression via up-regulation of DNA methyltransferases 1 and 3b. Under the condition, all-trans retinoic acid (ATRA) failed to activate p16 expression and thus could not inactivate the Rb-E2F pathway. Accordingly, Core-expressing cells exhibited resistance to ATRA-induced growth inhibition. Taken together, HCV Core antagonizes ATRA, a natural anti-cancer compound, to stimulate cell growth via epigenetic down-regulation of RAR-β2.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Down-Regulation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepacivirus; Hepatitis C Antigens; Humans; Liver Neoplasms; Methyltransferases; Neoplasm Proteins; Promoter Regions, Genetic; Receptors, Retinoic Acid; RNA Interference; RNA, Small Interfering; Tretinoin; Viral Core Proteins

Retinoid X receptor α [RXRα; nuclear receptor (NR)2B1] is a crucial regulator in the expression of a broad array of hepatic genes under both normal and pathologic conditions. During inflammation, RXRα undergoes rapid post-translational modifications, including c-Jun N-terminal kinase (JNK)-mediated phosphorylation, which correlates with a reduction in RXRα function. A small ubiquitin-like modifier (SUMO) acceptor site was recently described in human RXRα, yet the contributors, regulators, and consequences of SUMO-RXRα are not well understood. Inflammation and other stressors alter nuclear receptor function in liver and induce SUMOylation of several NRs as part of proinflammatory gene regulation, but linkages between these two pathways in liver, or for RXRα directly, remain unexplored. We sought to determine if inflammation induces SUMOylation of RXRα in human liver-derived (HuH-7) cells. Lipopolysaccharide, interleukin-1β, and tumor necrosis factor α (TNFα) rapidly and substantially stimulated SUMOylation of RXRα. Two RXRα ligands, 9-cis retinoic acid (9cRA) and LG268, induced SUMOylation of RXRα, whereas both inflammation- and ligand-induced SUMOylation of RXRα require the K108 residue. Pretreatment with 1,9-pyrazoloanthrone (SP600125), a potent JNK inhibitor, abrogates TNFα- and 9cRA-stimulated RXRα SUMOylation. Pretreatment with SUMOylation inhibitors markedly augmented basal expression of several RXRα-regulated hepatobiliary genes. These results indicate that inflammatory signaling pathways rapidly induce SUMOylation of RXRα, adding to the repertoire of RXRα molecular species in the hepatocyte that respond to inflammation. SUMOylation, a newly described post-translational modification of RXRα, appears to contribute to the inflammation-induced reduction of RXRα-regulated gene expression in the liver that affects core hepatic functions, including hepatobiliary transport.

Topics: Alitretinoin; Carcinoma, Hepatocellular; Cell Line, Tumor; Hepatocytes; Humans; Inflammation Mediators; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Ligands; Liver; Liver Neoplasms; Organic Chemicals; Protein Processing, Post-Translational; Receptors, Cytoplasmic and Nuclear; Retinoid X Receptor alpha; Signal Transduction; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Tretinoin; Tumor Necrosis Factor-alpha

Accumulating data from epidemiological and experimental studies have suggested that retinoids, which are vitamin A derivatives, exert antitumor activity in various organs. We performed a gene screening based on in silico analysis of retinoic acid response elements (RAREs) to identify the genes facilitating the antitumor activity of retinoic acid (RA) and investigated their clinical significance in hepatocellular carcinoma (HCC).. In silico analysis of RAREs was performed in the 5-kb upstream region of EST clusters. Chromatin immunoprecipitation analysis of the retinoic acid receptors and gene expression analysis were performed in HuH7, HepG2, and MCF7 cells treated with all-trans RA (ATRA). mRNA expression of RA-responsive genes was investigated using tumor and non-tumor tissues of clinical HCC samples from 171 patients. The association between gene expression and survival of patients was examined by Cox regression analysis.. We identified 201 candidate genes with promoter regions containing consensus RARE and finally selected 26 RA-responsive genes. Of these, downregulation of OTU domain-containing 7B (OTUD7B) gene, which was upregulated by ATRA, in tumor tissue was associated with a low cancer-specific survival of HCC patients. Functional analyses revealed that OTUD7B negatively regulates nuclear factor κB (NF-κB) signaling and decreases the survival of HCC cells.. We identified RA-responsive genes which are regulated by retinoid signal and found that low-OTUD7B mRNA expression is associated with a poor prognosis for HCC patients. OTUD7B-mediated inhibition of NF-κB signaling may be an effective target for antitumor therapy for HCC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Down-Regulation; Endopeptidases; Genes, Neoplasm; Humans; Liver Neoplasms; NF-kappa B; Prognosis; Response Elements; Signal Transduction; Tretinoin

Systemic chemotherapy serves as an adjuvant treatment for post-operation patients with hepatocellular carcinoma (HCC), and provides curative option for the patients with unresectable HCC. However, its efficiency is largely limited because of the high incidence of chemo-resistance. Increasing evidence has shown that tumor initiating cells (TICs) not only have the ability to self-renew and drive the initiation and progression of cancer, but also exhibit greater resistance to conventional chemo- and radio-therapies than non-TICs. It was the aim of this study to investigate the effects of ATRA with and without cisplatin on TIC differentiation and apoptosis in human HCC.. In the present study, we evaluated the TICs of HCC cell differentiation induced by all-trans retinoic acid (ATRA), and developed a novel chemotherapeutic approach to HCC, by characterizing the function of combinatorial treatment with cis-diammineplatinum(II) (cisplatin) and ATRA in vitro and in vivo.. ATRA effectively induced differentiation of TICs, which potentiated the cytotoxic effects of cisplatin. The combinatorial treatment of ATRA acid and cisplatin reduced protein kinase B (AKT) (Thr308) phosphorylation, and promoted apoptosis of HCC cells more significantly than treatment with cisplatin alone. In addition, the combined treatment with the two drugs exerted stronger inhibition on either HCC cell migration in vitro or metastasis in vivo, when compared to the treatment with either drug alone.. These results indicated that ATRA could significantly improve the effect of cisplatin, which is at least partially attributed to ATRA-induced differentiation of HCC TICs, and the subsequent decrease in this chemo-resistant subpopulation.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Differentiation; Cisplatin; Drug Synergism; Epithelial Cell Adhesion Molecule; Humans; Liver Neoplasms; Male; Mice; Neoplastic Stem Cells; Phosphorylation; Proto-Oncogene Proteins c-akt; Tretinoin

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

Schizophrenic patients show lower incidences of cancer, implicating schizophrenia may be a protective factor against cancer. To study the genetic correlation between the two diseases, a specific PPI network was constructed with candidate genes of both schizophrenia and hepatocellular carcinoma. The network, designated schizophrenia-hepatocellular carcinoma network (SHCN), was analysed and cliques were identified as potential functional modules or complexes. The findings were compared with information from pathway databases such as KEGG, Reactome, PID and ConsensusPathDB.. The functions of mediator genes from SHCN show immune system and cell cycle regulation have important roles in the eitology mechanism of schizophrenia. For example, the over-expressing schizophrenia candidate genes, SIRPB1, SYK and LCK, are responsible for signal transduction in cytokine production; immune responses involving IL-2 and TREM-1/DAP12 pathways are relevant for the etiology mechanism of schizophrenia. Novel treatments were proposed by searching the target genes of FDA approved drugs with genes in potential protein complexes and pathways. It was found that Vitamin A, retinoid acid and a few other immune response agents modulated by RARA and LCK genes may be potential treatments for both schizophrenia and hepatocellular carcinoma.. This is the first study showing specific mediator genes in the SHCN which may suppress tumors. We also show that the schizophrenic protein interactions and modulation with cancer implicates the importance of immune system for etiology of schizophrenia.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Databases, Genetic; Genetic Predisposition to Disease; Humans; Immune System; Liver Neoplasms; Metabolic Networks and Pathways; Schizophrenia; Tretinoin; United States; United States Food and Drug Administration; Vitamin A

As a therapeutic or chemopreventative agent for various cancers, all-trans retinoic acid (atRA) has been reported to inhibit growth, induce apoptosis or cause differentiation. It was found that atRA could protect hepatocellular carcinoma (HCC) cells against cell death induced by serum starvation. Furthermore, it was found that atRA could enhance cell adhesion, but had no effect on the cell cycle and apoptosis. Using an Illumina Human HT-12 v4 expression microarray, 207 upregulated and 173 downregulated genes were identified in HepG2 cells treated with atRA. The most upregulated genes are cytochrome P450 family 26 subfamily A polypeptide 1 (CYP26A1), histidine triad nucleotide binding protein 3 (HINT3), miR-1282 and cytochrome P450 family 26 subfamily B polypeptide 1 (CYP26B1), which showed more than fivefold greater expression. Using Gene Ontology analysis, the greatest significance was found in extracellular-matrix-related molecular functions and the cellular component in upregulated genes. The upregulation of collagen 8A2 (COL8A2) was further confirmed using quantitative RT-PCR and western blotting. Knockdown of COL8A2 blocked enhancement in the early stage of cell adhesion by atRA treatment. Re-expression of COL8A2 in COL8A2-knocked-down HCC cells reversed the effect of small interfering RNA-COL8A2. In addition, COL8A2 could increase HCC cell migration and invasion. Thus, COL8A2 was identified as the key protein involved in the enhancement of cell adhesion of atRA under serum-free conditions. In conclusion, atRA protects HCC cells against serum-starvation-induced cell death by enhancing cell adhesion, and COL8A2 plays an important role in HCC cell migration and invasion.

Topics: Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Cycle; Cell Movement; Collagen Type VIII; Culture Media, Serum-Free; Gene Expression Profiling; Humans; Liver Neoplasms; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Starvation; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Fenretinide is significantly more effective in inducing apoptosis in cancer cells than all-trans retinoic acid (ATRA). The current study uses a genome-wide approach to understand the differential role fenretinide and ATRA have in inducing apoptosis in Huh7 cells. Fenretinide and ATRA-induced gene expressions and DNA bindings were profiled using microarray and chromatin immunoprecipitation with anti-RXRα antibody. The data showed that fenretinide was not a strong transcription regulator. Fenretinide only changed the expressions of 1 093 genes, approximately three times less than the number of genes regulated by ATRA (2 811). Biological function annotation demonstrated that both fenretinide and ATRA participated in pathways that determine cell fate and metabolic processes. However, fenretinide specifically induced Fas/TNFα-mediated apoptosis by increasing the expression of pro-apoptotic genes i.e., DEDD2, CASP8, CASP4, and HSPA1A/B; whereas, ATRA induced the expression of BIRC3 and TNFAIP3, which inhibit apoptosis by interacting with TRAF2. In addition, fenretinide inhibited the expression of the genes involved in RAS/RAF/ERK-mediated survival pathway. In contrast, ATRA increased the expression of SOSC2, BRAF, MEK, and ERK genes. Most genes regulated by fenretinide and ATRA were bound by RXRα, suggesting a direct effect. This study revealed that by regulating fewer genes, the effects of fenretinide become more specific and thus has fewer side effects than ATRA. The data also suggested that fenretinide induces apoptosis via death receptor effector and by inhibiting the RAS/RAF/ERK pathway. It provides insight on how retinoid efficacy can be improved and how side effects in cancer therapy can be reduced.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; DNA; Extracellular Signal-Regulated MAP Kinases; Fenretinide; Gene Expression Profiling; Gene Expression Regulation; Humans; Liver Neoplasms; raf Kinases; ras Proteins; Retinoid X Receptor alpha; Signal Transduction; Transcriptome; Tretinoin

The aim of our study was to gain a better understanding of the molecular mechanism underlying the previously unrecognized role of cytoplasmic alpha fetoprotein (AFP) in retinoic acid receptors (RAR) mediated expression and biological effects of GADD153. Using microarray analysis, the expression of the GADD153 gene showed the greatest fold change among apoptosis/growth related genes in response to ATRA. AFP was able to interact with RAR in HepG2 cells, which was undetectable in HLE cells owing to absence of AFP. ATRA promoted nuclei entrance of RAR, expression of GADD153 and apoptosis, and these changes were reversed after transfection with the afp gene or addition of AGN193109. The level of GADD153 was gradually elevated as the effect of AFP was counteracted by increasing dose or prolonging treatment time with ATRA in HepG2 cells. Knockdown of AFP in siRNA-transfected HepG2 cells or over-expression of AFP in afp gene-transfected HLE cells was synchronously associated with up-regulation or down-regulation, respectively, of GADD153 expression. Both ATRA administration and AFP knockdown were each able to promote greater binding of RAR to its response element with consequent elevation of the proportion of apoptotic cells. Conversely, transfection of HLE cells with pcDNA3.1-afp resulted in apparent reduction of RAR binding to DNA and change of biological effect. These data taken together demonstrate the involvement of AFP in RAR-mediated expression and biological effects of GADD153. These findings provide a novel insight into the mechanism underlying the impact of AFP on the RAR signal network.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; DNA; Gene Expression Profiling; Hep G2 Cells; Humans; Liver Neoplasms; Receptors, Retinoic Acid; Signal Transduction; Transcription Factor CHOP; Tretinoin

Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Drug Combinations; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Liver Neoplasms; Microscopy, Confocal; Mitotic Index; Polymerase Chain Reaction; Rats; Tretinoin

Despite its well-defined role as a serum growth factor during fetal liver development and hepatic oncogenesis, the biological significance of cytoplasmic alpha-fetoprotein (AFP) remains incompletely understood. Here, we provide evidence to illustrate that cytoplasmic AFP may function as a regulator in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in human hepatocellular carcinoma cells. The results demonstrated colocalization and interaction of AFP and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the cytoplasm of AFP-producing Bel 7402 and HepG2 cells, with an interaction distance of 12.6 ± 2.7 Å as determined with the fluorescence resonance energy transfer technique. Knockdown of AFP mRNA or inhibition of AFP expression by all trans-retinoic acid resulted in enhancement of the PTEN level with a synchronous decrease in phosphorylated AKT. Transfection of the afp gene into HLE cells (originally AFP negative) led to a significant activation of AKT signaling. The inhibition of PI3K signaling by LY 294002 was simultaneously reversed by transfection, accompanied by diminution of all trans-retinoic acid-induced upregulation of PTEN and enhancement of cell growth. In conclusion, these results demonstrate that cytoplasmic AFP is involved in regulation of hepatocellular growth and tumorigenesis.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Chromosomes, Human, Pair 10; Fluorescence Resonance Energy Transfer; Gene Deletion; Homeostasis; Humans; Liver Neoplasms; Microfilament Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA Interference; Signal Transduction; Tensins; Transfection; Tretinoin; Tumor Suppressor Proteins; Up-Regulation

Hepatocellular carcinoma has a high mortality rate due to its rate of recurrence. Acyclic retinoid prevents recurrence of hepatocellular carcinoma in patients after surgical removal of their primary tumors by inducing apoptosis in hepatocellular carcinoma cells, although the molecular mechanisms of action are not understood.. Human hepatocellular carcinoma cells in culture, as well as nude mice transplanted with hepatocellular carcinoma cells and rats given with N-diethylnitrosamine were treated with acyclic retinoid. Changes in activated caspase 3 and transglutaminase 2 (TG2) levels, Sp1 cross-linking and its activities, expression of epidermal growth factor receptor, and apoptotic levels were measured.. Acyclic retinoid simultaneously stimulated the activation of caspase 3, and the expression, nuclear localization and crosslinking activity of TG2, resulting in crosslinking and inactivation of the transcription factor, Sp1, thereby reducing expression of epidermal growth factor receptor and cell death in three hepatocellular carcinoma cell lines. These effects were partially restored by a caspase inhibitor, transfection of antisense TG2, restoration of functional Sp1, or an excess of epidermal growth factor. Nuclear expression of TG2 and crosslinked Sp1, as also activated caspase 3 were found in both hepatocellular carcinoma cells transplanted into nude mice and cancerous regions within the liver in N-diethylnitrosamine-induced hepatocarcinogenesis model in rats, following treatment of animals with acyclic retinoid.. Treatment with acyclic retinoid produces a dual activation of caspase 3 and TG2 induced apoptosis of hepatocellular carcinoma cells via modification and inactivation of Sp1, resulting in reduced expression of epidermal growth factor receptor.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Diethylnitrosamine; Down-Regulation; ErbB Receptors; Gene Knockdown Techniques; GTP-Binding Proteins; Humans; Liver Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Protein Glutamine gamma Glutamyltransferase 2; Rats; Retinoid X Receptor alpha; Sp1 Transcription Factor; Transglutaminases; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Despite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G(1) arrest and senescence-associated β-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-2'-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; DNA Methylation; Down-Regulation; Hepatitis B virus; Humans; Liver Neoplasms; Neoplasm Proteins; Trans-Activators; Tretinoin; Viral Regulatory and Accessory Proteins

Recent genetic studies in mice have established that the nuclear receptor coregulator Trim24/Tif1α suppresses hepatocarcinogenesis by inhibiting retinoic acid receptor α (Rara)-dependent transcription and cell proliferation. However, Rara targets regulated by Trim24 remain unknown. We report that the loss of Trim24 resulted in interferon (IFN)/STAT pathway overactivation soon after birth (week 5). Despite a transient attenuation of this pathway by the induction of several IFN/STAT pathway repressors later in the disease, this phenomenon became more pronounced in tumors. Remarkably, Rara haplodeficiency, which suppresses tumorigenesis in Trim24(-/-) mice, prevented IFN/STAT overactivation. Moreover, together with Rara, Trim24 bound to the retinoic acid-responsive element of the Stat1 promoter and repressed its retinoic acid-induced transcription. Altogether, these results identify Trim24 as a novel negative regulator of the IFN/STAT pathway and suggest that this repression through Rara inhibition may prevent liver cancer.

Topics: Animals; Carcinoma, Hepatocellular; Cluster Analysis; Gene Dosage; Gene Expression Regulation; Humans; Interferons; Liver; Liver Neoplasms; Mice; Models, Biological; Nuclear Proteins; Receptors, Retinoic Acid; Reproducibility of Results; Retinoic Acid Receptor alpha; Signal Transduction; STAT Transcription Factors; Transcription Factors; Transcriptome; Tretinoin

Aberrant promoter methylation of retinoic acid receptor-beta(2) (RAR-beta(2)) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism of methylation and its biological significance are unknown. This study showed that HBx, the principal oncogene product of HBV, induced promoter hypermethylation of RAR-beta(2) via upregulation of DNA methyltransferases 1 and 3a, resulting in downregulation of its expression in human HCC cells. In addition, HBx abolished the potential of retinoic acid (RA) to downregulate levels of G(1)-checkpoint regulators including p16, p21 and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition compared with control cells. These effects almost completely disappeared when levels of RAR-beta(2) in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5-aza-2'-deoxycytidine. As RAR-beta(2) is a major executor of the anti-tumour potential of RA, its epigenetic downregulation by HBx is likely to be an important step during HBV-mediated tumorigenesis.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Proliferation; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Down-Regulation; Hep G2 Cells; Hepatitis B virus; Humans; Liver Neoplasms; Receptors, Retinoic Acid; Trans-Activators; Tretinoin; Viral Regulatory and Accessory Proteins

Acyclic retinoid (ACR) is currently under clinical trial as an agent to suppress the recurrence of hepatocellular carcinoma (HCC) through its ability to induce apoptosis in premature HCC cells. ACR has an anticancer effect in vivo as well, although it shows weak apoptosis-inducing activity against mature HCC cells, suggesting the existence of an additional action mechanism. In this study, we investigated the antiangiogenic activity of ACR. ACR inhibited angiogenesis within chicken chorioallantoic membrane (CAM) in as similar a manner as all-trans retinoic acid (atRA). Although suppression of angiogenesis by atRA was partially rescued by the simultaneous addition of angiopoietin-1, suppression of angiogenesis by ACR was not rescued under the same condition at all. Conversely, although suppression of angiogenesis by ACR was partially inverted by the simultaneous addition of vascular endothelial growth factor (VEGF), suppression of angiogenesis by atRA was not affected under the same condition. These results suggested that mechanisms underlying the suppression of angiogenesis by ACR and atRA were different. ACR selectively inhibited the phosphorylation of VEGF receptor 2 (VEGFR2) and of extracellular signal-regulated kinase (ERK) without changing their protein expression levels, and inhibited endothelial cell growth, migration, and tube formation. The inhibition of the phosphorylation of ERK, endothelial growth, migration, tube formation, and angiogenesis by ACR was rescued by the overexpression of constitutively active mitogen-activated protein kinase (MAPK). Finally, ACR, but not atRA, inhibited HCC-induced angiogenesis in a xenografted CAM model. These results delineate the novel activity of ACR as an antiangiogenic through a strong inhibition of the VEGFR2 MAPK pathway.

Topics: Angiogenesis Inhibitors; Animals; Aorta; Carcinoma, Hepatocellular; Cattle; Cell Division; Cell Movement; Cells, Cultured; Chick Embryo; Chorioallantoic Membrane; Endothelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms, Experimental; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Neovascularization, Pathologic; Neovascularization, Physiologic; Phosphorylation; Transplantation, Heterologous; Tretinoin; Umbilical Veins; Vascular Endothelial Growth Factor Receptor-2

SMMC-7721 hepatocellular carcinoma cells (HCC) were incubated with fucosylated glycoproteins that had been isolated from retinoic acid-treated cells by affinity chromatography. HCC migration was significantly inhibited by AAL- and LCA-glycoproteins. Glycopeptides, obtained by digestion of the glycoproteins with trypsin and papain, were found to have a similar inhibitory effect on HCC migration as the corresponding glycoproteins. The inhibitory actions of the glycoproteins were almost abolished after digestion with alpha-L-1,3/4- or alpha-L-1,2-fucosidase. Induction of HCC migration with chemokines including interleukin-8 (IL-8), lymphotactin, monocyte chemoattractant protein-1, and stroma cell-derived factor-1 was examined and IL-8 was found to be the most potent. Interestingly, the isolated glycoproteins significantly inhibited HCC migration and F-actin aggregation induced by IL-8, whereas the glycans themselves did not induce F-actin assembly. From receptor binding analysis AAL-glycan was found to bind IL-8 receptors especially CXCR2 directly and such binding could be blocked by 3'- or 2'-fucosyllactose. After CXCR2 silence by target RNAi, the cells almost lost the response to AAL-glycan inhibition. Our findings suggest that fucosylation plays an important role in the interaction between IL-8 and its receptors inducing HCC migration.

Topics: Carcinoma, Hepatocellular; Cell Movement; Drug Screening Assays, Antitumor; Fucose; Glycopeptides; Glycoproteins; Humans; Liver Neoplasms; Protein Binding; Receptors, Chemokine; Structure-Activity Relationship; Tretinoin

We previously reported that all-trans-retinoic acid (ATRA) induced apoptosis in N-acetylglucosaminyltransferase V (GnT-V) repressed human hepatocarcinoma 7721 (GnT-V-AS/7721) cells via endoplasmic reticulum (ER) stress. In addition to confirming these findings, we further found that ATRA repressed the expression of betaine-homocysteine methyltransferase (BHMT) and cystathionine-beta-synthase (CBS), which are key enzymes that are involved in homocysteine metabolism, increased the level of intracellular homocysteine, and decreased the glutathione (GSH) level in GnT-V-AS/7721 cells. To investigate the effect of ATRA on homocysteine metabolism, cells were challenged with exogenous homocysteine. In GnT-V-AS/7721 cells with ATRA treatment, a significant elevation of intracellular homocysteine levels suggests that ATRA perturbs homocysteine metabolism in GnT-V-AS/7721 cells and, therefore, sensitizes the cells to homocysteine-induced ER stress. An obvious increase in the levels of GRP78/Bip protein and spliced XBP1 mRNA were observed. Furthermore, we observed that ATRA blunted the homocysteine-induced increase of GSH only in GnT-V-AS/7721 cells. These results demonstrate that ATRA intensifies ER stress and induces apoptosis in GnT-V-AS/7721 cells by disturbing homocysteine metabolism through the down-regulation of CBS and BHMT, depleting the cellular GSH and, in turn, altering the cellular redox status. In addition, we showed that ATRA did not trigger ER stress, induce apoptosis, or affect homocysteine metabolism in L02 cells, which is a cell type that is derived from normal liver tissue. These results provide support for the hypothesis that ATRA is an anticancer agent.

Topics: Antineoplastic Agents; Apoptosis; Betaine-Homocysteine S-Methyltransferase; Carcinoma, Hepatocellular; Cell Line, Tumor; Cystathionine beta-Synthase; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Homocysteine; Humans; Liver Neoplasms; N-Acetylglucosaminyltransferases; Tretinoin

A 77-year-old man who developed pancytopenia was administered granulocyte colony-stimulating factor (G-CSF) by another doctor, and referred to us for the evaluation of pancytopenia. He had hepatocellular carcinoma and was treated with transcatheter arterial chemoembolization (TACE) containg epirubicin (total dose: 300 mg over the last two years). Bone marrow aspiration smear demonstrated hypercellular marrow with promyelocytes. Cytogenetic analysis demonstrated del(7), t(15;17)(q22;q12), and a fluorescence in-situ hybridization (FISH) study demonstrated chimeric fusion genes of PML-RAR-alpha. He was diagnosed with therapy-related acute promyelocytic leukemia (APL), and treated with all trans-retinoic acid (ATRA). He showed the progressive accumulation of ascites with liver damage, without pulmonary symptoms of ATRA differentiation syndrome. After 60 days of ATRA treatment, complete hematological and cytogenetic responses were achieved. However, the patient died of septic circulatory failure.

Topics: Aged; Antibiotics, Antineoplastic; Antineoplastic Agents; Ascites; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Epirubicin; Humans; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Male; Tretinoin

Nuclear receptor constitutive androstane/active receptor (CAR) is well known as a transcription factor regulating many genes that encode drug-metabolizing enzymes and factors modulating hepatic gluconeogenesis. However, there have been few studies on regulation of the CAR gene itself. In this study, we examined the involvement of retinoic acid receptor alpha (RAR alpha) in transcriptional regulation of the CAR gene in the liver. The expression levels of CAR mRNA in human primary hepatocytes and HepG2 cells were increased by all-trans retinoic acid. Activities of the human CAR promoter containing a region (termed cRARE) located at +1453/+1469 within intron 1 were increased by co-expression of RAR alpha in HepG2 cells. In addition, introduction of mutation into cRARE abolished transcriptional activation of the promoter by RAR alpha. The results of gel mobility shift assay and chromatin immunoprecipitation assay showed that RAR alpha was bound to cRARE. These results suggest that RAR alpha transactivated the human CAR gene by binding to cRARE located at +1453/+1469 within intron 1 of the gene. In contrast, the rat CAR gene was not activated by exposure to all-trans retinoic acid, probably due to the lack of a region corresponding to cRARE in the human CAR gene. Although the physiological significance of RAR alpha-dependent up-regulation of CAR in the human liver remains to be clarified, retinoid metabolism may be regulated by the up-regulation of CAR.

Topics: Androstanes; Carcinoma, Hepatocellular; Carrier Proteins; Cells, Cultured; Constitutive Androstane Receptor; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatocytes; Humans; Introns; Liver Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Transcription Factors; Tretinoin

Hypoxia induces survival signals in hepatocellular carcinoma (HCC). We attempted to find a hypoxia-induced signal that could be used for modulating HCC cell death. Cellular retinoic acid binding protein-II (CRABP-II) expression was significantly increased in hypoxic HCC cells. Treatment with retinoic acid (RA), a ligand for CRABP-II, induced HCC cell apoptosis more effectively in hypoxia than in normoxia, whereas hypoxia-induced CRABP-II expression attenuated RA-induced apoptosis. Inhibition of CRABP-II enhanced RA-induced apoptosis and sensitized RA-resistant HCC cells to RA cytotoxicity by attenuating p42/44 MAPK and Akt activation. Therefore, RA/CRABP-II signal modulation is therapeutically implicated in infiltrative HCCs exposed to hypoxia.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; Receptors, Retinoic Acid; Tretinoin; Unfolded Protein Response

To detect differentially expressed proteins in hepatocellular carcinoma cell line SMMC-7721 treated separately by eight telomerase inhibitors including antisense oligodeoxynuclectide of human telomerase RNA (hTR-ASODN), sense oligodeoxynuclectide of hTR (hTR-SODN), ASODN of human telomerase reverse transcriptase (hTERT-ASODN), SODN of hTERT (hTERT-SODN), epigallocatechin gallate (EGCG), 3'-azido-3'-deoxythymidine (AZT), all trans-retinoic acid (ATRA) and adriamycin (ADM) using surface enhanced laser desorption/ionization time of flight-mass spectrom (SELDI-TOF-MS) technology.. SELDI-TOF-MS technology and weak cation exchanger (WCX-2) protein chip were used to detect differentially expressed secretory and cytoplasmic proteins of SMMC-7721 cell treated separately by eight telomerase inhibitors. The control group was hepatocellular carcinoma SMMC-7721 cell without any disposal.. The results of WCX-2 protein chip showed that the secretory and cytoplasmic proteins were differentially expressed in SMMC-7721 cell treated separately by eight telomerase inhibitors. But some proteins were down-regulated or up-regulated together in all experimental groups. The molecular weight of these differential proteins were all less than 10,000 Da.. Differentially expressed and common changes of proteins in SMMC-7721 cell treated separately by eight telomerase inhibitors would associate with telomerase activity.

Topics: Carcinoma, Hepatocellular; Catechin; Cell Line, Tumor; Enzyme Inhibitors; Humans; Liver Neoplasms; Protein Array Analysis; Proteome; Telomerase; Tretinoin; Zidovudine

Hypoxia may activate survival signals in cancer cells. Moreover, hypoxic cells are less sensitive than normoxic cells to doxorubicin cytotoxicity, a potent activator of the p53 tumor suppressor gene. N-myc downstream-regulated gene-1 (NDRG1) is a hypoxia- and retinoic acid-inducible protein, and has been previously implicated in carcinogenesis. As this protein is also a downstream target of p53 and hepatocellular carcinoma (HCC) cells frequently evidence resistance to retinoic acid (RA) cytotoxicity, we attempted to determine whether the suppression of NDRG1 expression may sensitize HCC cells to doxorubicin and/or RA cytotoxicity. HCC cells expressed NDRG1 protein, and the expression of this protein was hypoxia- and RA-inducible. Doxorubicin treatment induced HCC cell cytotoxicity via the activation of mitochondrial apoptotic signals, including caspase-9 activation. Hypoxic HCC cells are less sensitive to doxorubicin-induced apoptosis. The suppression of NDRG1 expression either by siRNA or flavopiridol sensitized hypoxic HCC cells to doxorubicin cytotoxicity, and this was attributed to more profound augmentation of JNK and caspase-9 activation. The suppression of NDRG1 expression also sensitized RA-resistant HCC cells to RA-induced apoptosis, and this sensitization was more apparent in hypoxic HCC cells than in normoxic cells. Glutaredoxin2 expression was down-regulated in NDRG1-suppressed HCC cells. These results show that hypoxia- and RA-inducible NDRG1 expression is responsible for doxorubicin and RA resistance in HCC cells. Thus, the selective interruption of NDRG1 signaling may prove to be therapeutically useful in HCCs, particularly in the advanced infiltrative type of tumors exposed to hypoxic environments.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Hypoxia; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Microarray Analysis; Tretinoin

Carcinogen-DNA adducts could lead to mutations in critical genes, eventually resulting in cancer. Many studies have shown that retinoic acid (RA) plays an important role in inducing cell apoptosis. Here we have tested the hypothesis that levels of carcinogen-DNA adducts can be diminished by DNA repair and/or by eliminating damaged cells through apoptosis. Our results showed that the levels of total DNA adducts in HepG2 cells treated with benzo(a)pyrene (BP, 2 μM)+RA (1 μM) were significantly reduced compared to those treated with BP only (P=0.038). In order to understand the mechanism of attenuation of DNA adducts, further experiments were performed. Cells were treated with BP (4 μM) for 24h to initiate DNA adduct formation, following which the medium containing BP was removed, and fresh medium containing 1 μM RA was added. The cells were harvested 24h after RA treatment. Interestingly, the levels of total DNA adducts were lower in the BP/RA group (390 ± 34) than those in the BP/DMSO group (544 ± 33), P=0.032. Analysis of cell apoptosis showed an increase in BP+RA group, compared to BP or RA only groups. Our results also indicated that attenuation of BP-DNA adducts by RA was not primarily due to its effects on CYP1A1 expression. In conclusion, our results suggest a mechanistic link between cellular apoptosis and DNA adduct formation, phenomena that play important roles in BP-mediated carcinogenesis. Furthermore, these results help understand the mechanisms of carcinogenesis, especially in relation to the chemopreventive properties of nutritional apoptosis inducers.

Topics: Anticarcinogenic Agents; Benzo(a)pyrene; Carcinoma, Hepatocellular; Cytochrome P-450 CYP1A1; DNA Adducts; DNA Repair; Hep G2 Cells; Humans; Liver Neoplasms; Tretinoin

Hypoxia induces survival signals in hepatocellular carcinoma (HCC). Hypoxia and retinoic acid (RA) may also induce interleukin-2 (IL-2) receptor expression, and thus we evaluated if RA and IL-2 receptor-targeted therapy are indicated in hypoxic HCCs. RA induced IL-2 receptor expression (R alpha, R beta, R gamma) in HCC cells, whereas hypoxia specifically induced IL-2 R gamma expression. IL-2 stimulated hypoxic HCC cell growth via p42/44 MAPK activation. Combination of denileukin diftitox and RA significantly suppressed hypoxic HCC cell growth compared to single agent-treated or normoxic cells. Therefore, denileukin diftitox and RA may be therapeutically implicated in infiltrative HCCs which are exposed to hypoxic environments.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Division; Cell Hypoxia; Cell Line, Tumor; Diphtheria Toxin; Enzyme Activation; Humans; Interleukin-2; Liver Neoplasms; Mitogen-Activated Protein Kinases; Tretinoin

Mammalian class I aldehyde dehydrogenase (ALDH) plays an important role in the biosynthesis of the hormone retinoic acid (RA), which modulates gene expression and cell differentiation. RA has been shown to mediate control of human ALDH1 gene expression through modulation of the retinoic acid receptor alpha (RARalpha) and the CCAAT/enhancer binding protein beta (C/EBPbeta). The positive activation of these transcription factors on the ALDH1 promoter is inhibited by RA through a decrease of C/EBPbeta binding to the ALDH1 CCAAT box response element. However, the mechanism of this effect remains unknown. Here we report that the RARalpha/retinoid X receptor beta (RXRbeta) complex binds to the mouse retinaldehyde dehydrogenase 1 (Raldh1) promoter at a non-consensus RA response element (RARE) with similar affinity to that of the consensus RARE. We found that C/EBPbeta binds to a Raldh1 CCAAT box located at -82/-58bp, adjacent to the RARE. Treatment with RA increases GADD153 and GADD153-C/EBPbeta interaction resulting in a decreased cellular availability of C/EBPbeta for binding to the Raldh1 CCAAT box. These data support a model in which high RA levels inhibit Raldh1 gene expression by sequestering C/EBPbeta through its interaction to GADD153.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Line, Tumor; Conserved Sequence; Deoxyribonucleotides; DNA Primers; DNA, Complementary; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Liver Neoplasms; Mice; Mice, Inbred C57BL; Retinal Dehydrogenase; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Tretinoin

The farnesoid X receptor/retinoid X receptor-alpha (FXR/RXRalpha) complex regulates bile salt homeostasis, in part by modulating transcription of the bile salt export pump (BSEP/ABCB11) and small heterodimer partner (SHP/NR0B2). FXR is activated by bile salts, RXRalpha by the vitamin A derivative 9-cis retinoic acid (9cRA). Cholestasis is associated with vitamin A malabsorption. Therefore, we evaluated the role of vitamin A/9cRA in the expression of human and mouse bile salt export pump (hBSEP/mBsep), small heterodimer partner (hSHP/mShp), and mouse sodium-dependent taurocholate co-transporting polypeptide (mNtcp). HBSEP and hSHP transcription were analyzed in FXR/RXRalpha-transfected HepG2 cells exposed to chenodeoxycholic acid (CDCA) and/or 9cRA. BSEP promoter activity was determined by luciferase reporter assays, DNA-binding of FXR and RXRalpha by pull-down assays. Serum bile salt levels and hepatic expression of Bsep, Shp, and Ntcp were determined in vitamin A-deficient (VAD)/cholic acid (CA)-fed C57BL/6J mice. Results indicated that 9cRA strongly repressed the CDCA-induced BSEP transcription in HepG2 cells, whereas it super-induced SHP transcription; 9cRA reduced DNA-binding of FXR and RXRalpha. The 9cRA repressed the CDCA-induced BSEP promoter activity irrespective of the exact sequence of the FXR-binding site. In vivo, highest Bsep messenger RNA (mRNA), and protein expression was observed in CA-fed VAD mice. Shp transcription was highest in CA-fed vitamin A-sufficient mice. Ntcp protein expression was strongly reduced in CA-fed VAD mice, whereas mRNA levels were normal. CA-fed control and VAD mice had similarly increased serum bile salt levels.. We showed that 9cRA has opposite effects on bile salt-activated transcription of FXR/RXRalpha target genes. Vitamin A deficiency in CA-fed mice leads to high BSEP expression. Clearance of serum bile salts may, however, be limited because of post-transcriptional reduction of Ntcp. The molecular effects of vitamin A supplementation during cholestasis need further analysis to predict a therapeutic effect.

Topics: Alitretinoin; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Carcinoma, Hepatocellular; Cell Line, Tumor; Chenodeoxycholic Acid; Cholic Acid; DNA-Binding Proteins; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Organic Anion Transporters, Sodium-Dependent; Receptors, Cytoplasmic and Nuclear; Response Elements; Retinoid X Receptor alpha; Symporters; Transcription Factors; Tretinoin; Vitamin A; Vitamin A Deficiency

Although there is increasing evidence that alpha fetoprotein (AFP) may function as regulatory factor in the growth of tumor cells, the precise mechanism is still unclear. In the current study, we investigated the role of the cytoplasmic AFP in caspase-3-mediated signaling of apoptosis. Our results showed that low doses of TNF-related apoptosis-inducing ligand (TRAIL) elevated the activity of caspase-8, but not caspase-3. Caspase-3 colocalized and interacted with AFP in the cytoplasm of Bel 7402 cells, and translocated into nuclei in association with the occurrence of apoptosis while cells were under cotreatment with all-trans retinoic acid (ATRA) or TRAIL. AFP was able to form complexes with caspase-3 and block onward transmission of signaling from caspase-8. Knockdown of AFP increased the sensitivity of Bel 7402 cells to TRAIL, and thereby, triggered caspase-3 signaling. No intermolecule interaction occurred between AFP and caspase-8, nor was caspase-8 activity altered after AFP knockdown, demonstrating the selectivity of AFP in interfering with the apoptotic signaling pathway. The effect of AFP on caspase-3 was further confirmed by transfection of the AFP gene into HLE cells (AFP negative). We conclude that ATRA or TRAIL resistance in AFP producing hepatoma is at least, in part, attributable to the high level of the cytoplasmic AFP. Therefore, it is possible that the combination of AFP gene silencing together with ATRA/TRAIL cotreatment will benefit the enhancement of the chemotherapeutic efficiency of these agents on tumors.

Topics: alpha-Fetoproteins; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 8; Cell Proliferation; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Fluorescence Resonance Energy Transfer; Humans; Immunoprecipitation; Liver Neoplasms; Protein Transport; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tretinoin; Tumor Cells, Cultured

The purpose of this study was to characterize the properties in vitro, i.e. release, degradation, hemolytic potential and anticancer activity, and in vivo disposition of all-trans-retinoic acid (ATRA) in rats after administration of ATRA-loaded micelle-like nanoparticles. The amphiphilic block copolymers consisted of a micellar shell-forming mPEG block and a core-forming PLA block. The mPEG-PLA nanoparticles prepared by an acetone volatilization dialysis procedure were identified as having core-shell structure by (1)H NMR spectroscopy. Critical association concentration, drug contents, loading efficiency, particle size and xi potential were evaluated. The release of ATRA from the nanoparticles and the degradation of PLA were found to be mostly associated with the compositions of the nanoparticles. ATRA release was faster at smaller molecular weight of copolymer and lower drug contents. In vitro, the incorporation of ATRA in mPEG-PLA nanoparticles reduced the hemolytic potential of ATRA. Furthermore, anticancer activity of ATRA against HepG2 cell was increased by encapsulation, which showed an enhancement of tumor treatment of ATRA. In vivo, after intravenous injection to rats, the levels of ATRA in the blood stream and the bioavailability were higher for ATRA-loaded mPEG-PLA nanoparticles than those for ATRA solution. In conclusion, the structure of the mPEG-PLA diblock copolymer could be modulated to fit the demand of in vitro and in vivo characterizations of nanoparticles. The mPEG-PLA nanoparticles' loading ATRA have a promising future for injection administration.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Delayed-Action Preparations; Humans; Micelles; Nanoparticles; Polyesters; Polyethylene Glycols; Tretinoin

The role of AFP in the retinoic acid-RAR signaling pathway was investigated in human hepatoma Bel 7402 cells. The results showed that AFP and RAR-beta were co-localized and interacted in cytoplasm. AFP may inhibit translocation of RAR-beta into the nucleus via competitive binding to RAR-beta with ATRA, which was reversed by AFP-siRNA transfection. Our data suggest that the ATRA resistance of Bel 7402 cells is at least in part attributable to their high level of cytoplasmic AFP. Thus, by counteracting the effect of AFP, it may be possible to increase the sensitivity of tumor cells to ATRA.

Topics: alpha-Fetoproteins; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytoplasm; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Immunoprecipitation; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Microtubule-Associated Proteins; Protein Transport; Receptors, Retinoic Acid; Signal Transduction; Survivin; Transfection; Tretinoin

A malfunction of retinoid X receptor-alpha (RXRalpha) due to phosphorylation is associated with the development of hepatocellular carcinoma (HCC) and acyclic retinoid (ACR), which targets RXRalpha, can prevent the development of second primary HCC. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, induces apoptosis and cell cycle arrest in cancer cells. VPA can also enhance the sensitivity of cancer cells to retinoids. The present study examined the possible combined effects of ACR plus VPA in HepG2 human HCC cell line. The combination of 5muM ACR and 1mM VPA, about the IC(25) value for both compounds, synergistically inhibited the growth of HepG2 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VPA also acted synergistically to induce apoptosis and G(0)-G(1) cell cycle arrest in HepG2 cells. This combination further exerted a synergistic inhibition of the phosphorylation of RXRalpha, ERK, Akt and GSK-3beta proteins and caused an accumulation of acetylated histones H3 and H4 proteins. VPA enhanced the ability of ACR to raise the cellular levels of RARbeta and p21(CIP1). The combination of these agents markedly increased both the RARE and RXRE promoter activities in HepG2 cells. These results suggest that ACR and VPA cooperatively increase the expression of RARbeta and p21(CIP1), while inhibiting the phosphorylation of RXRalpha, and these effects were associated with induction of apoptosis and the inhibition of cell growth in HepG2 cells. This combination might therefore be an effective regimen for the chemoprevention and chemotherapy of HCC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Humans; In Situ Nick-End Labeling; Liver Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Valproic Acid

To investigate the expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines of SMMC-7721, Bel-7402, HepG2, MHCC-97 and normal hepatocellular cell line of L02, and to compare the response of these cell lines to all-trans retinoic acid.. RT-PCR was used to detect expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines and normal hepatocellular cell line. Real time-PCR was used to quantify the expression of the genes.. There are different levels of expression of the stem cell-related gene in hepatocellular carcinoma cell lines and control cell line (P less than 0.05). There are significant differences in HepG2 and L-02 for the response to all-trans retinoic acid (P less than 0.05).. The stem cell-related genes are differentially expressed in different hepatocellular carcinoma cell lines.

Topics: beta Catenin; Carcinoma, Hepatocellular; DNA Methylation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Homeodomain Proteins; Humans; Liver Neoplasms; Nanog Homeobox Protein; Octamer Transcription Factor-3; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smoothened Receptor; SOXB1 Transcription Factors; Stem Cells; Tretinoin; Wnt Proteins

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.

Topics: Activins; alpha-Fetoproteins; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Carcinoma, Hepatocellular; Cell Differentiation; Embryonic Stem Cells; Endoderm; Extracellular Matrix; Fibroblast Growth Factor 4; Genetic Vectors; Green Fluorescent Proteins; Hepatocyte Growth Factor; Humans; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Transgenes; Tretinoin

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.

Topics: Adenocarcinoma; Aldehydes; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Cells, Cultured; Gene Expression Regulation; Glutamate-Cysteine Ligase; Humans; Kidney; Lipid Peroxidation; Liver Neoplasms; Male; MAP Kinase Kinase 1; Mice; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-E2-Related Factor 2; Response Elements; Retinoid X Receptor alpha; Retinoid X Receptor beta; RNA, Small Interfering; Tretinoin

A malfunction of retinoid X receptor-alpha (RXRalpha) due to phosphorylation by the Ras/mitogen-activated protein kinase signaling pathway is associated with the development of hepatocellular carcinoma (HCC). The humanized anti-HER2 monoclonal antibody trastuzumab inhibits the activation of HER2 and its multiple downstream signaling pathways, including the Ras/mitogen-activated protein kinase pathway. In this study, the effects of phosphorylation of RXRalpha on the ability of RXRalpha ligand 9-cis-retinoic acid (9cRA) and trastuzumab to inhibit growth of HCC cells was examined.. The effects of a combination of 9cRA plus trastuzumab on the inhibition of cell growth in HLF human HCC cells which express constitutive activation of HER2 protein were examined.. The combination of 9cRA plus trastuzumab synergistically inhibited the growth of HLF cells without affecting the growth of Hc normal human hepatocytes. Combined treatment with these agents acted synergistically to induce apoptosis in HLF cells. The treatment of HLF cells with trastuzumab alone inhibited the phosphorylation of HER2, RXRalpha, ERK, Akt, and Stat3 proteins and these effects were enhanced when the cells were cotreated with 9cRA. Reporter assays indicated that the combination of 9cRA plus trastuzumab markedly increased both the retinoic acid responsive element and retinoid X responsive element promoter activities in HLF cells.. 9cRA and trastuzumab cooperatively inhibit the activation of HER2 and its downstream signaling pathways, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Synergism; ErbB Receptors; Hepatocytes; Humans; Phosphorylation; Promoter Regions, Genetic; Receptor, ErbB-2; Response Elements; Retinoid X Receptor alpha; Trastuzumab; Tretinoin

We previously demonstrated that endoplasmic reticulum (ER) stress was triggered in human hepatocarcinoma 7721 cells transfected with antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V-AS/7721) which were more susceptible to apoptosis induced by all-trans retinoic acid (ATRA). In the present study, we report that ATRA-induced apoptosis in GnT-V-AS/7721 cells is mediated through ER stress. We show here that ER stress is enhanced in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, which is evidenced by the increase of GRP78/Bip, C/EBP-homologous protein-10 (CHOP, also known as GADD153) and spliced XBP1. Additionally, activation of caspase-12, caspase-9, and -3 was detected, and apoptosis morphology was observed in GnT-V-AS/7721 cells with ATRA treatment. These results suggest that ATRA enhances the ER stress triggered in GnT-V-AS/7721 cells, which represents a novel mechanism of ATRA to induce apoptosis. We further observed that GnT-V was significantly repressed and the structure of N-glycans was changed in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, suggesting that repression of GnT-V by ATRA causes the enhanced ER stress and ER stress-mediated apoptosis in GnT-V-AS/7721 cells.

Topics: Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; DNA Primers; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Humans; Liver Neoplasms; N-Acetylglucosaminyltransferases; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

The constitutive over-expression of the retinol dehydrogenase 10 (RDH10) gene, involved in retinoic acid (RA) biosynthesis, produced in HepG2 cells a significant antiproliferative response, but not signs of apoptosis. An indirect assay based on the Chloramphenicol AcetylTransferase (CAT) reporter gene driven by a retinoic acid responsive element (RARE) suggests in genetically modified HepG2 cells an increase of the endogenous RA concentration. Furthermore, the growth arrest of HepG2 cells over-expressing the RDH10 gene was associated with the upregulation and downregulation of, respectively, RARbeta/p21(Cip1) and CycE/CdK2 mRNAs. These results indicated that forced expression of RDH10 produces antiproliferative effects highly comparable to those achieved by retinoids treatment and thus the development of a gene therapy, finalized at the restoration of the enzymatic and receptorial machinery of the RA pathway, could be a possible curative strategy for hepatocellular carcinoma (HCC).

Topics: Alcohol Oxidoreductases; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Tretinoin

In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy.. A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Extracellular Matrix; Hepatocytes; Humans; Integrin alpha3; Integrin beta1; Tretinoin

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, effective chemopreventive and chemotherapeutic agents for this cancer have not yet been developed. In clinical trials acyclic retinoid (ACR) and vitamin K(2) (VK(2)) decreased the recurrence rate of HCC. In the present study we examined the possible combined effects of ACR or another retinoid 9-cis retinoic acid (9cRA) plus VK(2) in the HuH7 human HCC cell line. We found that the combination of 1.0 microM ACR or 1.0 microM 9cRA plus 10 microM VK(2) synergistically inhibited the growth of HuH7 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VK(2) also acted synergistically to induce apoptosis in HuH7 cells. Treatment with VK(2) alone inhibited phosphorylation of the retinoid X receptor (RXR)alpha protein, which is regarded as a critical factor for liver carcinogenesis, through inhibition of Ras activation and extracellular signal-regulated kinase phosphorylation. Moreover, the inhibition of RXRalpha phosphorylation by VK(2) was enhanced when the cells were cotreated with ACR. The combination of retinoids plus VK(2) markedly increased both the retinoic acid receptor responsive element and retinoid X receptor responsive element promoter activities in HuH7 cells. Our results suggest that retinoids (especially ACR) and VK(2) cooperatively inhibit activation of the Ras/MAPK signaling pathway, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Humans; Liver Neoplasms; Tretinoin; Vitamin K 2

The role of alpha1,3fucosyltransferase-VII (alpha1,3 FucT-VII) in cell apoptosis was studied in human hepatocellular carcinoma H7,721 cells. After the cells were transfected with alpha1,3 FucT-VII cDNA, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB and phospho-Bad were increased as compared with mock (vector) transfected cells, indicating that alpha1,3FucT-VII is a potential anti-apoptotic factor in H7,721 cells. After "alpha1,3FucT-VII" cells were irradiated by UV to induce apoptosis, the anti-apoptotic potential of alpha1,3FucT-VII became more apparent, as evidenced by the less apoptotic cell % and active cleaved caspase-3, more phospho-p38 MAPK and JNK (two anti-apoptotic signaling molecules in H7,721 cells responsible to UV stress) when compared with the "Mock" cells. In contrast, "alpha1,3FucT-VII" cells facilitated the apoptosis induced by all-trans retinoic acid (ATRA), which was verified by the greater sub-G1 (apoptotic cells) peak in flow cytometry analysis, more expressions of active caspase-3 and pro-apoptotic protein Bax, as well as less expressions of anti-apoptotic proteins, Bcl-2 and Bcl-X(L). The up regulation of alpha1,3FucT-VII mRNA and cell surface SLe(x) (alpha1,3FucT-VII product) by UV and down regulation of them by ATRA was speculated to be one of the mechanisms that alpha1,3FucT-VII decreased and increased the susceptibility of apoptosis induced by UV and ATRA respectively.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cells, Cultured; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Oligosaccharides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialyl Lewis X Antigen; Signal Transduction; Transfection; Tretinoin; Ultraviolet Rays

After N-acetylglucosaminyltransferase V (GnT-V) activity was down-regulated by the transfection of its antisense cDNA(GnTV-AS), apoptosis of H7721 cells was appeared and the apoptosis induced by 80 microM all-transretinoic acid (ATRA) was facilitated, while ATRA itself could not induce apparent apoptosis in mock cells transfected with the vector. In the study of the molecular mechanism of this phenomenon, it was found that GnTV-AS reduced the expressions of anti-apoptotic proteins, such as phosphorylated protein kinase B and phosphorylated Bad as well as Bcl-2 and Bcl-X (L), and elevated those of pro-apoptotic proteins, including Bax, full length caspase-3 and its activated fragments as well as anti-oncoprotein p53. In the contrast, ATRA up regulated the expressions of Bax and activated caspase-3 fragments only. After the GnTV-AS transfected cells were treated with ATRA, phosphorylated PKB and Bad were further decreased, while Bax and activated caspase-3 fragment were further increased, leading to the enhanced apoptosis in flow-cytometry analysis when compared with GnTV-AS cells not treated with ATRA. It was speculated that the decreased phospho-Bad resulted from the reduced phospho-PKB and the up regulation of p53 caused the elevated activity of Bax. The increased active caspase-3 was the consequence of the elevated Bax/ Bcl-2(Bcl-X(L)) activity ratio in the cells.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; bcl-X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Line, Tumor; Down-Regulation; Humans; I-kappa B Proteins; N-Acetylglucosaminyltransferases; NF-KappaB Inhibitor alpha; Phosphorylation; Proto-Oncogene Proteins c-akt; Tretinoin; Tumor Suppressor Protein p53

Garlic extracts, either aqueous or oily, are commonly employed to prepare garlic derivative supplements used as nutraceuticals for the treatment of different pathologies. In this study, we investigated the effects of water garlic extracts from two different areas of Italy well known for garlic production, Latina (GEL) and Sulmona (GES), on cell cycle and death of HepG2 hepatoma cells. The effects of the treatments with GEL and GES were also compared with the oil-soluble sulfur compound of garlic, diallyl disulfide (DADS). GEL and GES induced a p53/p21-dependent cell cycle arrest in G2/M phase and apoptosis, although to a different extent, whereas DADS, under the experimental conditions used, was not detrimental to HepG2 cells. GEL and GES committed HepG2 cells to apoptosis by the activation of c-Jun-NH(2) terminal kinase (JNK)/c-Jun phosphorylative cascade without a detectable increase in the flux of reactive oxygen species. Moreover, differentiation of HepG2 cells induced by retinoic acid determined resistance to GEL and GES treatments without the activation of JNK signaling pathway. Overall, the results obtained indicate that water-soluble garlic extracts are more inhibitory of the growth of transformed hepatoma cells than the oil-soluble isolated compound DADS, and that their antiproliferative properties are different depending on the area of origin of the starting material.

Topics: Allyl Compounds; Biomarkers; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Disulfides; Enzyme Activation; Garlic; Humans; JNK Mitogen-Activated Protein Kinases; Plant Extracts; Selenium; Signal Transduction; Tretinoin; Tumor Suppressor Protein p53; Water

ATRA (all-trans retinoic acid), which is a major bioactive metabolite of vitamin A and a potent regulator of development and differentiation, mediates down-regulation of the human albumin gene. However, the mechanism of ATRA-mediated down-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrate that ATRA causes a marked decrease in the activity of the albumin promoter, the region between nt -367 and -167 from the transcription start site, where C/EBP (CCAAT/enhancer-binding protein)-binding sites are tightly packed, is indispensable for ATRA-mediated down-regulation. ChIP (chromatin immunoprecipitation) assays revealed that in vivo binding of C/EBPalpha to the region markedly decreases upon incubation with ATRA, whereas ATRA treatment marginally increases the recruitment of C/EBPbeta. We found that ATRA has the ability to differentially and directly induce expression of a truncated isoform of C/EBPbeta, which is an LIP (liver-enriched transcriptional inhibitory protein) that lacks a transactivation domain, and to increase the binding activity of C/EBPbeta-LIP to its response element. Overexpression of C/EBPbeta-LIP negatively regulates the endogenous expression of albumin, as well as the activity of the albumin promoter induced by C/EBP transactivators such as C/EBPalpha and full-length C/EBPbeta. In conclusion, we propose a novel model for down-regulation of the albumin gene, in which ATRA triggers an increase in the translation of C/EBPbeta-LIP that antagonizes C/EBP transactivators by interacting with their binding sites in the albumin promoter.

Topics: Albumins; Binding Sites; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation; Humans; Promoter Regions, Genetic; Protein Binding; Transcriptional Activation; Tretinoin

CYP4A11, the major fatty acid omega-hydroxylase in human liver is involved in the balance of lipids, but its role and regulation are both poorly understood. We studied the effects of retinoids on the regulation of CYP4A11 in the human hepatoma cell line HepaRG. Treatment of HepaRG cells with all-trans-retinoic acid resulted in a strong decrease in CYP4A11 gene expression and apoprotein content and, furthermore, was associated with a 50% decrease in the microsomal lauric acid hydroxylation activity. Such a strong suppression of CYP4A11 expression by retinoids could have a major impact on fatty acid metabolism in the liver.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Humans; Liver; Liver Neoplasms; PPAR alpha; RNA, Messenger; Tretinoin

Tumor metastasis is usually a serious problem in tumor patients because of the lack of therapeutic approaches. A new compound, N-all-trans-retinoyl-L-proline (ATRP), has been developed and its metastasis inhibition activity has been studied. Low concentrations of ATRP have already been found to inhibit hepatocellular carcinoma cells (HCC) in a dose- and time-dependent manner by inducing the expression of p27(kip). We found that ATRP inhibited metastasis-associated behaviors in Hep3B cells, such as cell migration, invasion, collagen adhesion and gelatinase expression, more significantly than retinoic acid. Further, such inhibitory activities were observed in the regulation of cellular surface fucosylated epitope functions, such as binding of ulex europaeus lectin, expression of Lewis x, y and b, and activity of alpha1,3 fucosyltransferase. Hep3B cells pretreated with ATRP showed a significantly reduced incidence of experimental intrahepatic metastasis in nude mice. We conclude that ATRP is an alternative inhibitor and potential therapeutic agent for HCC metastasis with a different mechanism of action from ATRP.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Adhesion; Cell Cycle; Cell Movement; Fenretinide; Fucose; Fucosyltransferases; Gelatinases; Humans; Liver Neoplasms, Experimental; Mice; Mice, Nude; Molecular Structure; Neoplasm Invasiveness; Neoplasm Transplantation; Proline; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Previous studies have shown that all-trans retinoic acid (ATRA) suppresses growth of hepatocarcinoma cell in vitro. To understand the underlying mechanisms, we investigated the protein expression profiles by 2-DE in hepatocarcinoma cell line SMMC-7721 treated with ATRA. Our results reveal that six proteins were differently expressed in response to ATRA. Using MS and database searching, they were identified as profilin 1, phosphoglycerate kinase 1, RuvB-like 1, alpha-enolase, pyridoxal kinase and F-actin capping protein. We selected the up-regulated protein, profilin 1 (PFN1), for further studies. The PFN1 expression was increased in response to ATRA in a dose- and time-dependent manner. The PFN1 expression was reduced dramatically in four hepatoma cell lines compared to L02 cell line of non-tumor origin. The PFN1 expression was also examined in 4 cases of primary hepatocarcinoma tissues by Western blot and 30 cases by tissues microarray. It was found that the protein level of PFN1 was lower in hepatocarcinoma tissues compared to that in the adjacent tissues. Similar to ATRA, overexpression of PFN1 led to inhibition of cell proliferation and migration. Furthermore, RNAi-based PFN1 knockdown could rescue the inhibitory effect of ATRA on cell proliferation and migration. In conclusion, ATRA inhibited cell proliferation and migration through up-regulation of PFN1.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Electrophoresis, Gel, Two-Dimensional; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Profilins; Proteomics; RNA Interference; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Transfection; Tretinoin; Up-Regulation

All-trans retinoic acid (ATRA) and 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] can inhibit the proliferation of tumor cells and induce their differentiation. They have been used to treat leukemia, but their effects on solid tumors remain unclear. This study was to investigate the effects of ATRA and 1,25(OH)2D3 on growth and cell cycle of human hepatoma cell line HepG2, and explore the molecular mechanism.. After HepG2 cells were treated with ATRA, 1,25(OH)2D3, or the combination of both chemicals, cell survival was assessed by MTT assay, morphologic changes were observed under light microscope, cell cycle and apoptosis were determined by flow cytometry (FCM) with dual staining of AnnexinV/PI, and the expression of p21(WAF/CIP1) and p27(KIP1) mRNA and protein were determined by reverse transcription-polymerase chain reaction (RT-PCR) and FCM.. ATRA and 1,25(OH)2D3, used alone or in combination, inhibited the growth of HepG2 cells in a time- and dose-dependent manner. The inhibition was more obvious in the combination group than in ATRA group and 1,25(OH)2D3 group (P<0.05). FCM analysis indicated that 10 nmol/L 1,25(OH)2D3, used alone or in combination with ATRA for 72 h, strongly induced G1 phase arrest of HepG2 cells [(54.27+/-3.69)% and (65.64+/-5.65)% vs. (40.40+/-1.91)% of the control, P<0.05], but 1 micromol/L ATRA did not show obvious effect. All of them induced apoptosis (P<0.05). The mRNA level of p21(WAF/CIP1) was enhanced by 35% and 56% of control in the cells treated with 10 nmol/L 1,25(OH)2D3 or 1,25(OH)2D3 combined with ATRA for 24 h. The combination of 1,25(OH)2D3 and ATRA markedly enhanced the protein levels of p21(WAF/CIP1) and p27(KIP1) as compared with 1,25(OH)2D3 alone, and 1 micromol/L ATRA did not enhance the protein levels of p21(WAF/CIP1) and p27(KIP1) in HepG2 cells.. ATRA and 1,25(OH)2D3 could inhibit growth and induce apoptosis of HepG2 cells, and the molecular basis of the cell cycle blockade by 1,25(OH)2D3 may be associated with the up-regulation of CDK inhibitors p21(WAF/CIP1) and p27(KIP1), which mediate G1 arrest. Furthermore, the combination of ATRA and 1,25(OH)2D3 can exert synergistic inhibitory effect on the growth of HepG2 cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Drug Synergism; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; RNA, Messenger; Tretinoin; Up-Regulation; Vitamin D

p62 is a cancer-associated antigen binding to mRNA encoding insulin-like growth factor II that was isolated by immunoscreening a cDNA expression library with autoantibodies from patients with hepatocellular carcinoma (HCC). In the present study, multiple methods including flow cytometry, confocal laser-scanning microscope, electron microscope were used to characterize the effect of ATRA on BGC-823 cells, which presented two phenotypes of differentiation and apoptosis in cells treated with 1.0 and 50 microM ATRA, respectively. Interestingly, we found that p62 was cytoplasmic in location, but it significantly decreased in cytoplasm and appeared in nucleus of cells when the cells were treated with 50 microM all-trans retinoic acid (ATRA) for 5 days. Furthermore, proteomics approach on differential nucleus proteins showed that the up-regulation and/or down-regulation of cell cycle proteins and IGF binding proteins were involved in the apoptosis of BGC-823 cells induced by ATRA. These results suggest that there is a significant association between expression and distribution of p62 and the growth arrest of tumor cells, in which p62 is associated with cell apoptosis induced by ATRA.

Topics: Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Electrophoresis, Gel, Two-Dimensional; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Image Processing, Computer-Assisted; Microscopy, Confocal; Microscopy, Electron; Neoplasm Proteins; Peptide Mapping; Proteomics; RNA-Binding Proteins; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stomach Neoplasms; Subcellular Fractions; Time Factors; Tretinoin

There is great medical need to develop novel therapies for treatment of human hepatitis C virus (HCV). By gene expression analysis of three HCV-subgenomic RNA replicon cell lines, we identified cellular proteins whose expression is affected by the presence of HCV and therefore may serve as drug targets. Data from cDNA array filter hybridization, as well as from Northern and Western blotting, revealed that the gastrointestinal-glutathione peroxidase (GI-GPx) was drastically down-regulated (up to 20-fold) in all replicon cell lines tested. Concomitantly, total cellular glutathione peroxidase activity was drastically reduced, which rendered these human liver cells more susceptible toward oxidative stress. Interferon alpha caused down-regulation of the HCV-replicon followed by recovery of GI-GPx expression to nearly normal levels. Furthermore, expression of GI-GPx in replicon cells by gene transduction caused down-regulation of HCV RNA in a dose-dependent manner. Moreover, activating the endogenous gene coding for GI-GPx by all-trans-retinoic acid (RA) was sufficient to cause down-regulation of the HCV replicon. A small interfering RNA duplex abrogated GI-GPx up-regulation by RA and concomitantly suppression of HCV. The RA effect was dependent on the presence of sodium selenite, was reversible, and was independent of RNA-activated protein kinase. Taken together, these results show that HCV inhibits the expression of GI-GPx in replicon cells to promote its intracellular propagation. Modulation of GI-GPx activity may open new avenues of treatment for HCV patients.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Survival; DNA Primers; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Viral; Glutathione Peroxidase; Hepacivirus; Humans; Liver; Liver Neoplasms; Paraquat; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Viral; Transfection; Tretinoin

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Its high mortality rate is mainly a result of high intrahepatic recurrence. The novel synthetic retinoid acyclic retinoid (ACR) has been reported to prevent the recurrence of human HCC after surgical resection of primary tumors, but the molecular mechanisms underlying its effects remain to be elucidated. In this study, we clarified the molecular targets of ACR.. The inhibitory effects by ACR on growth were examined. Intracellular signaling induced by ACR was comprehensively studied by a reporter assay. Gene expression changes by ACR were examined using a microarray. From these results, a candidate signaling pathway modulated by ACR was determined and whether antagonizing this pathway reverses the effect was examined.. We show that ACR inhibits the growth of HCC cells through the down-regulation of fibroblast growth factor (FGF) receptor 3 expression and FGF-mediated signaling, which in turn suppresses the activity of Rho and serum response factor-mediated transcription. Conversely, overexpression of the active form of FGF receptor 3 or the addition of FGF reverses the ACR-mediated inhibition of growth. In addition, silencing the FGF receptor 3 gene by RNA interference inhibits cell growth.. These studies show that ACR is a potent inhibitor of FGF signaling and that selective blocking of the FGF-mediated pathway could be a promising therapeutic approach for the management of patients with HCC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Fibroblast Growth Factors; Gene Expression; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Oligonucleotide Array Sequence Analysis; Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 3; Receptors, Fibroblast Growth Factor; rho GTP-Binding Proteins; Signal Transduction; Tretinoin

Hepatocellular carcinoma (HCC) is common worldwide and growing in importance in the West. HCC often occurs against a background of liver disease, tends to present at an advanced stage, and has a poor prognosis, suggesting that it is an ideal target for chemoprevention. We sought to identify in an animal model chemopreventive agents for HCC that might be tested in human subjects. To this end, we induced liver tumors by injecting ethyl-nitrosourea in 6-week-old male B6C3F1 mice. Two chemopreventive agents were administered over a period of 60 weeks: tamoxifen (420 mg/kg feed) and a retinoid, 13-cis-retinoic acid (200 mg/kg feed). Animals were killed at 60 weeks and their livers examined for HCC and premalignant lesions. All liver lesions (altered foci, adenomata, HCC) occurred significantly less frequently in the tamoxifen-treated group than the group given only ethylnitrosourea (HCC developed in 2 of 47 (4%) vs 11 of 44 (25%); P < .001). On the other hand, retinoic acid appeared to increase the number of liver tumors, and in 2 animals angiosarcoma developed. Tamoxifen significantly decreased the incidence of chemical hepatocarcinogenesis in this model, suggesting an important role for estrogens in the pathogenesis of HCC and suggesting that it should be tested in human beings as a chemopreventive agent against HCC.

Topics: Adenoma, Liver Cell; Animals; Anticarcinogenic Agents; Carcinoma, Hepatocellular; Chemoprevention; Disease Models, Animal; Ethylnitrosourea; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred Strains; Precancerous Conditions; Tamoxifen; Tretinoin

To investigate the effect of all-trans retinoic acid (ATRA) on the expression of connexin 26, 32 and 43 genes and the alteration of gap junction communication function in human hepatocellular carcinoma cells.. Human hepatocellular carcinoma cell of the lines SMMC-7721 and BEL-7404 were cultured in normal medium and medium containing ATRA at a concentration of 10(-5) mol/L for 24, 48 and 72 hours respectively. RT-PCR procedure was adopted to detect the mRNA expression of CX 26, 32 and 43. Scrape-loading and dye transfer procedure was performed to examine the gap junction communication function.. CX26 mRNA and CX32 mRNA were not expressed in the cell lines SMMC-7721, however, expression of CX26 mRNA and expression of CX32 mRNA were found 48 and 72 hours after being induction by ATRA respectively. CX26 mRNA and CX32 mRNA were not expressed in the cell lines BEL-7404, however, expression of CX26 mRNA and expression of CX32 mRNA were found 48 hours after induction by ATRA. Expression of CX43 mRNA was found in all cells, whether being induced by ATRA or not. Scrape-loading and dye transfer procedure showed that lucifer yellow was seen in only 1-2 lines by the delimited mark in the untreated SMMC-7721 cells and in 3-4 lines by the delimited mark in the SMMC-7721 cells treated by ATRA. But no dye transfer phenomenon was found in the BEL-7404 cells whether they were ATRA-treated or not.. ATRA is able to affect the expression of CX26 and CX32 in HCC cell lines SMMC-7721 and BEL-7404 by acting at the transcription level. Reinforcement of gap junction communication function is found in the SMMC-7721 cells and not in the BEL-7404 cells, which shows that ATRA modulates the gap junction intercellular communication, by acting in different mechanisms.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Communication; Cell Line, Tumor; Connexin 26; Connexin 43; Connexins; Gap Junction beta-1 Protein; Gap Junctions; Humans; Liver Neoplasms; RNA, Messenger; Tretinoin

To investigate the effect of all-trans-retinoic acid (ATRA) on arsenic trioxide (As(2)O(3))-induced apoptosis of human hepatoma, breast cancer, and lung cancer cells in an attempt to find a better combination therapy for solid tumors.. Human hepatoma cell lines HepG2, Hep3B, human breast cancer cell line MCF-7, and human lung adenocarcinoma cell line AGZY-83-a were treated with As(2)O(3) together with ATRA. Cell survival fraction was determined by MTT assay, cell viability and apoptosis were measured by annexin V-fluorescein isothiocyanate (FITC) and PI staining, and intracellular glutathione (GSH) and glutathione-S-transferase (GST) activities were determined using commercial kits.. Cytotoxicity of ATRA was low. ATRA (0.1, 1, and 10 micromol/L) could synergistically potentiate As(2)O(3) to exert a dose-dependent inhibition of growth and to induce apoptosis in each of the cell lines. HepG2 and Hep3B with low intracellular GSH or GST activities were remarkably sensitive to As(2)O(3) or As(2)O(3)+ATRA, while AGZY-83-a with higher GSH or GST activities was less sensitive to As(2)O(3) or As(2)O(3)+ATRA. Treatment with 2 micromol/L As(2)O(3) for 72 h significantly decreased intracellular GSH and GST levels in each of the cell lines, and 1 micromol/L ATRA alone reduced minimal intracellular GSH and GST levels. ATRA potentiated the effect of As(2)O(3) on intracellular GSH levels, but intracellular GST levels were not significantly affected by the combination of As(2)O(3) and ATRA for 72 h as compared to As(2)O(3) alone.. ATRA can strongly potentiate As(2)O(3)-induced growth-inhibition and apoptosis in each of the cell lines, and two drugs can produce a significant synergic effect. The sensitivity to As(2)O(3) or As(2)O(3)+ATRA is inversely proportional to intracellular GSH or GST levels in each of the cell lines. The GSH redox system may be the possible mechanism by which ATRA synergistically potentiates As(2)O(3) to exert a dose-dependent inhibition of growth and to induce apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Glutathione; Glutathione Transferase; Humans; Lung Neoplasms; Oxides; Tretinoin

Vimentin is a growth-related gene and often expressed when epithelial cells are stimulated to proliferate by growth factors. In cancer, vimentin expression is associated with a dedifferentiated malignant phenotype, increased motility, invasive ability and poor prognosis. We studied the regulation of vimentin mRNA and multistep invasion processes following treatment of 12-O-tetradecanoylphorbol 13-acetate (TPA) and all-trans-retinoic acid (RA) in Hep 3B hepatocellular carcinoma cells. TPA showed marked induction of vimentin mRNA, while RA decreased the mRNA level. TPA or RA did not affect cell proliferation, cell-matrix protein adhesion, and matrix metalloproteinases and urokinase plasminogen activator activities. In vitro invasion ability was significantly increased or decreased with TPA or RA treatment, paralleled to the in vitro motile activity, respectively. These findings suggest that TPA and RA could modulate the invasive potential of Hep 3B cells by altering cellular motility related to differential regulation of vimentin mRNA.

Topics: Carcinoma, Hepatocellular; Cell Movement; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinases; Neoplasm Metastasis; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vimentin

To study the effect of Zhengganfang (ZGF) drug serum in inducing differentiation of Bel-7402 human hepatocarcinoma cell (HHC) and its influence of the telomerase activity.. Rats were fed with ZGF decoction to prepare the drug-serum of ZGF, and the drug-serum was used for Bel-7402 HHC culture. Serum of normal rat, was taken as negative control and retinoic acid (RA) as the positive control. Cell proliferation was detected by MTT colorimetric assay, nucleocytoplasm ratio by image assay, alpha-fetoprotein (AFP) by RIA, alkaline phosphatase (ALP) activity and gamma-glutamyl transpeptidase (gamma-GT) by dynamic colorimetric assay and telomerase activity of Bel-7402 cell by PCR-ELISA.. Drug-serum of ZGF could inhibit the proliferation of Bel-7402 cell, markedly reduce the nucleocytoplasm ratio and the secretion of AFP, decrease gamma-GT activity, increase ALP activity and suppress the telomerase activity of Bel-7402 cell.. ZGF can induce the differentiation of Bel-7402 HHC, the main mechanism maybe through the suppression of telomerase activity.

Topics: Alkaline Phosphatase; alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Drugs, Chinese Herbal; gamma-Glutamyltransferase; Hepatocytes; Liver Neoplasms; Male; Rats; Rats, Sprague-Dawley; Serum; Telomerase; Tretinoin; Tumor Cells, Cultured

Effective therapy for advanced hepatocellular carcinoma (HCC) is lacking. Conventional chemotherapy was judged to be ineffective. We previously demonstrated that the histone deacetylase inhibitor Trichostatin A (TSA) blocks growth of HCC cells in vitro. The anti-tumoral effect of a combination of more than 2 classes of drugs remains unexplored. Four hepatoma cell lines were incubated with increasing concentrations of Tamoxifen (TAM), 9-cis retinoic acid (CRA), the methioninaminopeptidase inhibitor TNP-470 and TSA as single agents and in combination. Anti-proliferative and pro-apoptotic effects were assessed using BrdU-incorporation, FACS analysis and immunocytochemistry. Central pro- and anti-apoptotic proteins were measured by semi-quantitative Western blotting and substrate assays. All single substances inhibited proliferation and induced apoptosis in HCC cells only at high concentrations. The combination of TAM/CRA/TNP/TSA multiplied the anti-tumoral effects, reaching up to 93% inhibition of proliferation and 63% induction of apoptosis after 24 h in Hep1B cells. Pro-apoptotic factors bax and caspase 3 were highly increased with quadruple therapy, while anti-apoptotic bcl-2 decreased to undetectable levels. Fibroblasts remained largely unaffected. While the single substances were not effective on hepatoma cells in tolerable doses, their combination significantly increases anti-tumoral efficacy. Combination therapy with biomodulators is a promising treatment option for HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cyclohexanes; DNA; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Estrogen Antagonists; Humans; Hydroxamic Acids; Immunologic Factors; Mice; O-(Chloroacetylcarbamoyl)fumagillol; Proto-Oncogene Proteins c-bcl-2; Rats; Sesquiterpenes; Tamoxifen; Tretinoin

Retinoids can block cell proliferation and induce apoptosis in tumor cells. The antitumoral effect of synthetic retinoids like Adapalene (ADA) on hepatoma cells (HepG2, Hep1B) was investigated. Cell proliferation was assessed by measuring DNA synthesis and apoptosis by flow cytometry and immunocytochemistry. Cell cycle- and apoptosis-associated proteins were semi-quantified by Western Blotting and breakdown of mitochondrial membrane potentials was detected by JC-1 staining. ADA at 10(-4)M efficiently induced apoptosis, reaching 61.7% in HepG2 and 79.1% in Hep1B after 72 h incubation. This was accompanied by up-regulation of pro-apoptotic bax and caspase 3, while bcl-2 was down-regulated, shifting the bax/bcl-2 ratio to >2.3 in hepatoma cells. ADA inhibits hepatoma cell growth in vitro and is a powerful inducer of hepatoma cell apoptosis.

Topics: Adapalene; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Cycle; Flow Cytometry; Humans; Immunoenzyme Techniques; Liver Neoplasms; Membrane Potentials; Mitochondria; Naphthalenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

Activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in response to all-trans-retinoic acid (RA) or a glucocorticoid such as dexamethasone (Dex) requires a distinct arrangement of DNA-response elements and their cognate transcription activators on the gene promoter. Two of the accessory factor-binding elements involved in the Dex response (gAF1 and gAF3) coincide with the DNA-response elements involved in the RA response. We demonstrate here that the combination of Dex/RA has a synergistic effect on endogenous PEPCK gene expression in rat hepatocytes and H4IIE hepatoma cells. Reporter gene studies show that the gAF3 element and one of the two glucocorticoid receptor-binding elements (GR1) are most important for this effect. Chromatin immunoprecipitation assays revealed that when H4IIE cells were treated with Dex/RA, ligand-activated retinoic acid receptors (retinoic acid receptor/retinoid X receptor) and glucocorticoid receptors are recruited to this gene promoter, as are the transcription coregulators p300, CREB-binding protein, p/CIP, and SRC-1. Notably, the recruitment of p300 and RNA polymerase II to the PEPCK promoter is increased by the combined Dex/RA treatment compared with Dex or RA treatment alone. The functional importance of p300 in the Dex/RA response is illustrated by the observation that selective reduction of this coactivator, but not that of CREB-binding protein, abolishes the synergistic effect in H4IIE cells.

Topics: Animals; Antineoplastic Agents, Hormonal; Blotting, Western; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Chromatin; Dexamethasone; Drug Synergism; E1A-Associated p300 Protein; Gene Expression Regulation, Enzymologic; Genes, Reporter; Glucocorticoids; Hepatocytes; Humans; Ligands; Liver; Liver Neoplasms; Luciferases; Mutation; Nuclear Proteins; Phosphoenolpyruvate Carboxykinase (ATP); Plasmids; Precipitin Tests; Promoter Regions, Genetic; Protein Structure, Tertiary; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA Polymerase II; Trans-Activators; Transfection; Tretinoin

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.

Topics: Albumins; Calcitriol; Carcinoma, Hepatocellular; Cytarabine; Dimethyl Sulfoxide; Enzyme Activation; Humans; Liver Neoplasms; N-Acetylglucosaminyltransferases; Pyridines; Tretinoin

Gene33 and its human homologue, mitogen inducible gene-6/receptor-associated late transducer (mig-6, RALT), is a 53-kDa soluble protein that was identified as a hepatic gene regulated by glucocorticoids and insulin. Its mRNA is expressed in numerous tissues in addition to the liver. Mitogen inducibility of Gene33 mRNA has been described in several experimental systems. Recent reports have suggested a role for Gene33 in inhibition of proliferation induced by factors that bind to members of the ErbB family of receptors. In the present work, we examine the regulation of Gene33 protein by insulin in hepatoma cells of rat (H4IIE) and human (HepG2/Hep3B) origin. Inhibition of MEK1 significantly inhibited extracellularly regulated kinase (ERK)1/2 activation and insulin-regulated Gene33 transcription and protein levels in H4IIE cells. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity alone did not significantly alter transcription of Gene33. In Hep3B and HepG2 cells, insulin did not significantly induce either ERK1/2 activation or Gene33 expression. This work suggests that the MEK-ERK, but not the phosphatidylinositol 3-kinase (PI3-K), pathway plays a direct role in insulin regulation of Gene33 transcription and protein expression.

Topics: Adaptor Proteins, Signal Transducing; Androstadienes; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Chromones; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation; Humans; Immune Sera; Insulin; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Recombinant Fusion Proteins; Signal Transduction; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wortmannin

Although the PPARgamma agonist troglitazone has been shown to induce growth inhibition of hepatocellular carcinoma (HCC) cells at high concentration, this study indicates troglitazone does not significantly inhibit the growth of HCC cells at clinically achievable concentrations (1-10 microM), and this lack of activity could not be improved by the addition of 9-cis-retinoic acid. Furthermore, no synergistic effect was found between troglitazone and cytotoxic anticancer agents.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Cell Division; Chromans; Dose-Response Relationship, Drug; Drug Synergism; Humans; Liver Neoplasms; Thiazolidinediones; Tretinoin; Troglitazone; Tumor Cells, Cultured

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

The concentration of type I interferon receptor (IFN-Rc) in the liver is a crucial factor in determining the efficacy of interferon (IFN) therapy in patients with chronic hepatitis C. Retinoic acids (RAs) can enhance the expression of type I IFN-Rc expression. The aim of this study was to investigate whether RAs increase the anti-hepatitis C virus (HCV) effect of IFN through an increase in IFN-Rc. The hepatocellular carcinoma cell line HuH-7 was treated with 10(-7) mol/L all-trans RA (ATRA) and 9-cis RA (9-CRA). Expression of type I IFN-Rc was investigated at both the mRNA and protein levels with the use of real-time quantitative polymerase chain reaction and flow cytometry, respectively. We investigated the anti-HCV effect, using in vitro HCV transfection, by monitoring the level of HCV RNA in the culture medium. ATRA and 9-CRA enhanced the expression of type I IFN-Rc at both the mRNA and protein levels. After IFN-alpha treatment, the activity of 2,5'-oligoadenylate synthetase was enhanced by RAs, and this enhancement was abolished when blocking antibodies had previously been bound to the surface receptors. IFN treatment decreased the concentration of HCV RNA, and this effect was enhanced by treatment with RAs. Our findings suggest that RAs enhance the anti-HCV replication effect of IFN-alpha through up-regulation of type I IFN-Rc in HuH-7 cells.

Topics: 2',5'-Oligoadenylate Synthetase; Alitretinoin; Antibodies; Antiviral Agents; Carcinoma, Hepatocellular; Drug Interactions; Gene Expression; Hepacivirus; Humans; Interferon-alpha; Interferons; Liver Neoplasms; Membrane Proteins; Receptor, Interferon alpha-beta; Receptors, Interferon; RNA, Viral; Transfection; Tretinoin; Tumor Cells, Cultured; Virus Replication

Natural killer (NK) cells are important effector cells for the first line of defense against tumor, but the mechanisms by which they recognize and kill human hepatocellular carcinoma (HCC) remains to be elucidated. Distant MHC class I homologs MICA and MICB are recently identified human ligands for NK cell activating receptor NKG2D. In our present study, MICA or MICB transcript was detected in 6 of 10 human hepatocellular carcinoma tissues, but not in the surrounding non-cancerous tissues. Immunohistochemical analysis showed that MICA/B were expressed in the tumor cells of the cancerous tissues. Huh7 and HepG2 hepatoma cells, but not Hep3B cells, substantially expressed MICA/B on their cell surface. MICA/B expressed on hepatoma cells contributed to their NK sensitivity, because Huh7 and HepG2 were less susceptible to NK cytolysis when MAb against MICA/B was added during the cytolysis assay. Of interest is the finding that retinoic acid upregulated expression of MICA/B in Huh7 and HepG2 cells. Retinoic acid-treated hepatoma cells induced IFN gamma production from cocultured NK cells and rendered themselves more susceptible to NK cells. This was clearly dependent on upregulation of MICA/B, because both the enhanced IFN gamma production and NK cytolysis were completely abolished by MAb-mediated masking of MICA/B. These results suggest that MICA/B, expressed on a subset of human HCCs, may play an important role in their susceptibility to NK cells. Furthermore, retinoic acid can function as a modulator of MICA/B expression and thereby further activate NK cells.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; DNA Primers; Flow Cytometry; Gene Expression Regulation; Histocompatibility Antigens Class I; Humans; Immunoenzyme Techniques; Immunoglobulin G; Interferon-gamma; Killer Cells, Natural; Liver Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

We have reported previously that acyclic retinoid, a synthetic retinoid X receptor alpha (RXRalpha)-ligand, suppresses the development of hepatocellular carcinoma (HCC) in patients with chronic liver disease. On the other hand, HCCs become refractory to physiological concentrations of the natural RXRalpha-ligand, 9-cis retinoic acid (9cRA), due to extracellular signal-regulated kinase (Erk) 1/2-mediated phosphorylation and inactivation of RXRalpha. Here, we show that acyclic retinoid restores the function of RXRalpha in human HCC-derived HuH7 cells by inactivating the Ras-Erk 1/2 signaling system, thereby dephosphorylating RXRalpha. In contrast, 9cRA failed to suppress phosphoErk 1/2 levels and subsequent RXRalpha phosphorylation. Although 9cRA also suppressed Ras activity, it simultaneously down-regulated mitogen-activated protein kinase phosphatase-1, an enzyme that inactivates Erk, thereby leaving the phosphorylation status of Erk unchanged. A combination of 9cRA (a potent ligand) and acyclic retinoid (a weak ligand preventing phosphorylation) resulted in a marked cooperation in transactivation via the RXR-response element and in inhibiting the proliferation of HuH7 cells. These events provide a novel molecular basis for the antitumor activity of acyclic retinoid against HCC.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Division; Dual Specificity Phosphatase 1; Humans; Immediate-Early Proteins; Liver Neoplasms; Luciferases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Protein Phosphatase 1; Protein Tyrosine Phosphatases; ras Proteins; Receptors, Retinoic Acid; Response Elements; Retinoid X Receptors; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

To study the inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell line SMMC-7721 and to explore the mechanism of its effect.. SMMC-7721 cells were divided into two groups, one treated with all-trans retinoic acid (ATRA) for 5 days and the other as a control group. Light microscope and electron microscope were used to observe the morphological changes. Telomerase activity was analyzed with silver-stained telomere repeated assay protocal (TRAP). Expression of Caspase-3 was demonstrated with western blot.. ATRA-treated cells showed differentiation features including small and pyknotic nuclei, densely stained chromatin and fewer microvilli. Besides, ATRA could inhibit the activity of telomerase, promote the expression of Caspase-3 and its activation.. Telomerase activity and Caspase-3 expression are changed in human hepatocellular carcinoma cell line SMMC-7721 treated with all-trans retinioc acid. The inhibition of telomerase activity and the activation of Caspase-3 may be the key steps through which ATRA inhibits the proliferation of SMMC-7721 cell line.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Division; Humans; Liver Neoplasms; Telomerase; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (tRA, or tretinoin) can be metabolized through stereoisomerization to 13-cis retinoic acid (13-cRA) in vivo. We have developed a simple, sensitive and accurate method for analyzing tRA and 13-cRA in plasma with the addition of N-ethylmaleimide (NEM) and Vitamin C (Vit. C) to prevent the interconversion of cis/trans retinoic acid. All-trans RA, 9-cRA, and 13-cRA were well separated from each other in plasma by using a C18 precolumn and a column with a gradient solvent system of mobile phases A and B at a flow rate of 1.0 ml/min. In addition, thermal stability of tRA and cRA in plasma during the sample preparation under the temperature of 0 and 25 degrees C were studied. Our results showed that (1) the interconversion ratios (%) (cRA/tRA and tRA/cRA) were decreased with the addition of NEM and Vit. C and the minimum concentrations of NEM and Vit. C to inhibit the interconversion were 50 and 150 microM, respectively, (2) higher concentrations of NEM and Vit. C were required to prevent the interconversion at higher temperature, (3) the precision and accuracy of calibration curve with various concentration of tRA (1-1000 ng/ml) and 13-cRA (5-800 ng/ml) in plasma showed good linearity (r(2)=0.9992 and 0.9994), and between-day errors expressed by coefficient of variation (CV, %) for tRA and 13-cRA which were both less than 5.6%, (4) the mean recovery of the analytes were 78-94% for tRA and 80-92% for 13-cRA at concentration range from 1 to 1000 ng/ml and 5 to 800 ng/ml, respectively, and (5) the limit of quantitation of tRA and 13-cRA were 1 and 5 ng/ml, respectively. In addition, the HPLC method had been successfully applied to the tRA pharmacokinetic study in two hepatoma patients after receiving 45 mg/m(2) per day orally. Thus, our results suggest that the HPLC method for analyzing tRA and 13-cRA in plasma with the addition of NEM and Vit. C is a simple, sensitive and accurate method.

Topics: Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Humans; Liver Neoplasms; Pilot Projects; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Stereoisomerism; Tretinoin

Perturbation of folate and methyl group metabolism is associated with a number of pathological conditions, including cardiovascular disease and neoplastic development. Glycine N-methyltransferase (GNMT) is a key protein that functions to regulate the supply and utilization of methyl groups for S-adenosylmethionine (SAM)-dependent transmethylation reactions. Factors or conditions that have the ability to regulate GNMT and the generation of homocysteine, a product of transmethylation, have important implications in the potential perturbation of methyl group metabolism. We showed that retinoid compounds induce active hepatic GNMT, resulting in compromised transmethylation processes. Because retinoids can stimulate gluconeogenesis, a condition known to alter methyl group and homocysteine metabolism, the current study was undertaken to determine the relationship between all-trans-retinoic acid (RA) and gluconeogenic hormones on these metabolic pathways. Intact adrenal function was not required for RA to induce and activate hepatic GNMT; however, treatment of rats with dexamethasone (DEX) was as effective as RA in inducing GNMT in rat liver. The marked increase in plasma total homocysteine levels observed in adrenalectomized rats was reduced to normal levels by treatment with either RA or DEX, indicating that the transsulfuration and/or remethylation pathways may be enhanced. Moreover, coadministration of RA and DEX had an additive effect on GNMT induction. Similar findings were also observed in a rat hepatoma cell culture model using H4IIE cells. Taken together, these results demonstrate that both RA and DEX independently induce GNMT, thereby having substantial implications for the potential interaction of retinoid administration with diabetes.

Topics: Adrenalectomy; Animals; Bucladesine; Carcinoma, Hepatocellular; Dexamethasone; Enzyme Induction; Glucocorticoids; Glycine N-Methyltransferase; Homocysteine; Liver; Liver Neoplasms; Male; Methyltransferases; Rats; Rats, Sprague-Dawley; Tretinoin; Tumor Cells, Cultured

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

Acyclic retinoid, a synthetic retinoid analog, as well as interferon alfa (IFN-alpha) and IFN-beta induce apoptosis in hepatocellular carcinoma (HCC) cells and are used clinically in the prevention of HCC. Here, we show that acyclic retinoid acts synergistically with IFNs in suppressing the growth and inducing apoptosis (as characterized by DNA fragmentation and chromatin condensation) in 5 human HCC cell lines (JHH7, HuH7, PLC/PRF/5, HLE, and HLF). This synergism was only observed when cells were pretreated with the acyclic retinoid, whereas natural retinoic acids (all-trans and 9-cis retinoic acid) were ineffective. This promotion may be due to up-regulation of type 1 IFN receptor (IFNR) expression by the retinoid. Accordingly, incubation with antitype 1 IFNR antibody abolished the synergy. Enhanced IFNR expression was accompanied by increased expression and DNA-binding activity of STAT1, an intracellular signal transducing molecule of IFNR, and increased induction of 2', 5'-oligoadenyl-5'-triphosphate synthetase, which is a target gene of STAT1. Acyclic retinoid did not have any effects on the growth of normal human hepatocytes (Hc) probably because of a lack of IFNR and STAT1 up-regulation. In conclusion, these results provide a rationale for combined biochemoprevention of HCC using acyclic retinoid and IFN-beta.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; DNA-Binding Proteins; Drug Synergism; Gene Expression; Humans; In Vitro Techniques; Interferon-beta; Liver Neoplasms; Receptors, Interferon; RNA, Messenger; STAT1 Transcription Factor; Trans-Activators; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Gap junctional communication permits the direct exchange of small molecules and ions and has been implicated in tissue homeostasis/metabolite exchange. The lack of gap junctional intercellular communication (GJIC) plays important roles in the promotion and progression of carcinogenesis. In the present study, we demonstrate that treatment of human hepatoma Hep G2 cells with retinoic acid (RA) results in increased amounts and phosphorylation of connexins, their stabilisation in plasma membrane plaques and enhanced GJIC. In cultured fetal hepatocytes, which represent a non-transformed, proliferating and incompletely differentiated liver system, the effects of RA are limited to the establishment of connexin in areas of cell-cell contact and the improvement of GJIC. This suggests that modulation of cell-cell channel communication by RA occurs differently in these two experimental models: while RA is able to revert cell transformation in Hep G2 cells, in fetal hepatocytes it may induce the expression of a more differentiated phenotype.

Topics: Animals; Carcinoma, Hepatocellular; Cell Communication; Cell Differentiation; Cell Division; Female; Gap Junctions; Gestational Age; Hepatocytes; Humans; Liver; Liver Neoplasms; Phosphorylation; Phosphoserine; Pregnancy; Rats; Rats, Wistar; Tretinoin; Tumor Cells, Cultured

Chemotherapy does not have a prominent role in the treatment of hepatoma. However, an acyclic retinoid prevented tumor recurrence post-hepatectomy, and tamoxifen (TAM) induced apoptosis in tumor cells. Combination therapy of these agents on proliferation and apoptosis of hepatoma cells has not been explored. HepG2, Hep1B, Hepa1-6 and MH1C1 hepatoma cells were incubated with TAM, 9-cis- and all-trans retinoic acid (CRA, ATRA, respectively) alone or in combination. Proliferation rate was assessed and apoptosis was analyzed by flow cytometry, immunostaining, caspase activity assays and the expression of apoptosis- and/or cell cycle-related molecules. CRA and TAM, but not ATRA monotherapy were moderately effective. Apoptosis was accompanied by upregulation of caspase 3 and 8 activity, and increased p27, bax, caspase 3 expression, while the levels of p21cip/waf and bcl-2 were unchanged or decreased. Combination therapy enhanced apoptosis from a maximum of 60% after monotherapy to more than 90% after 96 h in all cell types. Pro-apoptotic effects were paralleled by inhibition of proliferation. Combination of TAM and CRA, but not ATRA, have an additive to synergistic anti-proliferative and pro-apoptotic effect on HCC cells. This justifies trials for HCC using combinations of these biological response modifiers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; DNA, Neoplasm; Flow Cytometry; Humans; Immunoblotting; Immunoenzyme Techniques; Liver Neoplasms; Mice; Neoplasm Proteins; Rats; Tamoxifen; Tretinoin; Tumor Cells, Cultured

Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level.. Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining.. Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis.. AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspases; Cell Differentiation; Cell Membrane Permeability; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Enzymologic; Humans; Liver Neoplasms; Mitochondria; RNA, Messenger; Telomerase; Tretinoin; Tumor Cells, Cultured

Isoverbascoside, a phenylethanoid glycoside, was isolated from Chinese folk medicine herb Pedicularis striata Pall. Here we report that isoverbascoside is capable of inducing differentiation in human hepatocellular carcinoma (HCC) cell line SMMC-7721. When treated with isoverbascoside, the proliferation of SMMC-7721 cells was markedly inhibited in a dose- and time-dependent manner, and the average cell population doubling time was delayed. Exposure of cells to 20 micromol/l isoverbascoside led to the decline of colony formation efficiency on soft agar, induced G0/G1 arresting, and resulted in the decrease of gamma-glutamyltransferase (gamma-GT) activity and the increase of tyrosine aminotransferase (TAT) activity, two marker enzymes, respectively, representing HCC malignance and differentiation stage. These results suggest that isoverbascoside possess the activity of inducing differentiation in SMMC-7721 cells.

Topics: Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; gamma-Glutamyltransferase; Glucosides; Humans; Magnoliopsida; Medicine, Chinese Traditional; Mitotic Index; Phenols; Tretinoin; Tumor Cells, Cultured; Tyrosine Transaminase

Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.

Topics: Antineoplastic Agents; Apoptosis; Biocompatible Materials; Carcinoma, Hepatocellular; Cell Adhesion; Collagen; Drug Combinations; Drug Interactions; Extracellular Matrix Proteins; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Laminin; Liver Neoplasms; Peptide Hydrolases; Protein-Tyrosine Kinases; Proteoglycans; Receptors, Fibronectin; Tretinoin; Tumor Cells, Cultured

The expressions of Lewis (Le) antigens, alpha-1,3/1,4 fucosyltransferases (alpha-1,3/1,4 FuTs), and metastatic potential after the treatment of 2 differentiation inducers, all- trans retinoic acid (ATRA), 8-bromo-cyclic 3',5'adenosine monophosphate (8-Br-cAMP); and 2 proliferation inducers, epidermal growth factor (EGF) and phobol-12-myristate-13-acetate (PMA), on 7721 human hepatocarcinoma cell line were studied. Cell adhesion to human umbilical vein endothelial cells (HUVEC), cell migration through transwell and invasion through matrigel were selected as the indexes of metastatic potential-related phenotypes. Using fluorescence-labelled antibodies and flow-cytometric analysis, it was found that 7721 cells mainly expressed sialyl Lewis X (SLe(x)) and a less amount of sialyl dimeric Lewis X (SDLe(x)) antigens on the cell surface. Their expressions were down-regulated by ATRA, and up-regulated by EGF. SLe(x)antigen was also decreased and increased by the treatment of 8-Br-cAMP and PMA respectively. With Northern blot to detect the mRNAs of alpha-1,3/1,4 FuTs, the main enzymatic basis for the change in SLe(x)expression was found to be the alteration of the expression of alpha-1,3 FuT-VII. It was evidenced by the observations that alpha-1,3 FuT-VII was the main alpha-1,3/1,4 FuT in 7721 cells, while alpha-1,3/1,4 FuT-III and alpha-1,3 FuT-VI were expressed rather low. The changes in the expressions of SLe(x)antigen and alpha-1,3 FuT-VII resulted in the altered cell adhesion to tumour necrosis factor-alpha stimulated HUVEC, since only the monoclonal antibody of the SLe(x), but not other monoclonal antibodies blocked the adhesion of 7721 cells to HUVEC. The migration and invasion of 7721 cells were also reduced by the treatment of ATRA or 8-Br-cAMP, and elevated by EGF or PMA. The above findings indicate that the metastatic potential of 7721 cells is suppressed by differentiation-inducers and promoted by proliferation-inducers.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Adhesion; Cell Differentiation; Cell Division; Cell Movement; Epidermal Growth Factor; Fucosyltransferases; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Liver Neoplasms; Neoplasm Metastasis; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The bile salt excretory pump (BSEP, ABCb11) is critical for ATP-dependent transport of bile acids across the hepatocyte canalicular membrane and for generation of bile acid-dependent bile secretion. Recent studies have demonstrated that the expression of this transporter is sensitive to the flux of bile acids through the hepatocyte, possibly at the level of transcription of the BSEP gene. To determine the mechanisms underlying the regulation of BSEP by bile acids, the promoter of the BSEP gene was cloned. The sequence of the promoter contained an inverted repeat (IR)-1 element (5'-GGGACA T TGATCCT-3') at base pairs -63/-50 consisting of two nuclear receptor half-sites organized as an inverted repeat and separated by a single nucleotide. This IR-1 element has been shown in several recent studies to serve as a binding site for the farnesoid X receptor (FXR), a nuclear receptor for bile acids. FXR activity requires heterodimerization with RXR alpha, and when bound by bile acids, the complex effectively regulates the transcription of several genes involved in bile acid homeostasis. Gel mobility shift assays demonstrated specific binding of FXR/RXR alpha heterodimers to the IR-1 element in the BSEP promoter. In HepG2 cells, co-transfection of FXR and RXR alpha is required to attain full transactivation of the BSEP promoter by bile acids. Two FXR transactivation-deficient mutants (an AF-2 deletion and a W469A point mutant) failed to transactivate, indicating that the effect of bile acids is FXR-dependent. Further, mutational analysis confirms that the FXR/RXR alpha heterodimer activates transcription through the IR-1 site in the human BSEP promoter. These results demonstrate a mechanism by which bile acids transcriptionally regulate the activity of the bile salt excretory pump, a critical component involved in the enterohepatic circulation of bile acids.

Topics: Alitretinoin; Amino Acid Substitution; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Base Sequence; Bile Acids and Salts; Binding Sites; Carcinoma, Hepatocellular; DNA Primers; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Hepatocytes; Humans; Liver Neoplasms; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Point Mutation; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Sequence Deletion; TATA Box; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Stearoyl-CoA desaturase (SCD) is a regulatory enzyme involved in the synthesis of the monounsaturated fatty acids palmitoleate and oleate. The regulation of SCD is of physiological importance because the ratio of saturated fatty acids to unsaturated fatty acids is thought to modulate membrane fluidity. Differential display analysis of retinal pigment epithelial (ARPE-19) cells identified SCD as a gene regulated by retinoic acid. Two SCD transcripts of 3.9 and 5.2 kilobases in size were found to be expressed in these cells by Northern blot analysis. All-trans-retinoic acid (all-trans-RA) increased SCD mRNA expression in a dose- and time-dependent manner; an approximately 7-fold increase was observed with 1 microm all-trans-RA at 48 h. SCD mRNA expression was also increased by 9-cis-retinoic acid (9-cis-RA) as well as 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB), a retinoic acid receptor (RAR)-specific agonist. AGN194301, a RAR alpha-specific antagonist, suppressed the SCD expression induced by all-trans-RA, TTNPB, and 9-cis-RA. These results indicate the involvement of RAR alpha in the induction of SCD expression by retinoic acid. However, AGN194204, a RXR (retinoid X receptor) pan agonist, also increased SCD mRNA expression. This increase was not blocked by AGN194301, suggesting that an RAR-independent mechanism may also be involved. Thus, SCD expression in retinal pigment epithelial cells is regulated by retinoic acid, and the regulation appears to be mediated through RAR and RXR.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Benzoates; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Chlorocebus aethiops; COS Cells; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Gingiva; HeLa Cells; Humans; Kinetics; Liver Neoplasms; Pigment Epithelium of Eye; Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; RNA, Messenger; Stearoyl-CoA Desaturase; Tetrahydronaphthalenes; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

In hepatoma Huh-7 cells, inhibition of sphingosine kinase (SphK) activity by N,N-dimethylsphingosine (DMS) resulted in up-regulated production of liver-specific serum proteins including albumin and alpha-fetoprotein (AFP). The changes in these protein levels coincided well with those of two liver-enriched transcription factors, hepatocyte nuclear factor (HNF)-1 and -4, which regulate a number of liver-specific genes at the transcriptional level. Moreover, DMS induced the expression of retinoic acid receptor-alpha and retinoid X receptor-alpha. In DMS-treated cells, 9-cis retinoic acid (RA) further enhanced HNF-4alpha and albumin expression but it inhibited AFP accumulation. These results suggest that activation of SphK disengages cells from their liver-specific phenotype, and that 9-cis RA further induces differentiation of hepatoma cells when SphK activity is inhibited.

Topics: Alitretinoin; alpha-Fetoproteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Carcinoma, Hepatocellular; Cell Differentiation; DNA-Binding Proteins; Enzyme Inhibitors; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 1-alpha; Hepatocyte Nuclear Factor 1-beta; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver; Lysophospholipids; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Serum Albumin; Sphingosine; Transcription Factors; Tretinoin; Tumor Cells, Cultured

We have shown previously that administration of acyclic retinoid to cirrhotic patients who had undergone curative treatment of preceding hepatocellular carcinoma (HCC) induced the disappearance of serum lectin-reactive alpha-fetoprotein (AFP-L3) and subsequently reduced the incidence of second liver cancers. AFP-L3 is a tumor marker that indicates the presence of occult tumors below the detection limit by diagnostic images. Therefore, we have proposed a new concept of 'clonal deletion' therapy with acyclic retinoid for the cancer chemoprevention against HCC. Such eradication of AFP-L3-producing latent malignant (or premalignant) cells from the liver suggested a new strategy to prevent HCC, which may be involved in the same category as cancer chemotherapy. In the present series of studies, we explored the molecular mechanism of 'clonal deletion' and found a novel mechanism of apoptosis induction by the retinoid. We have demonstrated a modification of a retinoid receptor, RXRalpha, by mitogen-activated protein (MAP) kinase-dependent phosphorylation, resulting in the loss of transactivating activity. This may lead HCC cells to be resistant to natural retinoic acid. However, acyclic retinoid restored the function of phosphorylated RXRalpha and induced its downstream pro-apoptotic genes including tissue transglutaminase, an enzyme that is implicated in apoptosis. Tissue transglutaminase-dependent apoptosis in HCC cells was independent of the activation of caspases. This novel mechanism of retinoid-induced apoptosis may give a clue to understand the molecular mechanism of clonal deletion.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Chemoprevention; Clonal Deletion; Humans; Liver Neoplasms; Tretinoin

Hepatocyte nuclear factor 4alpha (HNF-4alpha) (nuclear receptor 2A1) is an essential regulator of hepatocyte differentiation and function. Genetic and molecular evidence suggests that the tissue-restricted expression of HNF-4alpha is regulated mainly at the transcriptional level. As a step toward understanding the molecular mechanism involved in the transcriptional regulation of the human HNF-4alpha gene, we cloned and analyzed a 12.1-kb fragment of its upstream region. Major DNase I-hypersensitive sites were found at the proximal promoter, the first intron, and the more-upstream region comprising kb -6.5, -8.0, and -8.8. By the use of reporter constructs, we found that the proximal-promoter region was sufficient to drive high levels of hepatocyte-specific transcription in transient-transfection assays. DNase I footprint analysis and electrophoretic mobility shift experiments revealed binding sites for HNF-1alpha and -beta, Sp-1, GATA-6, and HNF-6. High levels of HNF-4alpha promoter activity were dependent on the synergism between either HNF-1alpha and HNF-6 or HNF-1beta and GATA-6, which implies that at least two alternative mechanisms may activate HNF-4alpha gene transcription. Chromatin immunoprecipitation experiments with human hepatoma cells showed stable association of HNF-1alpha, HNF-6, Sp-1, and COUP-TFII with the promoter. The last factor acts as a repressor via binding to a newly identified direct repeat 1 (DR-1) sequence of the human promoter, which is absent in the mouse homologue. We present evidence that this sequence is a bona fide retinoic acid response element and that HNF-4alpha expression is upregulated in vivo upon retinoic acid signaling.

Topics: Animals; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Binding Sites; Carcinoma, Hepatocellular; Cell Line; Chromatin; Cloning, Molecular; COS Cells; DNA-Binding Proteins; Dose-Response Relationship, Drug; Gene Expression Regulation; Hepatocyte Nuclear Factor 4; Humans; Ligands; Mice; Models, Biological; Models, Genetic; Molecular Sequence Data; Phosphoproteins; Plasmids; Precipitin Tests; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

It is extensively shown that integrin can regulate various cellular functions, including apoptosis, probably by contributing to signal transduction processes through interaction with extracellular matrix (ECM) proteins. In the present study, DNA flow cytometric analysis demonstrated that SMMC-7721 hepatocarcinoma cells treated with 80 microM all-trans-retinoic acid (atRA) showed an increased expression of the integrin alpha5beta1, which was associated with the growth inhibition of the cells. We found that atRA treated cells showed obvious apoptosis. Then, it was postulated that the enhanced content of integrin alpha5beta1 in the absence of ligation with fibronectin (Fn) would stop transducing survival signals, and lead to decreased cell growth and apoptosis. To elucidate this hypothesis, we cultured the atRA treated cell in L-poly-lysine-coated and Fn-coated flask, respectively. The results indicated that Fn binding prevented the cells from apoptosis induced by atRA, in contrast to L-poly-lysine binding. When the transfectant with enhanced expression of integrin alpha5beta1 at the same level of atRA treated cell was cultured in L-poly-lysine-coated flask, apoptosis was triggered. However, apoptotic cell was not detected when those cells were cultured in Fn-coated flask. Meanwhile, culturing the transfectant in the antibody (against integrin alpha5 subunit)-coated flask induced 18.33% of the cells into apoptosis, which is far more than the control group. Our study suggests that increased expression of integrin alpha5beta1 on the surface of SMMC 7721 hepatocarcinoma cell treated by atRA, when unbound to Fn, would stop transducing survival signals to lead to "anoikis", and can be reverted by the interaction of integrin alpha5beta1 with Fn.

Topics: Anoikis; Antigens, CD; Carcinoma, Hepatocellular; Cell Division; Fibronectins; Humans; In Situ Nick-End Labeling; Integrin alpha5; Integrin beta1; Kinetics; Liver Neoplasms; Protein Subunits; Receptors, Fibronectin; Recombinant Proteins; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

The effects of all-trans retinoic acid (ATRA) and epidermal growth factor (EGF) on the expression of nm23-H1, a metastasis suppressor gene, were studied in a human 7721 hepatocarcinoma cell line. It was discovered that the expression of nm23-H1 mRNA was up-regulated by ATRA. This was compatible with the observation that the metastasis-associated phenotypes, such as chemotaxic cell migration and invasion, were both reduced in the ATRA-treated and nm23-H1-cDNA-transferred 7721 cells. However, ability of cells to adhere to fibronectin and laminin was not altered identically in the ATRA-treated and nm23-H1-cDNA-transfected 7721 cells. In contrast, the expression of nm23-H1 mRNA in 7721 cells was down-regulated both by the treatment with EGF and by the transfection of c-erbB-2/neu cDNA, which codes a protein homologous to the EGF receptor. EGF is a compound with biological effects opposite to those of ATRA, and c-erbB-2/neu is known to be a metastasis-promoting gene. These results reveal that the metastasis-preventing effect of ATRA may partly result from the up-regulation of nm23-H1, and the metastasis-promoting effects of EGF and c-erbB-2/neu were probably mediated in part by the down-regulation of nm23-H1.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Liver Neoplasms; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The changes in N-acetylglucosaminyltransferase-V and -III (GnT-V, GnT-III) during the cell-cycle of synchronized 7721 human hepatocarcinoma cell line were investigated. Using an HPLC method to assay GnT and flow cytometry (FCM) for cell cycle analysis, it was found that GnT-V showed the highest activity, but GnT-III reached the lowest activity when G(2)/M cells were most abundant. In contrast, GnT-V declined to the minimum while GnT-III elevated to maximum when G(0)/G(1) cells were most predominant. The opposing changes were more obvious when the activities of GnT-V and GnT-III were expressed as relative activities (activity of GnT-V or GnT-III/the sum of activities of GnT-V plus GnT-IV plus GnT-III). These opposing changes of GnT-V and GnT-III during the cell cycle might result from the different regulatory mechanisms of GnT-V and GnT-III expression in the cell cycle. The alterations in the structures of cell surface N-glycans were compatible with the changes of the activities of GnTs. The results from immunocytochemistry and Northern blot showed that the protein and mRNA contents of GnT-V were not significantly changed during the cell cycle. The activity of a cell cycle regulating protein kinase, p34(cdc2) kinase, correlated to the activity of GnT-V. These findings suggested that the change of GnT-V activity in cell cycle was not the consequence of the alteration of gene transcription or enzyme protein synthesis, but might be caused by the post-translational regulation. The decrease in GnT-V and the corresponding increase in GnT-III activities were also found after the cells were treated with all-trans retinoic acid (ATRA), and the mechanism of this might be different from that in the cell cycle.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Flow Cytometry; G1 Phase; G2 Phase; Horseradish Peroxidase; Humans; Immunohistochemistry; Lectins; Mitosis; N-Acetylglucosaminyltransferases; Protein Serine-Threonine Kinases; Resting Phase, Cell Cycle; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Many patients with liver cirrhosis are in a state of protein and energy malnutrition and require careful nutritional support. Our research has revealed that approximately 30% of the patients have protein-energy malnutrition, 40% protein malnutrition, and 10% energy malnutrition; 20% are in a normal nutritional state. Supplementation with branched-chain amino acids alleviates chronic liver failure, improves the protein nutritional state, and subsequently prolongs survival. In contrast, therapeutic modalities for energy malnutrition have not yet been fully elucidated and await further studies. Improved survival of the cirrhotic patients essentially brings a higher incidence of hepatocellular carcinoma (HCC). A synthetic analogue of vitamin A (acyclic retinoid or 4,5-dehydrogeranyl geranoic acid) prevents at least the development of second primary tumors after curative treatment of preceding HCC. The mechanism of this cancer chemo-prevention is clonal deletion of premalignant and latent malignant cells by the retinoid. We describe our clinical experiences with these two nutritional pharmacotherapies of chronic liver diseases and review their basic mechanisms.

Topics: Amino Acids, Branched-Chain; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Combined Modality Therapy; Humans; Liver Cirrhosis; Liver Failure; Liver Neoplasms; Nutritional Support; Protein-Energy Malnutrition; Randomized Controlled Trials as Topic; Survival Rate; Treatment Outcome; Tretinoin

Retinoic acid (RA) induces apoptosis in Hep3B human hepatoma cells. 9-Cis-RA (c-RA) had a similar effect as all-trans-RA (t-RA) in inducing cell death in Hep3B cells. RA-induced Hep3B-cell death was associated with inhibited expression of the hepatocyte nuclear factor 4 (HNF-4) gene. Palmitoyl-CoA ((C16:0)-CoA), the reported HNF-4 ligand, prevented RA-induced apoptosis. The effect of (C16:0)-CoA was specific, since palmitic acid and co-enzyme A had no effect in preventing RA-induced apoptosis. Bovine serum albumin (BSA) also prevented RA-induced apoptosis. However, in contrast to BSA, which induced cell growth, (C16:0)-CoA alone had no effect on cell growth. Investigating the possible role of HNF-4 in apoptosis, the reported HNF-4 antagonist (C18:0)-CoA was employed, and it also prevented RA-induced apoptosis. By transient transfection, overexpression of HNF-4 did not prevent RA-induced apoptosis. The induction and prevention of apoptosis caused by RA and (C16:0)-CoA were associated, respectively with the induction and inhibition of the expression of transforming growth factor beta (TGFbeta), which is known to play a role in apoptosis. Furthermore, RA and (C16:0)-CoA can regulate AP-1, which is a key regulator of the TGFbeta gene. Our data indicate that fatty acyl-CoAs can prevent RA-induced apoptosis and that TGFbeta, rather than HNF-4, may play a role in these regulatory processes. Our data also suggest that (C16:0)-CoA and (C18:0)-CoA are not the agonist and antagonist for HNF4, respectively in the Hep3B cell system.

Topics: Alitretinoin; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Northern; Carcinoma, Hepatocellular; Culture Media, Serum-Free; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Fatty Acids; Hepatocyte Nuclear Factor 4; Humans; Ligands; Liver Neoplasms; Palmitoyl Coenzyme A; Phosphoproteins; RNA, Messenger; Time Factors; Transcription Factors; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-alpha. E-cadherin expression was only up-regulated by butyrate and interferon-alpha (IFN-alpha) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, beta-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-alpha. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that alpha- and y-catenin expression was not changed, while only the expression of beta-catenin increased. Beta-catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type beta-catenin are potentially modulated by butyrate and IFN-alpha, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.

Topics: Amino Acid Substitution; beta Catenin; Blotting, Western; Butyrates; Cadherins; Carcinoma, Hepatocellular; Cell Adhesion; Cell Differentiation; Cell Membrane; Culture Media; Cytoskeletal Proteins; Dexamethasone; Humans; Interferon-alpha; Liver Neoplasms; Mutation; Trans-Activators; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents - transforming growth factor beta1 (TGF-beta1), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) - on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-beta1; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-beta1, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinogens; Carcinoma, Hepatocellular; Cell Culture Techniques; Cycloheximide; Fluorescent Dyes; Hepatocytes; Humans; Indoles; Liver Neoplasms; Okadaic Acid; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured

A full-length cDNA clone encoding the retinol binding protein (RBP) was isolated from a mouse liver cDNA library by hybridization screening. The nucleotide sequence of murine RBP is 85 and 95% homologous to that of human and rat RBP, respectively, with a deduced amino acid sequence > or = 83% homologous to both species. Analysis of the tissue expression pattern of RBP mRNA in the female mouse indicated relatively abundant expression in the liver, with lesser amounts in extrahepatic tissues including adipose, kidney, spleen and uterus, suggesting that these tissues may have a significant role in retinol homeostasis. Mouse liver cell RBP regulation by retinoids was also investigated. Both all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9c-RA) induced RBP mRNA expression in a dose- and time-dependent manner. Maximal levels (up to 4-fold above controls) were observed at > or = 48 h following treatment of both mouse hepatoma cells in vitro and in vivo in mice receiving a single, oral dose of either retinoid. Interestingly, 9c-RA was more potent at RBP induction in both in vivo and in vitro systems. Given the extent and temporal pattern of RBP induction, we suggest that the RA-mediated increase in liver RBP is part of a cellular protection mechanism. Increased levels of RBP would facilitate sequestration and possibly cellular export of RA in cells receiving prolonged exposure to high levels of RA, thus minimizing toxicity.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Carcinoma, Hepatocellular; Cloning, Molecular; Dose-Response Relationship, Drug; Female; Gene Expression Regulation; Liver; Liver Neoplasms; Male; Mice; Molecular Sequence Data; Retinol-Binding Proteins; RNA, Messenger; Sequence Homology; Time Factors; Tretinoin; Tumor Cells, Cultured

The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21(CIPI/Waf1) and Bax proteins, as well as activation of p34(cdc2) kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-XL; Bcl-XS; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27(Kip1) expression. Olomoucine, a potent p34(cdc2) and Cdk2 inhibitor, effectively blocked RA-mediated p34(cdc2) kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21(CIP2/Waf1) and p34(cdc2) mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21(CIP2/Waf1) overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34(cdc2) kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21(CIP2/Waf1) and p34(cdc2) is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; CDC2 Protein Kinase; Cell Cycle; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; Humans; Mitosis; Oligonucleotides, Antisense; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

We observed that all-trans-retinoic acid (RA) down-regulated insulin-like growth factor binding proteins (IGFBPs) in cultured human hepatoma cells (Hep 3B, PLC/PRF/5, and Hep G2); therefore, we characterized the role of this down-regulation in cell growth. Treatment with 10 micromol/L RA revealed a rapid decrease in IGFBP-3 within 2 days, and continued treatment with RA for 6 days resulted in a time-dependent stimulation of Hep 3B cell growth. However, RA treatment decreased IGFBP-1 in PLC/PRF/5 cells and in Hep G2 cells, and the growth-stimulatory activity of RA was transient and less prominent, and was finally obliterated in both cell lines. The addition of 5 ng/mL or 50 ng/mL insulin-like growth factors (IGFs) did not change the growth effects elicited by RA. The addition of IGFBP-3 (1,000 ng/mL) inhibited the growth of Hep 3B cells and counteracted the growth-stimulatory activity of RA, but not completely, suggesting that RA has direct growth-stimulatory activity and that this is enhanced by autocrine down-regulation of IGFBP-3. IGFBP-3 also inhibited the growth of PLC/PRF/5 cells and of Hep G2 cells. Treatment with phosphorylated IGFBP-1 (1,000 ng/mL) alone or with RA did not affect the growth of PLC/PRF/5 cells or Hep G2 cells. However, addition of dephosphorylated IGFBP-1, derived from in vivo dephosphorylation of the phosphorylated form, stimulated the growth of both cell lines, independent of interaction with IGF-I. From these observations, we propose that RA down-regulates IGFBPs, which in turn causes autocrine modulation of cell growth independent of IGF in hepatoma cells in vitro or in vivo. In addition, RA regulates IGFBPs at the posttranscriptional (Hep 3B cells and Hep G2 cells) or transcriptional level (PLC/PRF/5 cells) in a cell-specific manner.

Topics: Carcinoma, Hepatocellular; Cell Division; Culture Media, Conditioned; Down-Regulation; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Liver Neoplasms; Phosphorylation; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Follow-Up Studies; Humans; Liver Neoplasms; Neoplasms, Second Primary; Survival Analysis; Tretinoin

To determine whether differentiation therapy is useful in treating patients with hepatoma, we assayed the effects of 25-hydroxycholesterol (25-OH) and/or all-trans retinoic acid (ATRA) on rat AH136B ascites hepatoma cells. Flow cytometric DNA analysis showed that treatment of cells with 25-OH alone induced entry into the sub-G1 phase in a dose-dependent manner. While ATRA alone was ineffective, it enhanced the activity of 25-OH. Condensed and fragmented nuclei occurred mainly in cells treated with 25-OH (4 microg/ml). When cells treated with 25-OH (4 microg/ml), or 25-OH (4 microg/ml) + ATRA (1 microM) were transplanted into Donryu rats, we found that tumor development was completely inhibited; in contrast, rats administered the methanol-treated AH136B cells developed tumors. These findings suggest that AH136B cells in the sub-G1 phase can not recover tumorigenicity, and that the administration of a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, such as 25-OH, and ATRA may be effective in treating patients with hepatoma.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transplantation; Diagnosis, Differential; Hydroxycholesterols; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Liver Neoplasms, Experimental; Male; Rats; Tretinoin; Tumor Cells, Cultured

The antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V, EC 2. 4.1.155) was constructed as pcDNA3/GnT-V-AS plasmid and transfected into 7721 cells, a human hepatocarcinoma cell line. The transfection was confirmed with Northern blot. By using HPLC and HRP-lectin staining, it was found that the cells transfected with pcDNA3/GnT-V-AS (GnT-V-AS/7721) expressed less GnT-V activity and beta-1,6-GlcNAc branching in the cell glycoproteins compared with the cells mock-transfected with the vector pcDNA3 (pcDNA3/7721). The growth rate of GnT-V-AS/7721 was decreased in serum-containing medium, while the cell death was accelerated in serum-free medium. The GnT-V-AS/7721 cells were more susceptible to the apoptosis induced by ATRA than the mock-transfected cells. This was evidenced by the obvious appearance of a hypoploid sub-G(1) fraction in the DNA histogram using FCM analysis, the more condensed new moon-type nuclei under morphological observation, and the more intensive TUNEL reaction for assaying the fragmented DNA. At the same time as GnT-V down-regulation by GnT-V-AS, an increase of another N-aceylglusaminyltransferase, GnT-III (EC 2.4.1.144), was observed, and the biological significance of this finding was discussed.

Topics: Apoptosis; Blotting, Northern; Carcinoma, Hepatocellular; Cell Division; Cell Survival; DNA, Antisense; Flow Cytometry; Horseradish Peroxidase; Humans; In Situ Nick-End Labeling; Liver Neoplasms; N-Acetylglucosaminyltransferases; Plasmids; Polysaccharides; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) up-regulates retinoic acid receptor beta (RAR beta) gene expression in a variety of cell lines. Whether up-regulation of the RAR beta gene reflects increased activity in a RAR beta-mediated biological process is unclear since RAR beta tends to heterodimerize with retinoid x receptor (RXR). In F9 teratocarcinoma cell line, RA-induced differentiation is accompanied by increased expression of the RAR beta, RXR alpha, and alpha-fetoprotein (AFP) genes. Previously, we have shown that the RA-mediated regulation of the AFP gene is through RXR alpha homodimers. In contrast to F9 cells, Hep3B is unique in that the AFP gene is down-regulated by RA in a manner reminiscent of down-regulation of AFP in postfetal liver. In this paper, we have examined the RA-mediated regulation of the RAR, RXR, peroxisome proliferator-activated receptor (PPAR), and AFP genes in Hep3B cells. RA induced the expression of RAR alpha, beta, and gamma mRNA in Hep3B cells. However, the expression of RXR alpha mRNA was down-regulated, and the levels of RXR beta and RXR gamma mRNA remained unchanged after RA treatment. In addition, the expression of the PPAR alpha, beta, and gamma genes was also unchanged. Gel retardation assays demonstrated that RA decreased the overall binding of nuclear receptors to the RA and PPAR response elements. By super-shift assays using specific anti-RAR and -RXR antibodies, RA treatment decreased the amount of RXR alpha while increasing the amount RAR beta bound to retinoic acid response element-DR1 (direct repeat with spacer of one nucleotide), indicating the levels of RAR/RXR heterodimer, RXR/RXR homodimer, or RAR/RAR homodimers were altered upon RA treatment of Hep3B cells. In addition, the RA-mediated reduction of RXR alpha in part results in down-regulation of the AFP gene. Our data indicates that RA exerts its effects by differentially regulating its own receptor gene expression.

Topics: Animals; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoid X Receptors; RNA, Messenger; Teratoma; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation

A 44-year-old female patient was admitted to our department with diagnosis of malignant lymphoma. The abdominal USG and CT showed multiple liver lesions with partial portal vein thrombosis, moderately increased alfa-fetoprotein (AFP), ASAT, ALAT (2x normal value), serology was negative for HBV and HCV. Liver transplantation was suggested but refused because of portal vein thrombosis. ATRA (45 mg/m2/day orally) was given on the basis of the assumption that HCC and acute promyelocytic leukaemia share similar oncogenic pathway (alter the RAR alpha and beta receptors). She was gained 15 kg-s and has resumed her work as a teacher for the last 20 months. Abdominal CT showed a complete regression of the intrahepatic tumour.

Topics: Adult; Antineoplastic Agents; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Lymphoma; Tomography, X-Ray Computed; Tretinoin; Ultrasonography

The expression of acute-phase protein genes is controlled by many factors, such as IL-1, IL-6, glucocorticoids, thyroid hormone (T3), and retinoic acids. We studied the interaction of T3, glucocorticoids, all-trans retinoic acid (RA), and 9-cis retinoic acid (9cRA) on the expression of the rat alpha 1-acid glycoprotein (AGP) gene in vitro. Dexamethasone (Dex) activated AGP gene expression in a rat liver derived cell line, RLN-10. Although T3, RA, and 9cRA by themselves had no effect on AGP production, they reduced the response to Dex of the AGP gene.

Topics: Alitretinoin; Animals; Carcinoma, Hepatocellular; Cell Line; Dexamethasone; Down-Regulation; Gene Expression Regulation; Liver; Orosomucoid; Rats; RNA, Messenger; Time Factors; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Genes containing peroxisome proliferator-activated receptor (PPAR) binding sites are both inducible by peroxisome proliferators and expressed in a tissue-specific fashion. A PPAR-responsive reporter gene cotransfected with a PPARalpha expression vector was highly expressed in H4IIEC3 hepatoma cells. Addition of clofibrate resulted in a modest further induction of the reporter gene. In CV-1 cells, high expression of the reporter required the addition of clofibrate. H4IIEC3 cells had higher levels of retinoid X receptor (RXRalpha) than CV-1 cells; cotransfection of CV-1 cells with PPARalpha plus RXRalpha expression plasmids abolished the cell line difference in basal and clofibrate-stimulated expression of the reporter. Lipid extracts of hepatoma cells or of liver or kidney stimulated expression of the reporter; extracts of CV-1 cells were far less effective. Chromatographic analysis of these extracts revealed high levels of three fractions of lipid in liver and H4IIEC3 cells that were lower in CV-1 cells. We conclude that (1) in cells expressing high levels of both RXRs and PPARalpha, such as hepatocytes and kidney cells, these factors are constitutively active; (2) activators of PPARalpha may increase its ability to form heterodimers with RXRs when the latter are limiting; and (3) hepatoma cells, liver, and kidney contain lipid-extractable compounds capable of activating PPARalpha.

Topics: Carcinoma, Hepatocellular; Clofibrate; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, Reporter; Liver; Luciferases; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; Humans; Tretinoin

The differential-display reverse transcription/polymerase chain reaction (DDRT-PCR) method was utilised to find genes differentially expressed at the messenger RNA level in SMMC-7721 human hepatocellular carcinoma cells treated and not treated with all-trans-retinoic acid, a differentiation inducer of the cell. Human transcription factor IIB (TFIIB) was discovered to be decreased on the 3rd day after the cells had been treated with retinoic acid and increased by phorbol 12-myristate 13-acetate, which stimulated the proliferation of human hepatocellular carcinoma cells. It was also found that the transcription of TFIIB in SMMC-7721 cell had no relationship to the cell cycle.

Topics: Antineoplastic Agents; Carcinogens; Carcinoma, Hepatocellular; DNA Primers; Humans; Liver Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA-Directed DNA Polymerase; Tetradecanoylphorbol Acetate; Transcription Factor TFIIB; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Topics: Anticarcinogenic Agents; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Tretinoin

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Fatty Acid Synthases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Liver Neoplasms; Luciferases; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Transcription, Genetic; Tretinoin

The biological significance of phosphatidylcholine-specific phospholipase C (PC-PLC) in hepatocarcinogenesis and the proliferation and differentiation of rat liver cancer cells was investigated. The Ca2+-dependent activities of PC-PLC gradually increased during N-nitrosodiethylamine (DEN)-induced hepatocarcinogenesis and peaked at weeks 18-20 when the tumour formed. There was a close relationship between Ca2+-dependent PC-PLC activities and cellular DNA content, membranous gamma-glutamyltranspeptidase (gamma-GT), and tyrosine protein kinase. In contrast, Ca2+-independent PC-PLC activities decreased during hepatocarcinogenesis. Similarly, when CBRH-7919 rat liver cancer cells were treated with phorbol 12-myristate 13-acetate, a proliferation stimulator of the cells, gamma-GT and Ca2+-dependent activities of PC-PLC and the expression of alpha-fetoprotein increased significantly. However, when these cells were induced by retinoic acid to differentiate, Ca2+-dependent PC-PLC and gamma-GT activities decreased significantly, together with alpha-fetoprotein expression. There was a close relationship between Ca2+-dependent PC-PLC and gamma-GT activities during differentiation as there was during proliferation. We suppose that Ca2+-dependent PC-PLC is involved in rat hepatocarcinogenesis induced by DEN and that it plays an important role in the phorbol ester-induced proliferation or retinoic acid-induced differentiation of liver cancer cells.

Topics: Animals; Antineoplastic Agents; Bucladesine; Carcinogens; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Liver Neoplasms; Male; Phorbol Esters; Rats; Rats, Wistar; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases

The role of retinoic acids (RA) on liver fatty acid-binding protein (L-FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9-cis-Retinoic acid (9-cis-RA) specifically enhanced L-FABP mRNA levels in a time- and dose-dependent manner. The higher induction was found 6 h after addition of 10(-6) M 9-cis-RA in the medium. RA also enhanced further both L-FABP mRNA levels and cytosolic L-FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR), which are known to be activated, respectively, by 9-cis-RA and long chain fatty acid (LCFA), co-operated to bind specifically the peroxisome proliferator-responsive element (PPRE) found upstream of the L-FABP gene. Our result suggest that the PPAR-RXR complex is the molecular target by which 9-cis-RA and LCFA regulate the L-FABP gene.

Topics: Alitretinoin; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Dimerization; Drug Synergism; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty Acids; Gene Expression Regulation; Liver; Microbodies; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

In order to elucidate the relation between cell signal transduction and cell differentiation, the effect of two differentiation inducers--all trans-retinoic acid (ATRA) and 13 cis-retinoic acid (13 cis-RA) on the specific activities (nmol/hr.mg protein) of phosphatidyl choline specific phospholipase D (PC-PLD) in 7721 human hepatocarcinoma cell line was studied. It was found that ATRA and 13 cis-RA increased the specific activities of membrane bound PC-PLD on 2nd and 4th day of cell culture respectively. If the PC-PLD activities were calculated as activity per bottle cells, the effect of ATRA on the 2nd day was also higher than that of 13 cis-RA, while the reverse was true on the 4th day, revealing a postponed effect of 13 cis-RA as compared with ATRA. The increase of PC-PLD was higher when the culture medium was refreshed every day than that when the medium was unrefreshed, suggesting that a factor might be existed in the fresh medium which could enhance the stimulating effect of retinoic acids on PC-PLD. The effect of ATRA on PC-PLD specific activity were higher on 2nd day than that on 4th day, owing to the accelerated proliferation of the cells on 4th day, leading to the increase of protein contents and decrease of the enzyme specific activity calculated on the base of protein contents. Whereas, the effect of 13 cis-RA on PC-PLD specific activity were higher on 4th day than that on 2nd day, because the increase of enzyme activity was higher than the increase of protein contents on 4th day. The relation between the elevation of PC-PLD and protein kinases was further studied and discovered that ATRA decreased both the specific activities of membrane bound and cytosolic protein kinase C (PKC) as well as tyrosine protein kinase (TPK) either in 2nd or 4th day of cell culture, which indicated that the increasing effect of ATRA on membrane bound PC-PLD was not resulted from the stimulating effect of PKC and TPK on PC-PLD, and its mechanism remains to be further investigated.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Isotretinoin; Liver Neoplasms; Phosphatidylcholines; Phospholipase D; Tretinoin; Tumor Cells, Cultured

We cultured human hepatoma Hep3B cells in the presence of RA (10(-5) M) for 30 days; the expression of both alpha-fetoprotein and hepatitis B virus surface antigen were suppressed over 70% at the transcriptional level by RA treatment. The doubling time of RA treated Hep3B cells was slightly different from the control cells when they were cultured in 5% fetal calf serum/DMEM medium. However, cultured under serum-free conditions, the control Hep3B cells still grow, but the RA treated cells could not attach to the substratum of the culture plate and stopped growing. In vivo assay indicated that RA treatment completely suppressed the tumorigenicity of Hep3B cells in nude mice.

Topics: alpha-Fetoproteins; Animals; Anticarcinogenic Agents; Carcinoma, Hepatocellular; Cell Division; Culture Media, Serum-Free; Female; Gene Expression; Hepatitis B Surface Antigens; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Tretinoin; Tumor Cells, Cultured

Synthetic 4,5-didehydro GGA (geranylgeranoic acid), a potent ligand both for cellular retinoic acid-binding protein and for nuclear retinoid receptors, induced apoptosis in human hepatoma-derived cell line HuH-7 but not in primary hepatocytes, although all-trans or 9-cis retinoic acid did not induce any growth inhibition. Either exogenous transforming growth factor-alpha (TGF alpha) or epidermal growth factor(EGF) prevented the cells from apoptosis in the presence of 4,5-didehydro GGA, but hepatocyte growth factor, insulin-like growth factor-II, insulin or triiodothyronine was essentially inactive. 4,5-Didehydro GGA down-regulated the cellular levels of TGF alpha mRNA as early as 30 min after the treatment. Either anti-TGF alpha or anti-EGF receptor monoclonal antibody induced apoptosis in HuH-7 cells without using the acid. Taken together, the present study strongly suggests that 4,5-didehydro GGA induced apoptosis in HuH-7 cells through the destruction of autocrine loop consisting of TGF alpha and EGF receptor, due to the down regulation of TGF alpha gene expression.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Insulin-Like Growth Factor II; Kinetics; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; Carrier State; Cell Transformation, Neoplastic; DNA, Viral; Hepatitis B virus; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Neoplasms, Second Primary; Retinoids; Tretinoin

Considering the link between plasma high-density lipoprotein (HDL) cholesterol levels and a protective effect against coronary artery disease as well as the suggested beneficial effects of retinoids on the production of the major HDL apolipoprotein (apo), apo A-I, the goal of this study was to analyze the influence of retinoids on the expression of apo A-II, the other major HDL protein. Retinoic acid (RA) derivatives have a direct effect on hepatic apo A-II production, since all-trans (at) RA induces apo A-II mRNA levels and apo A-II secretion in primary cultures of human hepatocytes. In the HepG2 human hepatoblastoma cell line, both at-RA and 9-cis RA as well as the retinoid X receptor (RXR)-specific agonist LGD 1069, but not the RA receptor (RAR) agonist ethyl-p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-l-pro penyl]-benzoic acid (TTNPB), induce apo A-II mRNA levels. Transient-transfection experiments with a reporter construct driven by the human apo A-II gene promoter indicated that 9-cis RA and at-RA, as well as the RXR agonists LGD 1069 and LG 100268, induced apo A-II gene expression at the transcriptional level. Only minimal effects of the RAR agonist TTNPB were observed on the apo A-II promoter reporter construct. Unilateral deletions and site-directed mutagenesis identified the J site of the apo A-II promoter mediating the responsiveness to RA. This element contains two imperfect half-sites spaced by 1 oligonucleotide. Cotransfection assays in combination with the use of RXR or RAR agonists showed that RXR but not RAR transactivates the apo A-II promoter through this element. By contrast, RAR inhibits the inductive effects of RXR on the apo A-II J site in a dose-dependent fashion. Gel retardation assays demonstrated that RXR homodimers bind, although with a lower affinity than RAR-RXR heterodimers, to the AH-RXR response element. In conclusion, retinoids induce hepatic apo A-II production at the transcriptional level via the interaction of RXR with an element in the J site containing two imperfect half-sites spaced by 1 oligonucleotide, thereby demonstrating an important role of RXR in controlling human lipoprotein metabolism. Since the J site also confers responsiveness of the apo A-II gene to fibrates and fatty acids via the activation of peroxisome proliferator-activated receptor-RXR heterodimers, this site can be considered a plurimetabolic response element.

Topics: Apolipoprotein A-II; Apolipoproteins E; Base Sequence; Benzoates; Bexarotene; Carcinoma, Hepatocellular; Cloning, Molecular; DNA-Binding Proteins; Female; Gene Expression Regulation; Genomic Library; HeLa Cells; Humans; Kinetics; Liver Neoplasms; Molecular Sequence Data; Nicotinic Acids; Oligodeoxyribonucleotides; Placenta; Pregnancy; Receptors, Retinoic Acid; Regulatory Sequences, Nucleic Acid; Retinoid X Receptors; Retinoids; RNA, Messenger; Tetrahydronaphthalenes; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Fatty Acid Synthases; Gene Expression Regulation, Enzymologic; Genes, Reporter; Humans; Liver Neoplasms; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Transfection; Tretinoin; Tumor Cells, Cultured

Previous studies have suggested that there is an interrelationship between responses mediated by retinoic acid (RA) and those to thyroid hormone (T3). These experiments have used transfected gene constructs, often in receptor-negative cells. To study the relationship between RA- and T3-mediated responses in intact human cells, we incubated HepG2 cells for 4 days in serum-free medium with T3 and/or RA or 9-cis-RA. Measured responses were stimulation of secreted sex hormone-binding globulin (SHBG) or inhibition of secreted T4-binding globulin (TBG). T3 induced a dose-responsive increase in SHBG secretion that was maximal at 10nM (206 +/- 24% of untreated value) and half-maximal at 0.36 +/- 0.16 nM T3. RA and 9-cis-RA, up to 100 nM, induced a slight fall in SHBG secretion to 79 +/- 9% and 88 +/- 9%, respectively. T3 induction of SHBG secretion was significantly attenuated in cells coincubated with T3(0-10nM) and RA. With T3 (10 nM) together with RA (3, 10, or 100 nM), the maximal SHBG responses were reduced to 193 +/- 24%, 151 +/- 5% and 132 +/- 30%, respectively. With T3 and 9-cis-RA (100 nM), maximal stimulation was 169 +/- 20%. Importantly, the effective half-maximal stimulatory concentration of T3 in the presence of either retinoid (3-100 nM) was unchanged at 0.3 nM T3. In addition, the inhibitory effect of 9-cis RA could not be overcome even with 300 nM T3. The threshold for the RA effect was between 0.3-1 nM, with half-maximal inhibition at 30 nM. 9-cis-RA was approximately 10-fold less potent than RA. Preliminary studies suggested that changes in SHBG messenger RNA levels were similar to those in secreted SHBG. No effect was observed with vitamin D or clofibrate, either alone or combined with T3. Conversely, T3 reduced TBG secretion, with maximal suppression to 74 +/- 5% of the control value at a T3 concentration of 10 nM. RA alone reduced TBG secretion to 76% of the control value. RA did not attenuate the effect of T3, and the two agents combined showed no synergism. Neither T3 nor RA, alone or in combination, influenced secreted total protein or albumin. RA did not alter the concentration of nuclear T3-binding sites. These data suggest that retinoids act via a gene-dependent mechanism to modulate maximal, but not half-maximal, responses to T3 in HepG2 cells with the specificity of RA greater than that of 9-cis-RA.

Topics: Carcinoma, Hepatocellular; Clofibrate; Dose-Response Relationship, Drug; Humans; Sex Hormone-Binding Globulin; Stereoisomerism; Thyroxine-Binding Proteins; Tretinoin; Triiodothyronine; Tumor Cells, Cultured; Vitamin D

We have investigated the biosynthesis, subcellular location and expression of insulin-degrading enzyme (IDE). a type-I peroxisomal protease, in semi-permeabilized hepatoma cells using pulse-chase experiments, non-denaturing immunoprecipitation protocols and Northern-blot analyses. In HcpG2 cell lysates prepared from cells radiolabelled with Tran[35S]-label, immunoprecipitated IDE was observed immediately after a 5 min pulse and subsequently declined during chase with t1/2 of approx. 33 h. In addition to the 110 kDa IDE protein, a protein of 70 kDa (p70) was identified in radiolabelled immunoprecipitates when using a monoclonal anti-IDE antibody 9B12 under non-denaturing conditions. This same antibody did not recognize p70 on Western blots of whole-cell lysates nor in sequential immunoprecipitates of immunocomplex-bead eluates from anti-IDE immunoprecipitations. Likewise, cross-linking studies performed on intact HepG2 and H35 hepatoma cells in vivo revealed the existence of a hetero-oligomeric complex of 180 kDa in which IDE and p70 were physically associated. Digitonin-permeabilization studies in normal and 35S-labelled HepG2 cells have defined a predominant association of IDE and its associated protein p70 with cytosol (supernatant); only a minor amount of the protein IDE was detected in peroxisomes (cellular pellet). Immunoprecipitation of IDE from 35S-labelled cell lysates of normal and stably transfected Chinese hamster ovary cells overexpressing IDE failed to detect p70. Treatment of HepG2 cells with clofibrate, a peroxisome proliferator, resulted in a dose-dependent increase of the two human IDE transcripts of 3.6 and 3.2 kb. This effect was not accompanied by a similar change at the protein level, nor by a change in the subcellular location of the proteins IDE and p70. Based on these findings we propose that in hepatoma cells: (1) IDE mainly exists in a stable cytoplasmic pool that is unchanged in cells undergoing peroxisomal proliferation; and (2) p70 binding to IDE may serve to maintain the dual cytosolic and peroxisomal pools of IDE in a stable equilibrium.

Topics: Animals; Blotting, Northern; Carcinoma, Hepatocellular; Cells, Cultured; CHO Cells; Clofibrate; Cricetinae; Cytosol; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Insulysin; Microbodies; Molecular Weight; Neoplasm Proteins; RNA, Messenger; Subcellular Fractions; Transfection; Tretinoin

Human hepatoma Hep 3B cells underwent apoptosis in response to 100 microM all-trans retinoic acid (RA) in full serum (10% fetal calf serum) condition in vitro. Cell death began approximately 24 h following treatment, with more than 80% of the cells dead after 60 h. The dead cells, mainly detached cells, exhibited condensed chromatin and DNA fragmentation, which are indicative of endonuclease activation and are the hallmarks of apoptosis in epithelial cells. Concurrent exposure to 1 microM cycloheximide (CX) prevented approximately 50% of cell death and DNA fragmentation induced by RA. Thus, other toxic injury to the cells as well as apoptosis might be involved in cell death. Sixty hours exposure of RA decreased the percentage of cells in G1 phase (16.3 +/- 0.4% versus 52.4 +/- 2.1%; P < or = 0.01) and in G2/M phase (13.4 +/- 1.2% versus 21.2 +/- 0.7%; P < or = 0.01), but did not change percent of cells in S phase (20.8 +/- 0.2% versus 20.7 +/- 0.5%) of the cell cycle compared with control. RA may have caused accumulation of Hep 3B cells before G1 phase, and that G0/G1 transition is a main check point in the active process of apoptosis. Electron micrographs of the cells treated with RA revealed typical morphologic changes of apoptosis, besides toxic injury to the cells. These data strongly indicate that RA is able to induce apoptosis and the induction of apoptosis may contribute to the antitumor activity of RA against hepatoma cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cycloheximide; DNA, Neoplasm; Humans; Liver Neoplasms; Microscopy, Electron; Protein Synthesis Inhibitors; Tretinoin

Apolipoprotein E (apoE), one of the major plasma lipoproteins, also is expressed in a variety of cell types, including the glial cells of the nervous system. apoE is involved in processes of degeneration and regeneration after nerve lesions as well as in the pathogenesis of Alzheimer's disease (AD). Glial synthesis of apoE is activated in response to injury both in the peripheral and central nervous system. We now report that the activity of the proximal apoE promoter in astrocytes is upregulated by cAMP and retinoic acid, which act synergistically. Sequence analysis of the apoE promoter indicated the presence of several AP-2 consensus sequences that could mediate the stimulatory effect of cAMP and retinoic acid. The possible functional role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with an apoE promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated apoE promoter activity. DNase I footprinting technique revealed the existence of two binding sites for recombinant AP-2 in regions from -48 to -74 and from -107 to -135 of the apoE promoter. Mutations in these regions markedly impaired the trans-stimulatory effect of AP-2. These results indicate the existence of functional AP-2 sites in the promoter region of apoE that could contribute to the complex regulation of this gene in developmental, degenerative, and regenerative processess of the nervous system.

Topics: Apolipoproteins E; Astrocytoma; Base Sequence; Carcinoma, Hepatocellular; Consensus Sequence; Cyclic AMP; DNA-Binding Proteins; Gene Expression Regulation; Humans; Molecular Sequence Data; Promoter Regions, Genetic; Transcription Factor AP-2; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Data Interpretation, Statistical; Diagnosis, Differential; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Neoplasms, Second Primary; Research Design; Tretinoin

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Neoplasms, Second Primary; Tretinoin; Vitamin A; Vitamin A Deficiency

A human hepatocellular carcinoma cell line, SMMC-7721, was treated with all-trans-retinoic acid (RA) and phorbol 12-myristate 13-acetate (PMA) to induce its differentiation and proliferation respectively. A biantennary sugar chain fluorescently labelled with 2-aminopyridine (PA), GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-PA, was used to detect the activity of beta-1,4-galactosyltransferase on the cell surface by HPLC. The results show that the activity of beta-1,4-galactosyltransferase on the cell surface increases when the cell is treated with RA, but decreases when it is treated with PMA, whereas the activities of the whole cell remain stable during the treatments.

Topics: Aminopyridines; Carbohydrate Conformation; Carbohydrate Sequence; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Cell Line; Cell Membrane; Humans; Kinetics; Liver Neoplasms; Molecular Sequence Data; N-Acetyllactosamine Synthase; Oligosaccharides; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

In order to study the effect of retinoic acid on the structure of N-glycans on the cell surface, the N-glycans of glycoproteins on the surface of 7721 human hepatocarcinoma cells were labelled with [3H] mannose, added to the culture medium. The 3H-labelled N-glycans were prepared from cell-surface glycoproteins, desialylated, and subjected to sequential chromatography on concanavalin A and Datura stranonium agglutinin affinity columns to separate the glycans into four fractions of different type and different antennary number. It was found that the percentage of C2C2 biantennary complex type N-glycans was increased, but the high-mannose type as well as the tri- and tetraantennary complex types, especially that with a C2.6 branched structure, were decreased after the cells had been treated with retinoic acid for 3-5 days. Using a Lens culinaris agglutinin affinity column, it was discovered that the core fucose in the biantennary glycan was also decreased. The enzymatic mechanisms of the above changes were revealed in further study to involve the decrease of N-acetylglucosaminyl-transferase V and core alpha-1,6-fucosyltransferase by retinoic acid.

Topics: Asparagine; Carbohydrate Sequence; Carcinoma, Hepatocellular; Chromatography, Affinity; Fucose; Fucosyltransferases; Humans; Liver Neoplasms; Membrane Glycoproteins; Molecular Sequence Data; Molecular Structure; N-Acetylglucosaminyltransferases; Polysaccharides; Tretinoin; Tritium; Tumor Cells, Cultured

HuH-7 cells, a human hepatoma-derived cell line, underwent apoptosis in response to all-trans 3, 7, 11, 15-tetramethyl- 2, 4, 6, 10, 14-hexadecapentaenoic acid, or acyclic retinoid. The retinoid-induced apoptosis was verified by a characteristic step-wise fragmentation of genomic DNA and chromatin condensation. The induction of apoptosis was detected as early as 8 hrs after the addition of the retinoid and was concentration dependent (0.1-10 microM). Neither the natural retinoid all-trans retinoic acid nor 9-cis retinoic acid induced apoptosis. These data strongly indicate that the antitumor activity of the acyclic retinoid may be partly explained by the induction of apoptosis in tumor cells.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line; Chromatin; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Kinetics; Liver Neoplasms; Time Factors; Tretinoin; Tumor Cells, Cultured

Thyroid hormone receptor (TR) and retinoic acid receptor (RAR) are closely related not only in their structures but also in their modes of action. TR and RAR share many amino acids in the functionally important DNA binding domain, ligand binding domain, and heterodimeric interface. To function, both receptors dimerize with retinoid X receptor, recognize their cognate hormone response elements, and then respond to the ligand, leading to the activation of the target genes. Genetic analysis has revealed mutated TRs in thyroid hormone resistance syndrome, which displays autosomal dominant inheritance. Eventually, the mutated TRs show the dominant-negative phenotype on the wild-type TR. The mutations have been observed mostly in conserved amino acids between TR and RAR in the ligand binding domain and produce hormone-insensitive receptors. In this report, we demonstrate that the dominant-negative phenotype is transferable to RAR by a single amino acid substitution identified in the syndromes of thyroid hormone resistance. The mutated RAR can suppress the wild-type RAR function, especially at the physiological concentration of retinoic acid. Consistently, the mutated RAR is an absolute requirement of intact dimeric and DNA binding capacities for the dominant-negative phenotype, indicating the necessity of the maintenance of a machinery for correct recognition of the targets. Thus, the hormone-insensitive receptor may be interfering with the access of functional receptors to the hormone response elements. The dominant-negative RAR will serve as a molecular tool to elucidate physiological roles of RAR by blocking RAR-mediated signaling pathways.

Topics: Base Sequence; Carcinoma, Hepatocellular; DNA-Binding Proteins; Genes, Dominant; Humans; In Vitro Techniques; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Oncogene Proteins v-erbA; Phenotype; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Retroviridae Proteins, Oncogenic; Sequence Alignment; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (RA) affects cell growth and regulates gene expression. We examined the expression of topoisomerase 11 gene in Hep3B cells treated with RA. At low RA concentration which did not significantly affect the growth rate of Hep3B cells, RA inhibited the synthesis of topoisomerase II mRNA as revealed by Northern analysis and nuclear run-on analysis. These results indicated that the repression of topoisomerase II gene expression could be directly induced by RA rather than was a secondary event which occurred after cell growth was inhibited by RA. An unexpected finding is that after up to 72 h continuous exposure to RA, the topoisomerase II protein concentration remained unchanged.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Division; DNA Topoisomerases, Type II; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Genes, fos; Genes, jun; Genes, p53; Humans; Immunoblotting; Liver Neoplasms; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoic acid treatment of the multipotent human embryonal carcinoma cell line GCT 27 X-1 induced differentiation into an epithelial cell type which morphologically resembled rodent visceral endoderm in vitro. The differentiated cells expressed some markers characteristic of yolk sac, such as cytokeratin 19 and extracellular matrix proteins, but none of the secreted serum proteins produced by normal yolk sac or liver. HNF-3 alpha expression increased upon retinoic acid-induced GCT 27 X-1 differentiation. This increase in HNF-3 alpha expression may characterize an early stage in the differentiation of extraembryonic endoderm. In support of this hypothesis, we found that a yolk sac carcinoma cell line with a phenotype similar to rodent visceral endoderm expressed transcripts for HNF-1 alpha, -1 beta, -3 alpha, -3 beta, -3 gamma, and 4, whereas yolk sac carcinoma cell lines resembling rodent parietal endoderm expressed transcripts for HNF-3 alpha. The results support the conclusion that hepatocytic transcription factors, particularly HNF-3 alpha, may play an important role in early endodermal differentiation in man.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antibody Specificity; Antigens, Surface; Biomarkers; Blotting, Northern; Carbohydrate Sequence; Carcinoma, Embryonal; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line; Cytoskeletal Proteins; DNA-Binding Proteins; Endoderm; Endodermal Sinus Tumor; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Gene Expression; Hepatocyte Nuclear Factor 3-alpha; Humans; Kinetics; Laminin; Liver Neoplasms; Models, Biological; Molecular Sequence Data; Nuclear Proteins; Restriction Mapping; RNA, Messenger; Rodentia; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The expression of the gene coding for retinol-binding protein has been studied in a system of cultured human hepatoma cells exposed to retinoids. We report that the gene is positively modulated by retinol and retinoic acid in a time- and dose-dependent fashion. The stimulation at the mRNA level is paralleled by an increase of the corresponding protein that is secreted in the presence of the physiological ligand. An RBP-CAT chimeric gene, introduced by transfection, is also responsive to the treatment, showing the gene dose-dependency as the endogenous gene. These results demonstrate that retinoids up-regulate the RBP gene and that the control takes place at transcriptional level.

Topics: Carcinoma, Hepatocellular; Chimera; Chloramphenicol O-Acetyltransferase; Gene Expression Regulation; Humans; Liver Neoplasms; Retinoids; Retinol-Binding Proteins; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured; Vitamin A

The effects of retinoic acid on the expression of the Ha-ras gene were studied in transformed rat hepatoma cells (H4IIE) and in rat aortic smooth muscle cells (ASMC) treated with benzo(a)pyrene (30 microM) in vitro. In H4IIE cells, a dose-dependent increase in steady state Ha-ras mRNA levels was observed upon exposure to retinoic acid for 24 hr. Exposure of ASMC to 10 microM retinoic acid under similar experimental conditions was also associated with increased Ha-ras expression. In contrast, retinoic acid (1 and 10 microM) inhibited benzo(a)pyrene-induced expression of Ha-ras in ASMC. These results suggest that retinoic acid modulates spontaneous and carcinogen-induced expression of Ha-ras in a differential manner.

Topics: Animals; Anticarcinogenic Agents; Aorta; Benzo(a)pyrene; Carcinoma, Hepatocellular; Cell Line; Gene Expression; Genes, ras; Liver Neoplasms; Muscle, Smooth, Vascular; Rats; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Transferrin and albumin, which are both secreted from the human hepatoma cell line Hep3B, were regulated transcriptionally by retinoic acid (RA) in a dose-dependent manner. The cell growth rate was little affected under the same conditions. The treatment of Hep3B cells with RA (10 microM for 48 h) resulted in an 8-fold increase in transferrin protein synthesis, a 10-fold increase in the steady-state transferrin mRNA level, and a 5-fold increase in its transcriptional rate. The same treatment led to 4-fold decrease in albumin synthesis, as well as a 7-fold decline in the steady-state albumin mRNA level and a 4-fold decrease in the transcriptional rate. Cycloheximide and actinomycin D blocked the action of RA, suggesting that RA may regulate transferrin and albumin gene expression indirectly in human liver cells.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cell Nucleus; Cycloheximide; Dactinomycin; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Liver Neoplasms; RNA, Neoplasm; Serum Albumin; Transcription, Genetic; Transferrin; Tretinoin

The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXR alpha together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXR alpha together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAI gene responsiveness to RXR alpha and RA and suggest that the magnitude of this responsi

Topics: Apolipoprotein A-I; Binding Sites; Carcinoma, Hepatocellular; Cell Line; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; COUP Transcription Factor II; COUP Transcription Factors; DNA-Binding Proteins; Humans; Kinetics; Liver Neoplasms; Macromolecular Substances; Plasmids; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Retinoic Acid; Receptors, Steroid; Retinoid X Receptors; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin

A retinoid X receptor (RXR) response element was located within the functionally defined hepatitis B virus (HBV) enhancer element. A short segment of the enhancer that contains this region has been shown with genetic analysis to play a key role in the regulation of enhancer function and to represent a major determinant of liver-specific activity. Both the full-length protein and the DNA-binding domain of the liver-specific receptor RXR alpha bound to the putative retinoic acid response element in the HBV enhancer. In vivo, an HBV enhancer-reporter gene construct responds to induction with retinoic acid when cotransfected with an RXR alpha expression vector. A single-base transition (G----A) in the HBV retinoic acid response element leads to a dramatic reduction both in the in vitro binding activity of RXR alpha and the in vivo activity of the HBV enhancer. Thus, retinoic acid and the RXR alpha are implicated as being significant determinants in the liver-specific regulation of HBV gene expression and the resultant disease pathogenesis.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Nucleus; DNA-Binding Proteins; Enhancer Elements, Genetic; Genes, Viral; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Liver Neoplasms; Molecular Sequence Data; Nuclear Proteins; Receptors, Cell Surface; Receptors, Retinoic Acid; Retinoid X Receptors; Trans-Activators; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Reversing effect of retinoic acid (RA) on some phenotypes of human hepatocarcinoma cell line was studied. It was found that the growth of SMMC-7721 human hepatocarcinoma cell line was markedly inhibited when cultured in 10 mumol/L RA. Morphology of cells treated with RA reversed to normal when observed under light and electron microscopes and by image analysis. The 3H-TdR incorporation declined. By flow cytometry, it was observed that cells in G0/G1 phase increased from 49.6% to 59.1%, whereas cells in S or G2 + M phase decreased from 50.4% to 40.9% after 3-day RA treatment. The major distribution of chromosomes changed from 48-56 to 44-50 with an increase in diploid (from 4% to 15%). The results indicate that RA could reverse some phenotypes of human hepatocarcinoma cell line.

Topics: Carcinoma, Hepatocellular; Cell Division; Diploidy; Flow Cytometry; Humans; Liver Neoplasms; Phenotype; Tretinoin; Tumor Cells, Cultured

cDNA probes for human retinoic acid receptors alpha and beta (RAR alpha and RAR beta) were modified for use as specific hybridization probes to study hepatocellular carcinomas (HCC) and cell lines, liver regeneration, and fetal development. RAR beta mRNA was detected at low levels in adult liver and rose markedly during the early phase of liver regeneration. RAR beta mRNA was present at very low levels in HCC and was not detected in fetal liver. In contrast, RAR alpha mRNA was present at low levels in normal liver, but showed a marked elevation in several HCCs and cell lines. Growth of cell lines was altered by retinoic acid (RA), but the effects could not be predicted by the levels of either RAR alpha or RAR beta mRNA. However, the response correlated with cell phenotype. Three cell lines with an adult phenotype (high albumin and low alpha-fetoprotein) were inhibited by RA, two undifferentiated lines showed moderate growth stimulation, and two of three cell lines that had high levels of alpha-fetoprotein were markedly stimulated by RA.

Topics: Adult; Carcinoma, Hepatocellular; Carrier Proteins; Cell Division; Cell Line; Cloning, Molecular; Gene Expression; Humans; Kinetics; Liver; Liver Neoplasms; Liver Regeneration; Nucleic Acid Hybridization; Receptors, Retinoic Acid; RNA; RNA, Neoplasm; Transcription, Genetic; Tretinoin

Acyclic retinoid (polyprenoic acid) has a slightly different structure from retinoic acid. However, acyclic retinoid acts similarly to retinoic acid, because both bind to cellular retinoic acid-binding protein and cellular retinoid-binding protein. F-type, with the same strong binding affinity. We studied the effects of acyclic retinoid, the 7-hydroxy derivative of acyclic retinoid (7OH-acyclic retinoid) and retinoic acid on a human hepatoma-derived cell line PLC/PRF/5 (Alexander cells). Acyclic retinoid inhibited cell growth with an ID50 value of 14 microM, and reduced cell viability with an LD50 value of 86 microM. The ratios of LD50 value to ID50 value were 6.1 for acyclic retinoid, 2.4 for 7OH-acyclic retinoid and 1.4 for all-trans-retinoic acid. Taking this ratio as a parameter of relative cytotoxicity, we concluded that acyclic retinoid is the least toxic compound. Growth inhibition of cells by acyclic retinoid was associated with the incorporation of 3H-thymidine in the logarithmic phase. Acyclic retinoid reduced secretion of alpha-fetoprotein (AFP) and reciprocally increased secretion of albumin in the culture media, suggesting that acyclic retinoid influences gene expression of these proteins. Thus, acyclic retinoid, one of the less toxic retinoids, inhibits cell growth of human cancer cell line PLC/PRF/5 and appears to alter gene expression of AFP and albumin toward a "normal" direction.

Topics: Acrylates; Albumins; alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Division; Cell Survival; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Thymidine; Tretinoin; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; DNA Transposable Elements; Genes, Viral; Genetic Code; Hepatitis B virus; Liver Neoplasms; Receptors, Drug; Tretinoin

Processes as diverse as growth, vision and reproduction depend on the presence of vitamin A and its metabolites (retinoids), but the molecular mechanisms which govern these diverse actions remain unclear (for reviews see refs 1,2). A crucial advance recently was the isolation of a specific nuclear receptor for retinoic acid, one of the physiologically active vitamin A derivatives. This nuclear receptor is a member of the steroid/thyroid hormone receptor family. Our analysis of an uncharacterized member of this class of intracellular receptors, encoded by a complementary DNA clone from a human placental library, has led us to discover a second retinoic acid receptor. This new receptor is expressed at high levels in a number of epithelial-type tissues. The gene for the receptor was first identified in a hepatocellular carcinoma where it surrounds a site of integration of hepatitis B virus. Activation by this virus may play a role in tumour development in liver cells, where it is normally not expressed.

Topics: Amino Acid Sequence; Carcinoma, Hepatocellular; Carrier Proteins; Genes; Humans; Liver Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Protein Biosynthesis; Receptors, Retinoic Acid; Transcription, Genetic; Transfection; Tretinoin

The human hap retinoic acid receptor RAR beta has been localized by in situ hybridization to the p24 band of chromosome 3.

Topics: Carcinoma, Hepatocellular; Carrier Proteins; Chromosome Banding; Chromosome Mapping; Chromosomes, Human, Pair 3; DNA; Genetic Markers; Hepatitis B virus; Humans; Karyotyping; Liver Neoplasms; Multigene Family; Nucleic Acid Hybridization; Receptors, Retinoic Acid; Tretinoin

We investigated the effects of polyprenoic acid, E5166, on the production and secretion of alpha-fetoprotein (AFP) and on cell kinetics in a human hepatoma cell line (HuH-7). The cellular AFP content, measured flow cytometrically for cells stained by an indirect immunofluorescence method, was decreased by treatment with E5166. AFP in the culture medium decreased exponentially during exposure of cells to the drug. These changes were dose-dependent. The growth of HuH-7 cells in vitro was clearly suppressed in the presence of E5166. The inhibition of growth depended on the concentration of the agent. The fraction of S phase cells decreased relatively in the cells treated with a high concentration of the drug, whereas it increased in the cells treated with lower doses.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cells, Cultured; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation; Humans; Liver Neoplasms; Tretinoin

Vitamin A level and the cytosol-binding proteins specific for vitamin A ere studied in human tumor and its surrounding tissue. The tissues examined were 10 hepatocellular carcinomas which were surgically removed, 4 other malignant tumors (2 metastatic liver cancer and one each of gastric cancer and glioma), and 3 human fetal livers. Compared with surrounding tissues, considerable decrease of vitamin A content was observed in the hepatocellular carcinoma suggesting local deficient state of the vitamin. In addition to cellular retinol-binding protein (CRBP) and retinoic acid-binding protein (CRABP), a new molecular species having affinity for both retinol and retinoic acid was detected in the cytosols obtained from hepatocellular carcinoma as well as glioma by means of gel filtration on Sephadex G-75. With regard to ligand specificity, the protein was found to be similar to cellular retinol-binding protein, F-type or CRBP(F) which was originally recognized in the fish eye cytosol. Since the protein was also demonstrated in human fetal liver, CRBP(F) is considered to be an oncofetal protein in nature. The present study further revealed that CRBP(F) was detected in 80% of hepatocellular carcinoma (whereas plasma alpha-fetoprotein was significantly elevated only in 50%), and hepatocellular carcinoma contained CRBP(F) in a larger amount than CRABP.

Topics: Adult; alpha-Fetoproteins; Carcinoma, Hepatocellular; Carrier Proteins; Chromatography, Gel; Fetus; Hepatitis B Surface Antigens; Humans; Liver; Liver Neoplasms; Middle Aged; Neoplasm Proteins; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Retinol-Binding Proteins, Plasma; Tretinoin; Vitamin A