trichostatin-a and Liver-Neoplasms

This work was designed to compare the sensitizing effects of epigenetic modifiers on cancer cells vs. that of glucocorticoids. Also, to evaluate their effects on genes involved in epigenetic changes and drug metabolism.. Hepatoma cells (HepG2) were treated with the anticancer drug (Taxol), with a histone deacetylase inhibitor (Trichostatin A [TSA]), DNA methyltransferase inhibitor (5-Aza-dC) or dexamethasone (DEX). Cytotoxicity was assessed by MTT assay and the apoptosis was determined by Annexin V-FITC. The expression levels of. The data reflects the sensitizing effect of acetylation modification by TSA on the responsiveness of hepatoma cells to anticancer therapy. The effect of histone deacetylase inhibition was more than hypomethylation and glucocorticoid effects. TSA exerts its role through its modulatory role on epigenetics and drugs metabolizing genes. Other modifiers (5-Aza-dC and DEX), however may adopt different mechanisms.

Topics: Carcinoma, Hepatocellular; Decitabine; Dexamethasone; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Paclitaxel

A multistep process with an accumulation of epigenetic alterations of tumor suppressor genes (TSGs) can induce cancer. Abnormal regional hypermethylation and histone deacetylation of several TSGs has been observed in hepatocellular carcinoma (HCC). Acetylation and deacetylation of histone are carried out by histone acetyltransferase (HAT) and histone deacetylase (HDAC) respectively. Besides, DNA methylation is carried out by DNA methyltransferases (DNMTs). Previously, we evaluated the effect of DNA demethylating agents and histone deacetylase inhibitors on HCC and colon cancer. This study aimed to evaluate the effect of curcumin (CUR) in comparison with trichostatin A (TSA) on estrogen receptor alpha (ERα) reactivation, apoptotic induction, and cell growth inhibition in HCC.. the cells were cultured and treated with various concentrations of CUR and TSA and the MTT assay, flow cytometry assay and Real-Time RT-PCR were achieved to determine cell viability, cell apoptosis, and ERα gene expression respectively.. CUR indicated dose and time-dependent antiproliferative effects (P < 0.035). A similar antiproliferative effect was observed by TSA (P < 0.001). Both compounds indicated significant apoptotic effects in all different periods (P < 0.001), CUR indicated a more significant apoptotic effect than TSA (P < 0.001). The ERα gene expression quantity was increased significantly by treatment with CUR and TSA (P <0.012).. CUR and TSA play important roles in restoring the ERα resulting in cell growth inhibition and apoptosis induction. Therefore, ERα may be a potential target for therapeutic intervention in the treatment of HCC.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Proliferation; Curcumin; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Tumor Cells, Cultured

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

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

Sarco(endo)plasmic reticulum Ca

Topics: Animals; Butyric Acid; Carcinoma, Hepatocellular; E1A-Associated p300 Protein; Gene Expression; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; Promoter Regions, Genetic; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tumor Cells, Cultured

Class I histone deacetylases (HDACs) are highly expressed and/or upregulated in hepatocellular carcinoma (HCC) and are associated with aggressiveness, spread, and increased mortality of HCC. Activation of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway was involved in the development of HCC and acquired resistance to sorafenib. A series of purine or 5H-pyrrolo[3,2-d]pyrimidine based hydroxamates were designed and developed as multitarget drugs to modulate both HDACs and the PI3K/Akt/mTOR pathway. Among 39 cell lines screened, the molecules (e.g., 20e, 20f, and 20q) were the most selective against leukemia, lymphoma, and HCC cells; they also demonstrated target modulation in cancer cell lines and in mice bearing MV4-11 and HepG2 tumors. Compound 20f in particular showed significant single agent oral efficacy in hypervascular liver cancer models (e.g., HepG2, HuH-7, and Hep3B) and was well-tolerated. These encouraging results, along with its favorable target profile and tissue distribution, warrant further development of 20f.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Screening Assays, Antitumor; Heterografts; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The effects of Histone deacetylase (HDAC) inhibition on epithelial-mesenchymal transition (EMT) differs in various types of cancers. However, its function in hepatocellular carcinoma (HCC) is not well-explored. In this study, we investigated the effect of HDAC inhibition on EMT in HCC cells by using trichostatin A (TSA) and valproic acid (VPA). The results showed that TSA/VPA significantly induced EMT phenotype, as demonstrated by the decreased level of E-cadherin, increased level of N-cadherin, vimentin, Twist and snail, and enhanced capacity of cell migration and invasion. In addition, CCR7 was speculated and confirmed as a function target of HDAC inhibition. CCR7 promotes the progression of HCC and is associated with poor survival. Knockdown of CCR7 significantly attenuated the effect of TSA on EMT. Moreover, our results demonstrated that HDAC inhibition up-regulates CCR7 via reversing the promoter hypoacetylation and increasing CCR7 transcription. Taken together, our study has identified the function of HDAC in EMT of HCC and suggested a novel mechanism through which TSA/VPA exerts its carcinogenic roles in HCC. HDAC inhibitors require careful caution before their application as new anticancer drugs.

Topics: Antigens, CD; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Neoplasm Staging; Nuclear Proteins; Promoter Regions, Genetic; Receptors, CCR7; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Survival Analysis; Twist-Related Protein 1; Valproic Acid; Vimentin

Histone deacetylase (HDAC) inhibitors show clinical promise for the treatment of cancers, including hepatocellular carcinoma (HCC). In this study, we investigated the effect of HDAC inhibitor treatment on hepatitis C virus (HCV) replication in Huh7 human liver cells and in a mouse model of HCV infection. Viral replication was markedly suppressed by the HDAC3 inhibitor at concentrations below 1 mmol/L, with no cellular toxicity. This was accompanied by upregulation of liver-expressed antimicrobial peptide 1(LEAP-1) and downregulation of apolipoprotein-A1 (Apo-A1), as determined by microarray and quantitative RT-PCR analyses. Moreover, HDAC3 was found to modulate the binding of CCAAT-enhancer-binding protein α (C/EBPα), hypoxia-inducible factor 1α (HIF1α), and signal transducer and activator of transcription 3 (STAT3) to the LEAP-1 promoter. HDAC3 inhibitor treatment also blocked HCV replication in a mouse model of HCV infection. These results indicate that epigenetic therapy with HDAC3 inhibitor may be a potential treatment for diseases associated with HCV infection such as HCC.

Topics: Acrylamides; Animals; Apolipoprotein A-I; Carcinoma, Hepatocellular; Cell Line, Tumor; Hepacivirus; Hepcidins; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Indoles; Liver Neoplasms; Mice; Mice, Transgenic; Phenylenediamines; Protein Binding; STAT3 Transcription Factor; Virus Replication

Epigenetic changes such as DNA methylation and histone acetylation play important roles in determining gene\ expression. Hypermethylation of CpG islands of the promoter region of tumor suppressor genes can greatly influence\ carcinogenesis through transcriptional silencing. Acetylation of lysine in histone tails causes relaxation of chromatin,\ which facilitates gene transcription, while deacetylation is associated with condensed chromatin resulting in gene\ silencing. DNA demethylating agents such as genistein (GE) and histone deacetylase inhibitors (HDACIs) such as\ trichostatin A (TSA) may strongly reactivate silenced genes and exposure to these two agents in combination is reported\ to enhance estrogen receptor alpha (ERα) reactivation and induction of apoptosis. The present study was designed to\ evaluate the effect of these compounds on ERα gene expression, cell viability and apoptosis in hepatocellular carcinoma\ (HCC) Hep G2 cells. GE exerted biphasic effects; it stimulated cell growth at a low concentration (1 μM) but inhibitory\ influence was noted with high concentrations (10, 20 and 40 μM). In contrast, TSA demonstrated inhibitory effects on\ growth at all of concentrations tested. Furthermore, GE and GE/TSA significantly induced apoptosis at all concentrations,\ but TSA only after 72 h. GE induced ERα re-expression and this was maximal in combined treatment groups treated\ with GE/TSA for 72 h. Discussion: Our finding clearly indicates that GE and TSA have an inhibitory cell growth,\ induce apoptosis and reactivate the ERα gene expression. Conclusion: GE and TSA can significantly inhibit the growth\ of HCC cells and play a significant role in apoptosis and reactivation of ERα gene.

Topics: Acetylation; Anticarcinogenic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; DNA Methylation; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Genistein; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Tumor Cells, Cultured

CYLD is a deubiquitinating enzyme that exerts a tumor suppressive function. Its downregulation or inactivation has been associated with the development of several types of malignancies including hepatocellular carcinoma (HCC). HCC cells display significantly lower Cyld expression compared to primary human hepatocytes, and Cyld downregulation can contribute to apoptotic resistance of HCC cells. Little is known about the mechanism of Cyld downregulation in human HCC cells. In the present study we explored the possible regulation of Cyld expression by histone deacetylases (HDACs) in human HCC cell lines. We demonstrated that the HDAC inhibitors suberoylanilide hydroxamic acid, sodium butyrate, and trichostatin A induced the upregulation of both mRNA and protein levels of CYLD in two different HCC cell lines, HepG2 and Huh7. Our results demonstrate the involvement of HDACs in the downregulation of Cyld expression in HCC cells and support and may improve the use of HDAC inhibitors for the treatment for HCC.

Topics: Butyric Acid; Carcinoma, Hepatocellular; Cell Line, Tumor; Deubiquitinating Enzyme CYLD; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Tumor Suppressor Proteins; Up-Regulation; Vorinostat

PTPRD (protein tyrosine phosphatase, receptor type, D) is a tumor suppressor gene, frequently inactivated through deletions or epigenetic mechanisms in several cancers with importance for global health. In this study, we provide new and functionally integrated evidence on genetic and epigenetic alterations of PTPRD gene in hepatocellular carcinomas (HCCs). Importantly, HCC is the sixth most common malignancy and the third most common cause of cancer-related mortality worldwide. We used a high throughput single nucleotide polymorphism (SNP) microarray assay (Affymetrix, 10K2.0 Assay) covering the whole genome to screen an extensive panel of HCC cell lines (N=14 in total) to detect DNA copy number changes. PTPRD expression was determined in human HCCs by Q-RT-PCR and immunohistochemistry. Promoter hypermethylation was assessed by combined bisulfite restriction analysis (COBRA). DNA methyl transferase inhibitor 5-azacytidine (5-AzaC) and/or histone deacetylase inhibitor Trichostain A (TSA) were used to restore the expression. We identified homozygous deletions in Mahlavu and SNU475 cells, in the 5'UTR and coding regions, respectively. PTPRD mRNA expression was downregulated in 78.5% of cell lines and 82.6% of primary HCCs. PTPRD protein expression was also found to be lost or reduced in HCC tumor tissues. We found promoter hypermethylation in 22.2% of the paired HCC samples and restored PTPRD expression by 5-AzaC and/or TSA treatments. In conclusion, PTPRD is homozygously deleted and epigenetically downregulated in HCCs. We hypothesize PTPRD as a tumor suppressor candidate and potential cancer biomarker in human HCCs. This hypothesis is consistent with compelling evidences in other organ systems, as discussed in this article. Further functional assays in larger samples may ascertain the contribution of PTPRD to hepatocarcinogenesis in greater detail, not to forget its broader importance for diagnostic medicine and the emerging field of personalized medicine in oncology.

Topics: Azacitidine; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Methylation; Down-Regulation; Enzyme Inhibitors; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Homozygote; Humans; Hydroxamic Acids; Liver Neoplasms; Microarray Analysis; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Receptor-Like Protein Tyrosine Phosphatases, Class 2; Sequence Deletion

The cancer stem cells (CSCs) have important therapeutic implications for multi-resistant cancers including hepatocellular carcinoma (HCC). Among the key pathways frequently activated in liver CSCs is NF-κB signaling.. We evaluated the CSCs-depleting potential of NF-κB inhibition in liver cancer achieved by the IKK inhibitor curcumin, RNAi and specific peptide SN50. The effects on CSCs were assessed by analysis of side population (SP), sphere formation and tumorigenicity. Molecular changes were determined by RT-qPCR, global gene expression microarray, EMSA, and Western blotting.. HCC cell lines exposed to curcumin exhibited differential responses to curcumin and were classified as sensitive and resistant. In sensitive lines, curcumin-mediated induction of cell death was directly related to the extent of NF-κB inhibition. The treatment also led to a selective CSC-depletion as evidenced by a reduced SP size, decreased sphere formation, down-regulation of CSC markers and suppressed tumorigenicity. Similarly, NF-κB inhibition by SN50 and siRNA against p65 suppressed tumor cell growth. In contrast, curcumin-resistant cells displayed a paradoxical increase in proliferation and expression of CSC markers. Mechanistically, an important component of the CSC-depleting activity of curcumin could be attributed to a NF-κB-mediated HDAC inhibition. Co-administration of the class I/II HDAC inhibitor trichostatine sensitized resistant cells to curcumin. Further, integration of a predictive signature of curcumin sensitivity with human HCC database indicated that HCCs with poor prognosis and progenitor features are most likely to benefit from NF-κB inhibition.. These results demonstrate that blocking NF-κB can specifically target CSC populations and suggest a potential for combined inhibition of NF-κB and HDAC signaling for treatment of liver cancer patients with poor prognosis.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Curcumin; Histone Deacetylases; Humans; Hydroxamic Acids; Liver Neoplasms; Mice; Neoplastic Stem Cells; NF-kappa B; Signal Transduction

Hepatocellular carcinoma (HCC) is one of the most deadly cancers. Aberrant oncogenic activation of the Wnt/β-catenin signaling pathway contributes to hepatocellular carcinogenesis. Various epigenetic modifications of the Wnt antagonist secreted frizzled-related protein (SFRP) family have been implicated in regulating Wnt signaling. Here, we report that Hepatitis C virus (HCV) core protein downregulates SFRP1 expression when it is expressed in Huh7 and HepG2 cells. SFRP1 expression can be effectively restored by using either a DNA methylation inhibitor alone or in combination with a histone deacetylase inhibitor. DNA methylation analysis of the SFRP1 promoter revealed that cytosine-phosphate-guanine (CpG) islands close to the transcriptional start site (TSS) in the SFRP1 promoter were hypermethylated in core-expressing Huh7 cells, suggesting that HCV core protein may downregulate SFRP1 expression by inducing hypermethylation of the SFRP1 promoter. Chromatin immunoprecipitation revealed that HCV core protein markedly increased the expression level and binding of DNA methyltransferase-1 (Dnmt1) and histone deacetylase-1 (HDAC1) to the TSS of the SFRP1 promoter region, resulting in repression of acetyl-histone H3-binding capacity to SFRP1 promoter and the eventual epigenetic silencing of SFRP1 expression. Furthermore, the core protein-promoted cell proliferation, migration and invasiveness were effectively abrogated either by Dnmt1 knockdown or restoration of SFRP1 expression in hepatoma cells. Dnmt1 knockdown or SFRP1 overexpression also inhibited HCV core-induced epithelial-mesenchymal transition (EMT) and significantly decreased the expression levels of activated β-catenin and Wnt/β-catenin target genes, c-Myc and cyclin D1. We further showed that knockdown of Dnmt1 and restoration of SFRP1 inhibited core-induced in vivo tumor growth and aggressiveness in a xenograft HCC model. Taken together, our results strongly suggest that the HCV core-induced epigenetic silencing of SFRP1 may lead to the activation of the Wnt signaling pathway and thus contribute to HCC aggressiveness through induction of EMT.

Topics: Acetylation; Animals; Azacitidine; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Decitabine; Disease Models, Animal; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Gene Knockdown Techniques; Gene Silencing; Histones; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Membrane Proteins; Promoter Regions, Genetic; Tumor Burden; Viral Core Proteins; Wnt Proteins; Xenograft Model Antitumor Assays

To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms.. HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of β-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for β-catenin, HDAC1and HDAC3 was tested by q-PCR. β-Catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit.. Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/ G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was 6.22±0.25%, which increased to 7.17±0.20% and 18.1±0.42% in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of β-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of β-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of β-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased.. TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/β-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

Topics: Acetylation; Apoptosis; beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Liver Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Wnt Signaling Pathway

DNA methylation-dependent transcriptional inactivation of tumor suppressor genes (TSGs) is critical for the pathogenesis of hepatocellular carcinoma (HCC). This study identifies potential TSGs in HCCs using methylation profiling and pharmacological unmasking of methylated TSGs.. Methylation profiling was performed on 22 pairs of HCCs and their corresponding noncancerous liver tissues using the Infinium HumanMethylation27 BeadChip. We also determined the gene reexpression after treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) in 5 HCC cell lines.. We selected CpGs that exhibited a significant increase in methylation in HCC tissues compared with that of the noncancerous control group. Two hundred and thirteen CpGs on different gene promoters with a mean difference in the β value ≥0.15 and a value of p < 0.05 were selected. Of the 213 genes, 45 genes were upregulated in 3 or more HCC cell lines with multiplier value of differences ≥2.0 after 5-Aza-dC and TSA treatment.. We identified several potential TSGs that participate in transcription inactivation through epigenetic interactions in HCC. The results of this study are important for the understanding of functionally important epigenetic alterations in HCC.

Topics: Aged; Azacitidine; Biopsy, Needle; Carcinoma, Hepatocellular; Case-Control Studies; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hydroxamic Acids; Liver Neoplasms; Male; Middle Aged; Reference Values; Sampling Studies; Tumor Cells, Cultured

Differential expression of long non-coding RNAs (lncRNAs) plays critical roles in hepatocarcinogenesis. Considerable attention has focused on the antitumor effect of histone deacetylase inhibitor (Trichostatin A, TSA) as well as the coding gene expression-induced apoptosis of cancer cells. However, it is not known whether lncRNA has a role in TSA-induced apoptosis of human hepatocellular carcinoma (HCC) cells. The global expression of lncRNAs and coding genes was analyzed with the Human LncRNA Array V2.0 after 24 h treatment. Expression was verified in cell lines and tissues by quantitative real-time PCR. The data showed that 4.8% (959) of lncRNA and 6.1% (1849) of protein coding gene were significantly differentially expressed. The differential expressions of lncRNA and protein coding genes had distinguishable hierarchical clustering expression profiling pattern. Among these differentially expressed lncRNAs, the greatest change was noted for uc002mbe.2, which had more than 300 folds induction upon TSA treatment. TSA selectively induced uc002mbe.2 in four studied HCC cell lines. Compared with normal human hepatocytes and adjacent noncancerous tissues, uc002mbe.2 expression level was significantly lower in the HCC cell lines and liver cancer tissues. The TSA-induced uc002mbe.2 expression was positively correlated with the apoptotic effect of TSA in HCC cells. In addition, knockdown the expression of uc002mbe.2 significantly reduced TSA-induced apoptosis of Huh7cells. Therefore, TSA-induced apoptosis of HCC cells is uc002mbe.2 dependent and reduced expression of uc002mbe.2 may be associated with liver carcinogenesis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Hydroxamic Acids; Liver Neoplasms; RNA, Long Noncoding

To gain insight into the role of peptidylarginine deiminase type 4 (PADI4), we determined the relationship between PADI4 mRNA and global hypomethylation during HBV exposure in hepatocarcinogenesis. We analyzed Line-1 methylation by MSP, and CD133 mRNA by real-time PCR in 74 HCC. The HCC cancer lines (7721, Huh7, and Hep-G2) were treated with 5-Aza-CdR and TSA. PADI4 mRNA were lower in HCC tissues (Mean(-∆Ct) = 1.41) than that in Non-Hcc tissues (Mean(-∆Ct) = 3.10). Expression of PADI4 was elevated in only 20 (27 %) of the 74 HCC patients but decreased in 54 (73 %) of the patients. The declined PADI4 mRNA was significantly associated with Line-1 demethylation in HCC patients. PADI4 mRNA were lower in HCC patients with Line-1 ∆MI <-0.15 (Mean(-∆∆Ct) = -2.66) than those with Line-1 ∆MI >= -0.15 (Mean(-∆∆Ct) = -1.02). The results suggested that HCC showing hypomethylation of Line-1 is considered to be silencing PADI4 mRNA. And the lower PADI4 mRNA was also found in HCC patients with HBV >= 10(5) (copy/ml) than those with HBV < 10(5) (copy/ml). After treated by 5-Aza-CdR and TSA, we found that PADI4 mRNA induced significantly by TSA in Huh7 and Hep-G2 cells, but not in 7721 cells. Meanwhile, PADI4 mRNA induced by the combination of 5-Aza-CdR and TSA in HCC cells, and it could no effect for exposure to 5-Aza-CdR alone. The results suggested that decreased PADI4 mRNA is associated with global hypomethylation in HCC during HBV exposure.

Topics: Adult; Aged; Azacitidine; Carcinoma, Hepatocellular; Cell Line, Tumor; Decitabine; DNA Methylation; DNA Modification Methylases; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatitis B; Hepatitis B virus; Histone Deacetylase Inhibitors; Host-Pathogen Interactions; Humans; Hydrolases; Hydroxamic Acids; Immunohistochemistry; Liver Neoplasms; Long Interspersed Nucleotide Elements; Male; Middle Aged; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Analysis

Growing evidence indicates that some tumor suppressive miRNAs are subject to epigenetic modifications during carcinogenesis. Here, we found that a large miRNA cluster of C19MC was upregulated in HCC cells after combined treatment with DNA methylation inhibitor and histone deacetylase inhibitor. MiR-517a and miR-517c were strikingly different from the remaining 41 miRNAs in C19MC. Ectopic expression of MiR-517a and miR-517c inhibited cell proliferation by blocking G2/M transition, whereas down-regulation of miR-517a and miR-517c facilitated cell growth. We further showed Pyk2 is a target of miR-517a and miR-517c and both the miRNAs are downregulated in HCC samples. These data collectively suggest that down-regulation of both miR-517a and miR-517c contribute to HCC development through regulating Pyk2.

Topics: Azacitidine; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA Modification Methylases; Down-Regulation; Focal Adhesion Kinase 2; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; MicroRNAs; Multigene Family; Oligonucleotide Array Sequence Analysis; RNA Interference; Transcriptome

Non-small-cell lung cancer (NSCLC) represents approximately 80% of all types of lung cancer. Here, we report the chemotherapeutic effect of honokiol, a phytochemical from Magnolia grandiflora, on NSCLC cells and the molecular mechanisms underlying these effects using in vitro and in vivo models. Treatment of NSCLC cells (A549, H1299, H460 and H226) with honokiol (20, 40 and 60 µM) inhibited histone deacetylase (HDAC) activity, reduced the levels of class I HDAC proteins and enhanced histone acetyltransferase activity in a dose-dependent manner. These effects of honokiol were associated with a significant reduction in the viability of NSCLC cells. Concomitant treatment of cells with a proteasome inhibitor, MG132, prevented honokiol-induced degradation of class I HDACs, suggesting that honokiol reduced the levels of HDACs in NSCLC cells through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of NSCLC cells. Treatment of A549 and H1299 cells with honokiol resulted in an increase in G 1 phase arrest, and a decrease in the levels of cyclin D1, D2 and cyclin dependent kinases. Further, administration of honokiol by oral gavage significantly inhibited the growth of subcutaneous A549 and H1299 tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death and marked inhibition of class I HDACs proteins and HDAC activity in the tumor xenograft tissues. Together, our study provides new insights into the role of class I HDACs in the chemotherapeutic effects of honokiol on lung cancer cells.

Topics: Acetylation; Animals; Biphenyl Compounds; Bronchi; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Epithelial Cells; G1 Phase Cell Cycle Checkpoints; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Hydroxamic Acids; Lignans; Liver Neoplasms; Mice; Mice, Nude; Proteasome Endopeptidase Complex; Proteolysis; Valproic Acid; Xenograft Model Antitumor Assays

An exciting recent approach to targeting transcription factors in cancer is to block formation of oncogenic complexes. We investigated whether interfering with the interaction of the transcription factor SALL4, which is critical for leukemic cell survival, and its epigenetic partner complex represents a novel therapeutic approach. The mechanism of SALL4 in promoting leukemogenesis is at least in part mediated by its repression of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) through its interaction with a histone deacetylase (HDAC) complex. In this study, we demonstrate that a peptide can compete with SALL4 in interacting with the HDAC complex and reverse its effect on PTEN repression. Treating SALL4-expressing malignant cells with this peptide leads to cell death that can be rescued by a PTEN inhibitor. The antileukemic effect of this peptide can be confirmed on primary human leukemia cells in culture and in vivo, and is identical to that of down-regulation of SALL4 in these cells using an RNAi approach. In summary, our results demonstrate a novel peptide that can block the specific interaction between SALL4 and its epigenetic HDAC complex in regulating its target gene, PTEN. Furthermore, targeting SALL4 with this approach could be an innovative approach in treating leukemia.

Topics: Animals; Carcinoma, Hepatocellular; Drug Design; Endometrial Neoplasms; Epigenesis, Genetic; Female; Gene Expression Regulation, Leukemic; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia, Monocytic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Liver Neoplasms; Male; Mice; Mice, Inbred NOD; Mice, SCID; Peptide Fragments; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Transcription Factors; Xenograft Model Antitumor Assays

Histone deacetylation regulates chromatin remodeling and transcriptional down-regulation of specific genomic regions; it is altered in many types of cancer cells. We searched for microRNAs (miRs) that are affected by histone deacetylation and investigated the effects in hepatocellular carcinoma (HCC) cells.. HCC cell lines (HepG2, HLE, HLF, and Huh7) and immortalized liver cell lines (THLE-2 and THLE-3) were incubated with the histone deacetylase inhibitor trichostatin A. Differentially expressed messenger RNAs (mRNAs) and miRs were identified by expression profiling. Small interfering RNAs were used to reduce levels of histone deacetylases (HDAC)1-3, and HCC cell lines were transfected with miR-449. We evaluated growth of xenograft tumors from modified cells in nude mice. Cells were analyzed by immunoblot and luciferase reporter assays. We analyzed HCC samples from 23 patients.. HDAC1-3 were up-regulated in HCC samples from patients. In cell lines, inhibition of HDAC significantly increased levels of hsa-miR-449a. c-MET mRNA, which encodes the receptor tyrosine kinase for hepatocyte growth factor, is a target of miR-449. Incubation of HCC cells with trichostatin A or transfection with miR-449 reduced expression of c-MET and phosphorylation of extracellular signal-regulated kinases 1 and 2 (downstream effectors of c-MET), increased apoptosis, and reduced proliferation. Huh-7 cells transfected with miR-449 formed tumors more slowly in mice than cells expressing control miRs. HCC samples from patients had lower levels of miR-449 and higher levels of c-MET than human reference.. In HCC cells, up-regulation of HDAC1-3 reduces expression of miR-449. miR-449 binds c-MET mRNA to reduce its levels, promoting apoptosis and reducing proliferation of liver cells. Expression of miR-449 slows growth of HCC xenograft tumors in mice; this miR might function as a tumor suppressor.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Binding Sites; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Reporter; HEK293 Cells; Hep G2 Cells; Hepatocyte Growth Factor; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Liver Neoplasms; Mice; Mice, Nude; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Proto-Oncogene Proteins c-met; RNA Interference; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Tumor Burden

DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

Topics: Azacitidine; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Down-Regulation; Female; Hep G2 Cells; Humans; Hydroxamic Acids; Liver; Liver Neoplasms; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Survival Analysis

The synthetic retinoid fenretinide is one of the most promising clinically tested retinoids. Previously, we have shown that fenretinide induces apoptosis of Huh7 cells, but HepG2 cells are relatively resistant to fenretinide-induced apoptosis. This study examines the interactive role of fenretinide and histone deacetylase inhibitors (HDACi) in inducing apoptosis of human hepatocellular carcinoma (HCC) cells and the underlying mechanism. Trichostatin A and scriptaid can either enhance fenretinide-induced apoptosis in the fenretinide sensitive HCC cells (Huh7 and Hep3B) or sensitize the fenretinide resistant cells (HepG2) to become sensitive to the apoptotic effect of fenretinide in a cancer cell-specific manner. The sensitivity of cells to fenretinide-induced apoptosis was not associated with reactive oxygen species production nor with antioxidant gene expression. However, the level of retinoic acid receptor β (RARβ) and Nur77 (NR4A1) was important for inducing apoptosis. Upon fenretinide and HDACi treatment, the expression of RARβ and Nur77 were induced and colocalized in the cytosol. The induction of Nur77 protein level, but not the messenger RNA level, was RARβ-dependent. In addition, RARβ interacted with Nur77. Nur77 was essential for fenretinide-induced and HDACi-induced apoptosis of Huh7 cells. Induction of the expression, the interaction, and the nuclear export of RARβ and Nur77 mediate fenretinide-induced and HDACi-induced apoptosis.. Our findings suggest that targeting Nur77 and RARβ simultaneously provides an effective way to induce HCC cell death.

Topics: Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Fenretinide; Hep G2 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Hydroxylamines; Liver Neoplasms; Nuclear Receptor Subfamily 4, Group A, Member 1; Quinolines; Reactive Oxygen Species; Receptors, Retinoic Acid; RNA, Messenger

Agents commonly used in cancer chemotherapy rely on the induction of cell death via apoptosis, mitotic catastrophe, premature senescence and autophagy. Chemoresistance is the major factor limiting long-term treatment success in patients with hepatocellular carcinoma (HCC). Recent studies have revealed that the hepatitis B virus X protein (HBx) exerts anti-apoptotic effects, resulting in an increased drug resistance in HCC cells. In this study, we showed that etoposide treatment activated caspase-8 and caspase-3, leading to cleavages of p53, Bid and PARP, which subsequently induced apoptosis. Furthermore, p53 and Bid were accumulated in cytoplasm following etoposide treatment. However, HBx significantly attenuated etoposide-induced cell death. In HBx-expressing cells, despite the translocation of p53 and Bid to cytoplasm, the activation of caspases was inhibited. Furthermore, the phosphorylation of extracellular-signal-regulated kinase (ERK) was markedly increased in HBx-expressing cells. Moreover, the pretreatment with trichostatin A (TSA, a histone deacetylase inhibitor) or TSA in combination with etoposide significantly sensitized HCC cells to apoptosis by inhibiting ERK phosphorylation, reactivating caspases and PARP, and inducing translocation of p53 and Bid to cytoplasm. Collectively, HBx reduces the sensitivity of HCC cells to chemotherapy. TSA in combination with etoposide can significantly overcome the increased resistance of HBx-expressing HCC cells to chemotherapy.

Topics: BH3 Interacting Domain Death Agonist Protein; Caspases; Cell Death; Cell Nucleus; Drug Screening Assays, Antitumor; Enzyme Activation; Etoposide; Extracellular Signal-Regulated MAP Kinases; Green Fluorescent Proteins; Hep G2 Cells; Humans; Hydroxamic Acids; Liver Neoplasms; Phosphorylation; Protein Transport; Trans-Activators; Tumor Suppressor Protein p53; Viral Regulatory and Accessory Proteins

Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas.. SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions.. We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases.. Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis.

Topics: Adult; Aged; Aged, 80 and over; Azacitidine; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Methylation; DNA Modification Methylases; DNA Mutational Analysis; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Liver Neoplasms; Male; Middle Aged; Molecular Sequence Data; Promoter Regions, Genetic; Repressor Proteins; Restriction Mapping; Transcription, Genetic; Young Adult; Zinc Finger E-box Binding Homeobox 2

To investigate the role of hepatopoietin Cn (HPPCn) in apoptosis of hepatocellular carcinoma (HCC) cells and its mechanism.. Two human HCC cell lines, SMMC7721 and HepG2, were used in this study. Immunostaining, Western blotting and enzyme linked immunosorbent assay were conducted to identify the expression of HPPCn and the existence of an autocrine loop of HPPCn/HPPCn receptor in SMMC7721 and HepG2. Apoptotic cells were detected using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide.. The HPPCn was highly expressed in human HCC cells and secreted into culture medium (CM). FITC-labeled recombinant human protein (rhHPPCn) could specifically bind to its receptor on HepaG2 cells. Treatment with 400 ng/mL rhHPPCn dramatically increased the viability of HCC-derived cells from 48.1% and 36.9% to 85.6% and 88.4%, respectively (P < 0.05). HPPCn silenced by small-interfering RNA reduced the expression and secretion of HPPCn and increased the apoptosis induced by trichostatin A. Additionally, HPPCn could up-regulate the expression of myeloid cell leukemia-1 (Mcl-1) in HCC cells via mitogen-activated protein kinase (MAPK) and sphingosine kinase-1.. HPPCn is a novel hepatic growth factor that can be secreted to CM and suppresses apoptosis of HCC cells by up-regulating Mcl-1 expression.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Hepatocyte Growth Factor; Humans; Hydroxamic Acids; Liver Neoplasms; Mitogen-Activated Protein Kinase Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Phosphotransferases (Alcohol Group Acceptor); Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-met; Recombinant Proteins; Up-Regulation

The xenobiotic metabolizing enzymes Cyp1a1 and Cyp1b1 can be induced by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-rho-dioxin (dioxin) via the aryl hydrocarbon receptor (AhR). These genes are differentially induced by dioxin in different mouse cell lines. In the mouse hepatoma cell line Hepa1c1c7 (Hepa-1), the Cyp1a1 gene is induced to very high levels by dioxin, but the levels of Cyp1b1 mRNA are extremely low and are not inducible by dioxin. The reverse is the case for the mouse embryonic fibroblast cell line C3H10T1/2, in which Cyp1b1 is induced to very high levels by dioxin, but the levels of Cyp1a1 mRNA are extremely low and not inducible by dioxin. However, dioxin treatment leads to the recruitment of AhR to the enhancer regions of both genes in both cell lines. Somatic cell hybrid clones generated between the two cell lines display high levels of induction of both genes in response to dioxin. Strong reactivation of the Cyp1a1 gene was also observed in C3H10T1/2 cell line after treatment with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (5-AzadC) and the histone deacetylase inhibitor, trichostatin-A (TSA). However, only modest reactivation of Cyp1b1 was observed in Hepa-1 cells after 5-AzadC or TSA treatment. These data demonstrate that the presence or absence of binding of AhR to regulatory regions is not responsible for determining the differences in levels of induction of the two genes in these cell lines and indicate that DNA methylation plays a major role in silencing of Cyp1a1 gene expression in C3H10T1/2 cells, but appears to play only a minor role in silencing Cyp1b1 gene expression in Hepa-1 cells, which likely occurs principally because Hepa-1 cells lack a factor required for high levels of induction of this gene.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Azacitidine; Carcinoma, Hepatocellular; Cell Line; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Decitabine; Dioxins; Enzyme Induction; Fibroblasts; Gene Expression Regulation, Enzymologic; Hybrid Cells; Hydroxamic Acids; Liver Neoplasms; Mice; Receptors, Aryl Hydrocarbon; RNA, Messenger

The incidence of hepatocellular carcinoma is rising due to alcohol drinking, hepatitis C viral infection and metabolic syndrome. Differential expression of CYP2E1 may play a pleiotropic role in the multistep process of liver carcinogenesis. Considerable attention has focused on the antitumor effect of trichostatin A (TSA) as well as CYP2E1 expression-induced apoptosis of cancer cells. However, very few studies have examined the mechanisms by which TSA has an antitumor effect and its association to CYP2E1 expression. The current study examined the action of TSA on CYP2E1 expression and the role of CYP2E1 in inducing apoptosis of HepG2 cells. Our data showed that TSA selectively induced CYP2E1 in four studied human hepatocellular carcinoma (HCC) cell lines (Huh7, PLC/PRF/5, Hep3B and HepG2), but not in normal primary human hepatocytes. TSA-mediated up-regulation of CYP2E1 expression was associated with histone H3 acetylation and the recruitment of HNF-1 and HNF-3beta to the CYP2E1 promoter in HepG2 cells. siRNA-mediated knockdown experiments showed that TSA-induced caspase-3 cleavage was decreased due to reduced expression of CYP2E1 in HepG2 cells. Moreover, down-regulation of CYP2E1 was accompanied by decreased production of mitochondrial reactive oxygen species. These results suggest that histone modification is involved in CYP2E1 gene expression and that CYP2E1-dependent mitochondrial oxidative stress plays a role in TSA-induced apoptosis.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Caspase 3; Cell Line; Cell Line, Tumor; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP2E1 Inhibitors; DNA Primers; Enzyme Induction; Gene Expression; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 3-beta; Hepatocytes; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; Oxidative Stress; RNA, Small Interfering

S100A6 has been implicated in a variety of biological functions as well as tumorigenesis. In this study, we investigated the expression status of S100A6 in relation to the clinicopathological features and prognosis of patients with gastric cancer and further explored a possible association of its expression with epigenetic regulation. S100A6 expression was remarkably increased in 67.5% of gastric cancer tissues as compared with matched noncancerous tissues. Statistical analysis demonstrated a clear correlation between high S100A6 expression and various clinicopathological features, such as depth of wall invasion, positive lymph node involvement, liver metastasis, vascular invasion, and tumor-node metastasis stage (P < 0.05 in all cases), as well as revealed that S100A6 is an independent prognostic predictor (P = 0.026) significantly related to poor prognosis (P = 0.0004). Further exploration found an inverse relationship between S100A6 expression and the methylation status of the seventh and eighth CpG sites in the promoter/first exon and the second to fifth sites in the second exon/second intron. In addition, the level of histone H3 acetylation was found to be significantly higher in S100A6-expressing cancer cells. After 5-azacytidine or trichostatin A treatment, S100A6 expression was clearly increased in S100A6 low-expressing cells. In conclusion, our results suggested that S100A6 plays an important role in the progression of gastric cancer, affecting patient prognosis, and is up-regulated by epigenetic regulation.

Topics: Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Azacitidine; Base Sequence; Cell Cycle Proteins; Cell Line, Tumor; DNA Methylation; Epigenesis, Genetic; Female; Humans; Hydroxamic Acids; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Prognosis; Promoter Regions, Genetic; Protein Synthesis Inhibitors; S100 Calcium Binding Protein A6; S100 Proteins; Stomach Neoplasms; Up-Regulation

The retinoblastoma-interacting zinc finger gene RIZ1 is inactivated in many cancers, but the underlying mechanisms remain unknown. This study aimed to investigate the epigenetic mechanisms of RIZ1 inactivation by analyzing the relationship between DNA methylation and histone modifications during regulation of RIZ1 expression.. Methylation-specific PCR, RT-PCR, and immunohistochemistry were performed to examine RIZ1 methylation and expression. Dynamic changes in histone H3 lysine 9 (H3K9) modifications and histone deacetylases (HDACs) associated with the promoter were analyzed by chromatin immunoprecipitation (ChIP).. RIZ1 methylation was detected in 66.7% (32/48) HCC tissues, 6.3% (3/48) corresponding non-cancerous tissues, and 66.7% (4/6) HCC cell lines. All 32 HCC tissues with promoter methylation showed complete loss of RIZ1 protein, whereas RIZ1 protein was present in all the corresponding non-cancerous tissues. Neither 5-aza-2-deoxycitidine (5-Aza-dC) nor Trichostatin A (TSA) reversed promoter methylation, but did restore RIZ1 mRNA and resulted in the downregulation of HDAC1 but not HDAC3. However, 5-Aza-dC+TSA induced a partial reversal of promoter methylation and a markedly synergistic reactivation of RIZ1. Moreover, both HDAC1 and HDAC3 were downregulated. The ChIP assays showed 5-Aza-dC and/or TSA also contributed to the dynamic conversion of trimethylated to acetylated H3K9 at the promoter. Furthermore, a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation.. Our results suggest that promoter methylation and H3K9 modifications work together to silence the RIZ1 gene in HCC. 5-Aza-dC can restore the expression of RIZ1, as reflected by its effects on histone modification levels. This finding indicates that cooperative effects between these epigenetic modifications exist.

Topics: Acetylation; Azacitidine; Carcinoma, Hepatocellular; Cell Line, Tumor; Decitabine; DNA Methylation; DNA-Binding Proteins; Epigenesis, Genetic; Genes, Tumor Suppressor; Histone-Lysine N-Methyltransferase; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; Nuclear Proteins; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors

Scavenger receptor class B type I (SR-BI) and its human homologue CLA-1 plays an important role in reverse cholesterol transport (RCT). Using a previously established cell-based CLA-1 up-regulator screening assay, one of the positive strains, 04-9179, presented potent activity in elevating CLA-1 transcriptional level. We report here the identification of an active compound 9179A as a known compound trichostatin A (TSA), and its effects on CLA-1/SR-BI expression both in HepG2 human hepatoma cells and RAW 264.7 murine macrophage cells in vitro. The results showed that the mRNA and protein level of CLA-1/SR-BI were significantly up-regulated by 9179A both in HepG2 and RAW 264.7 cells. Corresponding to this, the uptake of DiI-HDL by both cells and the efflux of [(3)H]cholesterol by RAW 264.7 cells were increased by 9179A in dose-dependent manner. ABCA1 was also increased but SR-A decreased by 9179A in RAW 264.7 cells. Using a combination of reporter assays with various deletion in CLA-1 promoter and electrophoretic mobility shift assay, we demonstrated that -419/-232 bp fragment of the CLA-1 promoter mediated the effects of 9179A (i.e., TSA). Together, these studies identified TSA as a novel up-regulator of CLA-1/SR-BI both in HepG2 and RAW 264.7 cells.

Topics: Animals; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Cell Line; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Liver Neoplasms; Macrophages; Mice; Promoter Regions, Genetic; RNA, Messenger; Scavenger Receptors, Class A; Scavenger Receptors, Class B; Transcriptional Activation; Transfection; Up-Regulation

T-cadherin is an atypical cadherin and growing evidence has indicated that T-cadherin exerts tumor-suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T-cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T-cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T-cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT-PCR analysis also showed that the T-cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32-38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5-aza-2'-deoxycytidine or histone deacetylase inhibitor trichostatin A, T-cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T-cadherin induced G(2)/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage-independent growth in soft agar and increased sensitivity to TNFalpha-mediated apoptosis in HCC cells. Intriguingly, we found that T-cadherin significantly suppressed the activity of c-Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T-cadherin was differentially expressed in human HCCs. The underexpression of T-cadherin in HCC cells suggests it may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC development.

Topics: Acetylation; Adult; Aged; Anticarcinogenic Agents; Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Blotting, Western; Cadherins; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Decitabine; DNA Methylation; DNA Modification Methylases; Down-Regulation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Liver Neoplasms; Loss of Heterozygosity; Male; Middle Aged; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

The ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a carboxyl-terminal ubiquitin hydrolase regulating cellular ubiquitin levels, recently suggested as a tumor suppressor. However, the role of UCHL1 in hepatocellular carcinoma (HCC) is not clear. We investigated the expression and DNA methylation of the UCHL1 in primary HCC, liver metastases from digestive carcinomas, and primary digestive cancers. UCHL1 is expressed in all normal tissues and immortalized normal epithelial cell lines, but was low or silenced in 77% (10/13) of HCC cell lines, which is well correlated with its promoter methylation status. Methylation was further detected in 44% (12/27) of HCCs, but less in metastatic tumors generated from colorectal and stomach in the liver (19%, 3/16; P < 0.05). Methylation was also detected in primary digestive tumors, including 71% (22/31) of colon, 77% (53/69) of gastric, and 40% (18/45) of esophageal carcinomas, but none or occasionally in paired adjacent nontumor tissues. Detailed methylation analysis of 49 CpG sites at a 540-bp promoter region by bisulfite genomic sequencing confirmed the methylation. UCHL1 silencing could be reversed by chemical or genetic demethylation of the promoter, indicating direct epigenetic silencing. Restoring UCHL1 expression in silenced cell lines significantly inhibited their growth and colony formation ability by inhibiting cell proliferation, causing cell cycle arrest in G2/M phase and inducing apoptosis through the intrinsic caspase-dependent pathway. Moreover, UCHL1 directly interacts with p53 and stabilizes p53 through the ubiquitination pathway.. Epigenetic inactivation of UCHL1 is common in primary HCCs and other digestive tumors. UCHL1 appears to be a functional tumor suppressor involved in the tumorigenesis of HCCs and other digestive cancers.

Topics: Azacitidine; Biomarkers, Tumor; Carcinoma, Hepatocellular; Caspases; Cell Division; Cell Line, Tumor; Cell Proliferation; CpG Islands; Decitabine; Digestive System Neoplasms; DNA Methylation; Epigenesis, Genetic; G2 Phase; Gene Silencing; Humans; Hydroxamic Acids; Liver Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Transfection; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ubiquitin Thiolesterase

This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Benzothiazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Damage; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Toluene; Tumor Suppressor Protein p53; Vorinostat

We have been using polarized hepatic WIF-B cells to examine ethanol-induced liver injury. Previously, we determined microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells. We proposed that the ethanol-induced alterations in microtubule dynamics may explain the ethanol-induced defects in membrane trafficking that have been previously documented. To test this, we compared the trafficking of selected proteins in control cells and cells treated with ethanol or with the histone deacetylase 6 inhibitor trichostatin A (TSA). We determined that exposure to 50 nM TSA for 30 minutes induced microtubule acetylation ( approximately 3-fold increase) and stability to the same extent as did ethanol. As shown previously in situ, the endocytic trafficking of the asialoglycoprotein receptor (ASGP-R) was impaired in ethanol-treated WIF-B cells. This impairment required ethanol metabolism and was likely mediated by acetaldehyde. TSA also impaired ASGP-R endocytic trafficking, but to a lesser extent. Similarly, both ethanol and TSA impaired transcytosis of the single-spanning apical resident aminopeptidase N (APN). For both ASGP-R and APN and for both treatments, the block in trafficking was internalization from the basolateral membrane. Interestingly, no changes in transcytosis of the glycophosphatidylinositol-anchored protein, 5'-nucleotidase, were observed, suggesting that increased microtubule acetylation and stability differentially regulate internalization. We further determined that albumin secretion was impaired in both ethanol-treated and TSA-treated cells, indicating that increased microtubule acetylation and stability also disrupted this transport step.. These results indicate that altered microtubule dynamics explain in part alcohol-induced defects in membrane trafficking.

Topics: Acetylation; Animals; Carcinoma, Hepatocellular; Ethanol; Histone Deacetylase Inhibitors; Hydroxamic Acids; Kinetics; Liver; Liver Neoplasms; Microtubules; Proteins; Rats; Serum Albumin

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells.. Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.

Topics: Azacitidine; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Expression Profiling; Gene Silencing; Genes, Tumor Suppressor; Glycoproteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Liver Neoplasms; Neoplasm Invasiveness; Promoter Regions, Genetic; Serine Proteinase Inhibitors

DNA methylation and histone modifications have emerged as key mechanisms in transcriptional regulation. The target of methylation-induced silencing 1 (TMS1) is a bipartite protein. Recent studies have indicated that methylation-associated silencing of TMS1 occurs in many cancers. However, whether and how TMS1 is regulated by epigenetic mechanisms in cancers remains unknown. In this study we showed that methylation of the TMS1 promoter occurred in five of six hepatocellular carcinoma (HCC) cell lines. TMS1 expression was reduced in four HCC cell lines and correlated with methylation status. Furthermore, the TMS1 promoter was completely methylated and mRNA expression was undetectable. TMS1 expression could be restored by 5-aza-2'-deoxycitidine (5-Aza-dC) (a DNA methyltransferase inhibitor) or trichostatin A (TSA) (a histone deacetylase inhibitor) alone and the promoter methylation was partially reversible. TSA was more efficient than 5-Aza-dC in inducing TMS1 expression, and the combination of 5-Aza-dC and TSA resulted in markedly synergistic reactivation of the gene and completely reversed promoter methylation. Interestingly, TMS1 promoter methylation-associated gene silencing was accompanied by histone H3 Lysine 9 (H3K9) hypoacetylation and trimethylation. 5-Aza-dC and/or TSA also had some effect on conversion of methylated to acetylated H3K9 in restoring TMS1. This conversion was dynamic at the TMS1 promoter and a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation after 5-Aza-dC and/or TSA treatment. Our results thus suggest that epigenetic inactivation of TMS1 expression is regulated by promoter hypermethylation and H3K9 modifications in a coordinated way.

Topics: Acetylation; Antimetabolites, Antineoplastic; Azacitidine; Carcinoma, Hepatocellular; CARD Signaling Adaptor Proteins; Cell Line, Tumor; Cytoskeletal Proteins; Decitabine; DNA Methylation; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; Methylation; Promoter Regions, Genetic; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic

Multiple regions on the chromosome arm 3p are frequently affected by loss of heterozygosity in human cancers. A candidate tumor suppressor gene is TMEM7, at 3p21.3, which encodes a transmembrane protein. TMEM7 is expressed specifically in the liver, and the encoded protein shares substantial sequence homology with human and mouse 28-kDa interferon-alpha (IFN-alpha) responsive protein. In investigation of the possible role of TMEM7 in development of hepatocellular carcinoma (HCC), we examined TMEM7 expression in 20 primary HCC and 18 HCC cell lines and found recurrent functional alterations. Although TMEM7 mRNA was expressed in normal hepatic cells, downregulation or inactivation of the gene was detected in 85% of primary HCC and 33% of HCC cell lines. To identify the mechanisms responsible, we examined genomic deletion and mutation, and also the effect of inhibitors of DNA methyltransferase and histone deacetylase on cells with low or no endogenous TMEM7 expression. Homozygous deletion of TMEM7 was not detected in 17 pairs of human HCC and corresponding noncancerous liver tissues, nor in any of the 18 HCC cell lines. TMEM7 mutation was not detected in the 18 HCC cell lines (low or normal TMEM7 expression). Treatment of two of six cell lines exhibiting downregulation or loss of TMEM7 with 5-aza-2'-deoxycytidine and trichostatin A yielded additive increase in TMEM7 expression, implicating aberrant DNA methylation and histone deacetylation in transcriptional silencing of this gene. Ectopic expression of TMEM7 in two TMEM7-deficient HCC lines suppressed cell proliferation, colony formation, and cell migration in vitro and reduced tumor formation in nude mice. Treatment of two highly invasive HCC cell lines with IFN-alpha for 7 days significantly increased TMEM7 expression and inhibited cell migration. These findings implicate loss of TMEM7 expression in hepatocarcinogenesis and suggest that modification of TMEM7 expression by IFN-alpha may have therapeutic relevance in a subset of HCC.

Topics: Amino Acid Sequence; Animals; Azacitidine; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Interferon-alpha; Liver Neoplasms; Male; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Invasiveness; Promoter Regions, Genetic; Sequence Homology, Amino Acid

Inhibition of cholesterol 7alpha-hydroxylase (CYP7A1) by bile acids and inflammatory cytokines provides an important mechanism to protect hepatocytes from bile acid toxicity during cholestasis. Transforming growth factor beta1 (TGFbeta1) released by hepatic stellate cells during chronic liver injury plays a critical role in liver inflammation and fibrogenesis. The objective of this study is to investigate the role of TGFbeta1 in hepatic bile acid synthesis.. mRNA expressions in primary human hepatocytes and HepG2 cells were measured by quantitative real-time polymerase chain reaction. Reporter assay, glutathione-S-transferase pull-down assay, adenovirus-mediated gene transduction, and chromatin immunoprecipitation assay were used to study the mechanism of TGFbeta1 regulation of CYP7A1 gene transcription.. TGFbeta1 inhibited the mRNA expression of CYP7A1 and bile acid synthesis in HepG2 cells and primary human hepatocytes. Mothers against decapentaplegic homolog (Smad3) inhibited both CYP7A1 promoter activity and mRNA expression by inhibiting DNA-binding activity of hepatocyte nuclear factor 4alpha (HNF4alpha). The histone deacetylase (HDAC) inhibitor Tricostatin A partially blocked the TGFbeta1 inhibition of CYP7A1 mRNA expression, whereas TGFbeta1 decreased histone 3 acetylation in the CYP7A1 chromatin. TGFbeta1 treatment and adenovirus Smad3 reduced HNF4alpha binding but increased the recruitment of Smad3, HDAC1, and a repressor mSin3A to the CYP7A1 chromatin.. This study provides the first evidence that TGFbeta1 represses CYP7A1 gene transcription in human hepatocytes by a mechanism involving Smad3-dependent inhibition of HNF4alpha and HDAC remodeling of CYP7A1 chromatin. The TGFbeta1/Smad3 signaling may reduce bile acid synthesis in the liver and prevent hepatocyte injury in cholestatic liver disease.

Topics: Bile Acids and Salts; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Cholesterol 7-alpha-Hydroxylase; Enzyme Inhibitors; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Hydroxamic Acids; Liver Neoplasms; RNA, Messenger; Signal Transduction; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta1

The human class I alcohol dehydrogenase (ADH) genes (ADH1A, ADH1B, and ADH1C) differ in expression during development and in various tissues. They are repressed in the HepG2 human hepatoma cell line. We hypothesized that epigenetic modifications play a role in this repression and that class I ADH gene expression would be enhanced upon global inhibition of DNA methylation and histone deacetylation.. Southern blotting was used to assess the methylation status of each class I ADH gene. HepG2 and HeLa cells were treated with either the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the histone deacetylase inhibitor Trichostatin A (TSA), or both in combination, and class I ADH gene expression was analyzed. Chromatin immunoprecipitation assays were performed to analyze histone H3 acetylation. Transient transfections and gel mobility shift assays were used to analyze the role that methylation plays in inhibiting transcription factor binding and promoter function.. We show that the upstream regions of ADH1A, ADH1B, and ADH1C are methylated in HepG2 cells. 5-Aza-2'-deoxycytidine treatment enhanced expression of both ADH1B and ADH1C. Trichostatin A treatment elevated expression of ADH1C. ADH1A expression was not stimulated by either 5-aza-dC or TSA. H3 histones associated with a methylated upstream region of ADH1B were hyperacetylated in TSA-treated, but not in 5-aza-dC-treated, HepG2 cells. A methylated upstream region of ADH1C achieved histone H3 hyperacetylation upon either 5-aza-dC or TSA treatment. Methylation of the ADH1B proximal promoter in vitro decreased its activity to 54% and inhibited the binding of the upstream stimulatory factor.. These findings suggest that the class I ADH genes are regulated by epigenetic mechanisms in human hepatoma cells. The temporal and tissue-specific expression of these genes may in part result from differences in epigenetic modifications and the availability of key transcription factors.

Topics: Alcohol Dehydrogenase; Azacitidine; Carcinoma, Hepatocellular; Cell Line, Tumor; Decitabine; DNA Methylation; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation, Enzymologic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Liver Neoplasms; Promoter Regions, Genetic

We examined expression of maspin and the epigenetic status of its gene in 40 primary hepato-biliary tract carcinomas and 11 cell lines originating from hepato-pancreatico-biliary tract carcinomas. Aberrant maspin expression was frequently observed immunohistochemically in biliary tract carcinomas (22/25, 88%) but not in hepatocellular carcinomas (HCCs) (0/15, 0%). Aberrant maspin expression by five pancreatico-biliary tract carcinoma cell lines was closely associated with demethylation at the maspin promoter. Five of six HCC cell lines were maspin-negative and exhibited extensive hypomethylation and hypoacetylation at the maspin promoter. Treatment with 5-aza-2'-deoxycytidine did not activate maspin expression in these five maspin-negative HCC cell lines, whereas treatment with Trichostatin A (TSA) activated maspin expression in two of them. Treatment with TSA increased histone acetylation in some HCC cell lines. These results suggest that aberrant maspin expression in biliary tract carcinomas is closely associated with demethylation at the promoter region, but that some HCC cell lines additionally require histone acetylation. In addition, the fact that maspin-negative HCC cell lines remain after treatment with TSA suggests the existence of other repressive factors controlling maspin expression.

Topics: Acetylation; Bile Duct Neoplasms; Carcinoma; Cell Line, Tumor; Chromatin; DNA Methylation; Epigenesis, Genetic; Female; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Pancreatic Neoplasms; Precipitin Tests; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Proteins; RNA, Messenger; Sequence Analysis, DNA; Serpins

The objective of this study was to investigate the separate and combined effects of antioxidants, trichostatin A (TSA) and their combination on the in vitro cytotoxicity of Ni2+ in human hepatoma cells. Cell viability, lactate dehydrogenase (LDH) release, and DNA fragmentation were measured as indicators of cell damage. Reactive oxygen species (ROS) generation and histone acetylation were also measured. The cytotoxicity of Ni2+ increased in a time- and dose-dependent manner. In the presence of 2 mM Ni2+, ROS generation increased 365% (p<0.05), while histone acetylation decreased 37% (p<0.05). Although antioxidants, ascorbic acid (AA, 0.5 or 1 mM), reduced glutathione (GSH, 100 or 200 microM) and N-acetyl-cysteine (NAC, 0.5 or 1 mM) strongly inhibited ROS generation, their effect on Ni2+-caused histone hypoacetylation was not so obvious. On the contrary, TSA (100 nM) showed no inhibition on ROS generation but significantly increased histone acetylation in both control and Ni2+-exposed cells. As expected, the combination of antioxidants and TSA possessed the activity of both diminishing ROS generation and increasing histone hypoacetylation caused by Ni2+. Further studies found that both antioxidants and TSA could diminish the cytotoxicity of Ni2+, and their combined effects obviously improved each of their protection roles, indicating that both ROS generation and histone hypoacetylation are involved in the cytotoxicity of Ni2+, and the combination of antioxidants and TSA may act as a useful strategy to protect against this cytotoxicity.

Topics: Acetylation; Acetylcysteine; Antioxidants; Apoptosis; Ascorbic Acid; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Glutathione; Hepatocytes; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; Nickel; Reactive Oxygen Species

Curcumin (Cur), a well-known dietary pigment derived from Curcuma longa, is a promising anticancer drug, but its in vivo target molecules remain to be clarified. Here we report that exposure of human hepatoma cells to Cur led to a significant decrease of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cur treatment resulted in a comparable inhibition of histone acetylation in the absence or presence of trichostatin A (the specific HDAC inhibitor), and showed no effect on the in vitro activity of HDAC. In contrast, the domain negative of p300 (a most potent HAT protein) could block the inhibition of Cur on histone acetylation; and the Cur treatment significantly inhibited the HAT activity both in vivo and in vitro. Thus, it is HAT, but not HDAC that is involved in Cur-induced histone hypoacetylation. At the same time, exposure of cells to low or high concentrations of Cur diminished or enhanced the ROS generation, respectively. And the promotion of ROS was obviously involved in Cur-induced histone hypoacetylation, since Cur-caused histone acetylation and HAT activity decrease could be markedly diminished by the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) or their combination, but not by their heat-inactivated forms. The data presented here prove that HAT is one of the in vivo target molecules of Cur; through inhibiting its activity, Cur induces histone hypoacetylation in vivo, where the ROS generation plays an important role. Considering the critical roles of histone acetylation in eukaryotic gene transcription and the involvement of histone hypoacetylation in the lose of cell viability caused by high concentrations of Cur, these results open a new door for us to further understand the molecular mechanism involved in the in vivo function of Cur.

Topics: Acetylation; Acetyltransferases; Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Catalase; Cell Cycle Proteins; Cell Line, Tumor; Curcumin; Dose-Response Relationship, Drug; Flow Cytometry; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; p300-CBP Transcription Factors; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Superoxide Dismutase; Time Factors; Transcription Factors

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.

Topics: Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Kidney Neoplasms; Liver Neoplasms; Nasopharyngeal Neoplasms; Neoplasms; Oligonucleotide Array Sequence Analysis; Tumor Cells, Cultured; Vorinostat

Epigenetics is the key factor in the regulation of gene expression. We conducted cDNA microarray analysis to screen for genes induced by histone deacetylase (HDAC) inhibition and examined epigenetic alterations.. Microarray analysis was performed in six hepatoma cell lines and primary hepatocytes treated with trichostatin A (TSA). mRNA expression of several genes was examined by reverse transcription-polymerase chain reaction in TSA-treated cells and hepatoma samples. Acetylated histones and methylation status in 5'CpG islands was assessed by chromatin immunoprecipitation (ChIP) assay and bisulfite genomic sequencing, respectively.. Fifty-seven genes showed greater than 2-fold change after TSA treatment in multiple cell lines. Among them, four genes including p21(WAF1) exhibited substantial induction (greater than 5-fold changes). Decreased mRNA levels of these genes in hepatoma tissues were observed in more than half of patients. ChIP assay, in general, demonstrated a good correlation between mRNA expression and histone acetylation, but only a limited correlation with the methylated DNA in the promoter region.. We identified 57 up-regulated genes by TSA treatment in hepatoma cells and some of them appeared to be cancer-related genes in hepatomas. The alterations in acetylated histones are likely closely associated with gene expression.

Topics: Acetylation; Base Sequence; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Connective Tissue Growth Factor; CpG Islands; Cyclin-Dependent Kinase Inhibitor p21; DNA Methylation; DNA Primers; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Profiling; Hepatocytes; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Up-Regulation

Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA.. To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody.. The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, alpha2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, alpha7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes.. This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromatin; Coloring Agents; DNA, Complementary; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; In Situ Nick-End Labeling; Liver Neoplasms; Oligonucleotide Array Sequence Analysis; Precipitin Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tetrazolium Salts; Thiazoles; Transcription, Genetic; Up-Regulation

Remodeling of the chromatin template by inhibition of HDAC activities represents a potential transcriptional therapy for neoplastic disease. A number of HDAC inhibitors that modulate in vitro cell growth and differentiation have been developed. We analyzed the effects of TSA, a specific and potent HDAC inhibitor, on the human hepatoma cell lines HepG2 and Huh-7. TSA increased levels of acetylated histones H3 and H4 in both HepG2 and Huh-7. It inhibited cell proliferation in vitro and induced G(0)/G(1) arrest in HepG2 and apoptosis in Huh-7. Gene expression of liver-specific functions and liver-enriched transcription factors was upregulated by TSA. TSA upregulated the ammonia removal rate and the albumin synthesis rate of HepG2 and Huh-7. Our results indicate that TSA can induce cell-cycle arrest/apoptosis and hepatocyte differentiation in human liver cancer cell lines.

Topics: Albumins; Ammonia; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Hepatocytes; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Liver Neoplasms; Neoplasm Proteins; Tumor Cells, Cultured

Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown.. The hepatoma cells HepG2, MH1C1, Hepa1-6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10(-6) to 10(-9)M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21(cip/waf), bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay.. 10(-6)M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24h, inducing a G(2)/M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l(cip/waf), bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected.. TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Proteins

Methionine adenosyltransferase (MAT), an essential enzyme that catalyzes the formation of S-adenosylmethionine (SAM), is encoded by two genes, MAT1A (liver-specific) and MAT2A (non-liver-specific). We showed a switch from MAT1A to MAT2A expression in human liver cancer, which facilitates cancer cell growth. The present work examined the role of methylation in MAT2A transcriptional regulation. We found that the human MAT2A promoter is hypomethylated in hepatocellular carcinoma, in which the gene is upregulated transcriptionally, but hypermethylated in normal liver, in which the gene is minimally expressed. Luciferase activities driven by in vitro methylated MAT2A promoter constructs were 75-95% lower than activities driven by unmethylated constructs. SAM treatment of Hep G2 cells reduced MAT2A endogenous expression by 75%, hypermethylated the MAT2A promoter, and reduced luciferase activities driven by MAT2A promoter constructs by 65-75% while not affecting MAT1A's promoter activity. Treatment of adult rat and human hepatocytes with trichostatin A, an inhibitor of histone deacetylase, upregulated MAT2A expression by more than fourfold. Collectively, these results suggest that MAT2A expression is regulated by promoter methylation and histone acetylation.

Topics: Animals; Cells, Cultured; Enzyme Inhibitors; Hepatocytes; Humans; Hydroxamic Acids; Isoenzymes; Liver Neoplasms; Methionine Adenosyltransferase; Methylation; Promoter Regions, Genetic; Rats; RNA, Messenger; S-Adenosylmethionine

The presence of telomerase has been demonstrated recently in many different malignancies. Several reports documented that in human hepatocellular carcinoma, the level of telomerase activity parallels its differentiation stage. In the present study, the effect of the differentiation-inducing agent sodium butyrate on telomerase activity in four human liver cancer cell lines was investigated using the telomeric repeat amplification protocol. We assayed telomerase activity before and after butyrate treatment and in cell cycle synchronized non-dividing quiescent cells. In addition, telomerase reverse transcriptase levels were measured at the mRNA level. All four cell lines possessed high but not identical levels of telomerase activity. Telomerase activity was significantly reduced by treatment with sodium butyrate as well as trichostatin A in a dose- and time-dependent fashion, paralleling the reduction of cell proliferation. Although methotrexate, hydroxyurea, and colchicine synchronized the cell cycle at G1, S, and G2/M, respectively, and thereby also caused proliferating cells to cease dividing and become quiescent, in this case telomerase activity remained essentially unaltered compared to the control cultures. Moreover, levels of mRNA encoding telomerase reverse transcriptase were not always significantly altered by either sodium butyrate treatment or cell cycle synchronization. These results suggest that sodium butyrate, as a histone deacetylase inhibitor, effectively reduces telomerase activity without affecting transcription levels of the reverse transcriptase component.

Topics: Antineoplastic Agents; Butyrates; Cell Cycle; Cell Differentiation; Cell Division; Colchicine; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Hydroxyurea; Liver Neoplasms; Methotrexate; RNA; RNA, Messenger; Telomerase; Tumor Cells, Cultured

There is a strong correlation between the acetylation status of nucleosomal histones and transcriptional activity. Here we show that the histone deacetylase inhibitor trichostatin A (TSA) activates reporter gene constructs driven by the human platelet-derived growth factor B (PDGF-B) gene promoter. This activation showed an inverse correlation with the cell type-specific transcriptional activities of the promoter. The TSA response was minimal in three tumor cell lines that exhibit high-level promoter activity. In JEG-3 choriocarcinoma cells, however, where the basal promoter activity is considerably lower, there was a strong response to TSA. This was in contrast to constructs that included a PDGF-B enhancer, which were refractory to TSA effects, indicating a possible function of the enhancer in modulating acetylation status. Analysis of PDGF-B promoter mutants with respect to TSA induction revealed no specific TSA-responsive element, but suggested that association of nonacetylated histones to the PDGF-B promoter may be a default process in the absence of enhancer activation. TSA treatment of JEG-3 cells, either alone or in combination with the demethylating agent 5-azacytidine, failed to activate the silenced endogenous PDGF-B transcript, however, which appears to be repressed by additional mechanisms.

Topics: Adenocarcinoma; Breast Neoplasms; Carcinoma, Hepatocellular; Choriocarcinoma; Chromosomes; DNA Methylation; Enhancer Elements, Genetic; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Introns; Liver Neoplasms; Mutagenesis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-sis; Rhabdomyosarcoma; Transcription, Genetic; Tumor Cells, Cultured

The Hep3B cell line analyzed in the present study is a widely used in vitro model in studies characterizing pathogenetic, functional, and therapeutic aspects of human hepatocellular carcinoma (HCC). Here we have determined the chromosomal composition using a combination of cytogenetic techniques. In agreement with the original description for this cell line, Hep3B was found to have a hypotriploid chromosome content carrying 59-63 chromosomes and no cytogenetic differences were demonstrated between early and late passages suggesting that this cell line has remained stable after repeated subculturing. Mutations and alterations of the IGF-axis as well as of chromosome 1p34, where the genes for histone deacetylase 1 (HDAC1) and transforming growth factor beta receptor interacting protein-1 (TRIP-1) map, are frequent events in hepatocarcinogenesis. This study characterizes the Hep3B cell line in detail at the karyotypic level, using comparative genomic hybridization (CGH), spectral karyotyping (SKY), G-banding and FISH techniques. We have also examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on members of the IGF-axis, and analysed them with regard to the karyotype. The results show that expression of one member of the IGF-axis, IGFBP-3, is greatly upregulated by treatment of Hep3B cells with TSA. As IGFBP-3 has been shown to induce apoptosis, these results suggest a possible use for histone deacetylase inhibitors and/or IGFBP-3 in the treatment of HCC.

Topics: Carcinoma, Hepatocellular; Chromosome Banding; Chromosome Mapping; Eukaryotic Initiation Factor-3; Humans; Hydroxamic Acids; In Situ Hybridization, Fluorescence; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor II; Karyotyping; Liver Neoplasms; Ploidies; Proteins; Receptor, IGF Type 2; Ribonucleases; Tumor Cells, Cultured

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent basic helix-loop-helix-PAS-containing transcription factor which is activated by chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Constitutive expression of the AhR gene occurs in a tissue- and developmentally specific manner and appears to be altered by chemicals which affect histone deacetylase (HDAC) activity in cells in culture. Here we have directly characterized the effects of two HDAC inhibitors, n-butyrate and trichostatin A, on the promoter activity of the murine AhR gene. HDAC inhibitors increased the constitutive activity of the AhR gene promoter in a luciferase reporter construct by five- to sevenfold in a dose- and time-dependent manner in several cell lines and was correlated with an increase in endogenous AhR activity in an AhR-deficient cell line. Deletion analysis of the upstream region of the AhR gene localized the HDAC inhibitor effect to a 167-bp region encompassing -77 to +90 of the AhR gene promoter. Cotransfection of an AhR promoter-luciferase reporter plasmid with a vector expressing the E1A(12s) oncoprotein, a negative regulator of p300, a protein with histone acetylase activity, decreased AhR promoter activity fivefold. Overall, our results support a role for histone acetylation in the transcriptional activity of the AhR gene promoter.

Topics: Animals; Breast Neoplasms; Butyrates; Carcinoma, Hepatocellular; COS Cells; Enzyme Inhibitors; Female; Gene Expression Regulation; Genes, Reporter; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kinetics; Liver Neoplasms; Luciferases; Mice; Okadaic Acid; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Transfection; Tumor Cells, Cultured

Cell growth and division are controlled through the actions of cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CKIs). Treatment of cell lines with Trichostatin A leads to induction of one of these CKIs, p21, and growth arrest. Induction of p21 can also occur through the actions of TGFbeta1. Latent TGFbeta1 can be activated by the M6P/IGF2R. In the present study we have examined the effect of TSA on members of the IGF axis, the CKIs p21 and p27, and also TGFbeta1 in Hep3B cells. The only member of the IGF axis to be affected by treatments was IGF2. Expression of another gene from the same chromosomal location, H19, was also affected. TGFbeta1 expression was greatly enhanced by TSA. In addition, both CKIs, p21 and p27, were upregulated by TSA. Effects of adding IGF-II or TGFbeta1 to TSA-treated cells on p21 induction were examined. The results show that the induction of p21 by TSA can be modulated by additions of IGF-II whereas addition of TGFbeta1 affects its own expression but not p21. In conclusion, the results indicate that the induction of p21 and cell growth arrest caused by Trichostatin A may involve multiple signaling pathways.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Hydroxamic Acids; Insulin-Like Growth Factor II; Liver Neoplasms; Protein Binding; Receptor, IGF Type 2; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured