trichostatin-a and Esophageal-Neoplasms

Histone deacetylases (HDACs) have been demonstrated to be aberrantly activated in tumorigenesis and cancer development. Thus, HDAC inhibitors (HDACIs) are considered to be promising anti-cancer therapeutics. However, recent studies have shown that HDACIs promote the migration of many cancer cells. Therefore, there is a need to elucidate the underlying mechanisms of HDACIs on cancer cell migration to establish a combination therapy that overcomes HDACI-induced cell migration.. KYSE-150 and EC9706 cells were treated differently. Effects of drugs and siRNA treatment on tumor cell migration and cell signaling pathways were investigated by transwell migration assy. Gene expression for SNAI2 was tested by RT-qPCR. Western blot analysis was employed to detect the level of E-cadherin, β-catenin, vimentin,Slug，ERK1/2, H3, PAI-1 and BRD4. The effect of drugs on cell morphology was evaluated through phase-contrast microscopic images.. TSA promotes epithelial-mesenchymal transition (EMT) in ESCC cells by downregulating the epithelial marker E-cadherin and upregulating mesenchymal markers β-catenin, vimentin, Slug, and PAI-1. Knockdown of Slug by siRNA or inhibition of PAI-1 clearly suppressed TSA-induced ESCC cell migration and resulted in the reversal of TSA-triggered E-cadherin, β-catenin, and vimentin expression. However, no crosstalk between Slug and PAI-1 was observed in TSA-treated ESCC cells. Blocking ERK1/2 activation also inhibited TSA-induced ESCC cell migration, EMT, and upregulation of Slug and PAI-1 levels in ESCC cells. Interestingly, inhibition of BRD4 suppressed TSA-induced ESCC cell migration and attenuated TSA-induced ERK1/2 activation and upregulation of Slug and PAI-1 levels.. Our data indicate the existence of at least two separable ERK1/2-dependent signaling pathways in TSA-mediated ESCC cell migration: an ERK1/2-Slug branch and an ERK1/2-PAI-1 branch. Both branches of TSA-induced ESCC cell migration appear to favor the EMT process, while BRD4 is responsible for two separable ERK1/2-dependent signaling pathways in TSA-mediated ESCC cell migration.

Topics: beta Catenin; Butadienes; Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Shape; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Flavonoids; Gene Expression; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Nitriles; Plasminogen Activator Inhibitor 1; Protein Kinase Inhibitors; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors; Vimentin

Protein acetylation modification controlled by acetyltransferases (HATs) and histone deacetylases (HDACs) regulates multiple biologic processes including cell proliferation and migration. HDAC inhibitors (HDACi) are currently used as a promising epigenetic-based therapy for cancer treatment. Of the anticancer activity, accumulating evidence has shown that HDACi can enhance cell migration in subset of cancer cells. Thus, there is a critical need to identify such counter anticancer activity to HDACi in different cancer cell types and elucidate the rational in order to develop appropriate combination therapies in cancer treatment. In seeking to address the effect of HDACi on esophageal squamous cell carcinoma (ESCC) cells migration, trichostatin A (TSA), a canonical HDACi targeting class I and class II HDACs, was used. Here, we report the discovery that TSA augmented ESCC cells migration by increasing the acetylation of nuclear factor-κB/RelA at lysine 310 (K310). To elucidate the mechanism by which TSA promotes the migration of ESCC cells, plasmid of RelA K310R, a mutant precluding acetylation at K310, was transfected into ESCC cells. Blocking acetylation of RelA at K310 significantly arrogated TSA-induced cell migration. Mechanistic investigations revealed that TSA increased the level of acetylated RelA at K310 (RelA K310ac), thereby increasing the level of epithelia-mesenchymal transition (EMT) transcription factor slug mRNA, which in turn induced EMT. Overall, this study indicates that TSA promotes ESCC cells migration by RelA K310ac-slug-EMT pathway. Our findings provide a strategy to eradicate HDACi-induced ESCC cells migration by targeting RelA as a combination therapy with nonspecific HDACi in ESCC treatment.

Topics: Acetylation; Apoptosis; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lysine; Transcription Factor RelA; Tumor Cells, Cultured

Esophageal squamous cell carcinoma (ESCC) is a common malignancy without effective therapy. Histone deacetylase inhibitors (HDACIs) have been demonstrated as an emerging class of anticancer drugs for a range of haematological and solid tumours. However, the effect of HDACIs has not yet been investigated on ESCC cells. In this study, HDACIs were initially considered to have anticancer activity for ESCC, due to the high expression of HDAC genes in ESCC cell lines by analysing expression data of 27 ESCC cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia. Next, we used five ESCC cell lines and one normal immortalized esophageal epithelial cell line to screen three HDACIs, panobinostat (LBH589), vorinostat (SAHA), and trichostatin A (TSA), for the ability to inhibit growth. Here, we report that LBH589 more effectively suppressed cell proliferation of ESCC cell lines, in a dose-dependent manner, than TSA and SAHA, as well as had lower toxicity against the SHEE normal immortalized esophageal epithelial cell line. Further experiments indicated that LBH589 treatment significantly inhibited TP53 (mutated TP53) expression, both at the mRNA and protein level, and simultaneously increased p21 and decreased cyclin D1 expression. Taken together, we propose that LBH589 inhibits ESCC cell proliferation mainly through inducing cell cycle arrest by increasing p21 and decreasing cyclin D1 in a p53-independent manner. SIGNIFICANCE OF THE STUDY: In this study, the antitumor activity of HDACIs LBH589, SAHA, and TSA on ESCC was characterized, with LBH589 displaying the most potent anti-proliferative activity while not harming normal immortalized esophageal epithelial cells. Furthermore, we propose that LBH589 exerts its anti-proliferative effect by inducing cell cycle arrest. The ability to specifically target cancer cells indicates therapeutic potential for use of LBH589 in the treatment of ESCC.

Topics: Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Panobinostat; Tumor Suppressor Protein p53; Up-Regulation

Histone deacetylase (HDAC) inhibitors have recently emerged as a new class of anticancer agents. As a classical HDAC inhibitor, trichostatin A (TSA) has been shown to possess many anticancer activities such as induction of cell cycle arrest, promotion of cell death, and enhancement of radiosensitity. In our previous work, we found that TSA treatment induced Rad9 gene expression, which suggested that Rad9 might play a role in TSA-induced biological effects. As Rad9 is involved in maintaining genomic integrity, we further analyzed the DNA damage induced by TSA and combined with Rad9 knockdown in esophageal cancer cells (ESCCs). Our results showed that TSA treatment alone induced significantly DNA damage in ESCC cells. Simultaneously, TSA also induced Rad9 gene expression both at transcriptional and translational levels in EC109 cells, but not in KYSE150 cells. Further, the induction of Rad9 by TSA was accompanied with increased level of histone H3K9 acetylation in Rad9 promoter region. To understand the role of Rad9 in TSA-induced DNA damage, Rad9 gene expression was efficiently knocked down by small interfering RNA (siRNA), which led to enhanced DNA damage and cell death induced by TSA. Our data suggested that Rad9 plays an important role in DNA damage, which is related to the biological effects of TSA.

Topics: Acetylation; Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; DNA Damage; Esophageal Neoplasms; Gene Expression; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; RNA, Small Interfering

As a result of alternative splicing and differential promoter usage, RASSF5 exists in at least three isoforms (RASSF5A-RASSF5C), which may play different roles in tumorigenesis. The present study was to detect the role of RASSF5A, B and C in esophageal squamous cell carcinoma (ESCC) and clarify the critical CpG sites of RASSF5A, in order to clarify more information on the role of RASSF5 with regard to the pathogenesis of ESCC. Frequent silencing of RASSF5A but not RASSF5B and RASSF5C were found in esophageal cancer cell lines and the silencing of RASSF5A may be reversed by 5-Aza-dC or TSA treatment. The aberrant CpG island 1 methylation of RASSF5A induces silencing of its expression in TE13 cell line. Decreased mRNA and protein expression of RASSF5A was observed in ESCC tumor tissues and was associated with RASSF5A CpG island 1 methylation status. Unlike RASSF5A, expression variation of RASSF5B and RASSF5C was not found in ESCC tissues. Aberrant promoter methylation of RASSF5C was also not found in ESCC. RASSF5A methylation and protein expression were independently associated with ESCC patients' survival. These data indicated that the inactivation of RASSF5A through CpG island 1 methylation may play an important role in ESCC carcinogenesis, RASSF5A may be a functional tumor suppressor and may serve as a prognostic biomarker for ESCC.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Alternative Splicing; Apoptosis; Apoptosis Regulatory Proteins; Azacitidine; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cytidine Triphosphate; DNA Methylation; Esophageal Neoplasms; Esophagus; Female; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Humans; Hydroxamic Acids; Male; Middle Aged; Monomeric GTP-Binding Proteins; Neoplasm Invasiveness; Prognosis; Promoter Regions, Genetic; Protein Isoforms; Protein Synthesis Inhibitors; RNA, Messenger, Stored

Histone deacetylase (HDAC)‑mediated epigenetic modification plays crucial roles in numerous biological processes, including cell cycle regulation, cell proliferation and apoptosis. HDAC inhibitors demonstrate antitumor effects in various cancers, including glioblastoma and breast cancer. HDAC inhibitors are therefore promising antitumor drugs for these tumors. The tumorigenesis and development of esophageal squamous cell carcinoma (ESCC) involve genetic and epigenetic mechanisms. However, the effects of the HDAC inhibitor on ESCC are not fully investigated. In the present study, ESCC cells were treated with trichostatin A (TSA) and its antitumor effects and related mechanisms were investigated. The results indicated that TSA suppressed the proliferation of ESCCs and caused G1 phase arrest by inducing the expression of p21 and p27. TSA also induced cell apoptosis by enhancing the expression of pro‑apoptotic protein Bax and decreasing the expression of anti‑apoptotic protein Bcl‑2. Furthermore, TSA inhibited the expression of phosphatidylinositol‑3‑kinase (PI3K) and reduced the phosphorylation of Akt and extracellular signal‑regulated kinase (ERK)1/2 in EC9706 and EC1 cell lines. High levels of acetylated histone H4 were detected in TSA‑treated ESCC cell lines. Overall, these results indicate that TSA suppresses ESCC cell growth by inhibiting the activation of the PI3K/Akt and ERK1/2 pathways. TSA also promotes cell apoptosis through epigenetic regulation of the expression of apoptosis‑related protein.

Topics: Acetylation; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; G1 Phase Cell Cycle Checkpoints; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2

F-box protein 32 (FBXO32) (also known as atrogin-1), a member of the F-box protein family, has recently been identified as a transforming growth factor beta (TGF-β)/Smad target gene involved in regulating cell survival, and it may be transcriptionally silenced by epigenetic mechanisms in some kinds of carcinomas, yet its role in esophageal squamous cell carcinoma (ESCC) has not been defined.. The role of FBXO32 in ESCC and the correlation of FBXO32 methylation with a series of pathologic parameters were studied in a large cohort of patients with ESCC.. Decreased messenger RNA (mRNA) expression and protein expression of FBXO32 were observed in esophageal cancer cell lines, and the silencing of FBXO32 could be reversed by treatment with 5-aza-2'-deoxycytidine or trichostatin A in the TE13 cell line. In addition, aberrant methylation of FBXO32 and histone deacetylation was capable of suppressing FBXO32 mRNA and protein expression in TE13 cells. Decreased mRNA and protein expression of FBXO32 was observed in ESCC tumor tissues and was associated with FBXO32 promoter methylation status. A positive correlation between FBXO32 and phosphorylated SMAD family members 2 and 3 expression and Smad4 protein expression also was observed in clinical specimens. FBXO32 methylation status and protein expression were independently associated with survival in patients with ESCC.. FBXO32 may be a functional tumor suppressor. Its inactivation through promoter methylation could play an important role in ESCC carcinogenesis, and reactivation of the FBXO32 gene may have therapeutic potential and might be used as a prognostic marker for patients with ESCC.

Topics: Adult; Aged; Azacitidine; Calmodulin-Binding Proteins; Carcinoma, Squamous Cell; Cell Growth Processes; Decitabine; DNA Methylation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Silencing; Humans; Hydroxamic Acids; Immunohistochemistry; Male; Middle Aged; Muscle Proteins; Promoter Regions, Genetic; RNA, Messenger; SKP Cullin F-Box Protein Ligases; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has been shown to act as an anti-tumor agent; however, the effect and mechanism of TSA on the invasion of esophageal squamous cell carcinoma (ESCC) remains unknown.. To determine whether TSA suppresses the invasiveness of ESCC cell via HDAC2, the expression of HDAC2 in ESCC tissues and adjacent non-tumor tissues were compared using Western blot and immunohistochemistry. Cells were transfected with HDAC2 siRNAs and non-targeting control siRNA using Lipofectamine TM 2000. Cell invasion was investigated using a transwell assay. The protein levels of matrix metalloproteinase-2/9 (MMP-2/9) were examined by Western blot analysis.. Expression of HDAC2 was significantly higher in ESCC than in adjacent non-tumor tissues. Additionally, the in vitro invasion assay found that both downregulation of HDAC2 expression and TSA treatment inhibited ESCC cell invasion by approximately 75%. Also, an MMP2/9-specific inhibitor sharply suppressed ESCC cell invasion. Furthermore, both downregulation of HDAC2 and treatment with TSA decreased MMP-2 and MMP-9 protein levels in ESCC cells.. These results suggest that the inhibitory effect of TSA on cancer invasion is mediated through the suppression of HDAC2 expression, and that the reduction of MMP-2 and MMP-9 expression induced by HDAC2 may be involved in the anti-invasive effect of TSA.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Esophageal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Small Interfering

Esophageal cancer is an intractable disease due to late diagnosis, high incidence of post-surgical locoregional recurrence and frequent distant metastasis. Oncolytic adenovirus (Ad) vectors are a promising method for cancer treatment. The H101 virus is a recombinant Ad which has replication-selective properties and replicates only in tumor cells. The coxsackievirus and adenovirus receptor (CAR) is considered a surrogate marker that monitors the outcome of Ad-mediated gene therapy. Accumulating evidence indicates that CAR expression levels are lower in various types of tumors such as ovarian, lung, breast and bladder when compared to their normal counterparts. In this study, we reported that trichostatin A (TSA) induced the expression of CAR in esophageal squamous cell carcinoma (ESCC) cell lines through the MAPK/ERK1/2 signaling pathway. The expression levels of CAR were positively related with the antitumor activity of H101. Our results suggest that TSA increases the antitumor activity of the oncolytic adenovirus H101 through the MAPK/ERK pathway.

Topics: Adenoviridae; Carcinoma, Squamous Cell; Cell Survival; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Esophageal Neoplasms; Gene Expression; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Oncolytic Viruses; Transcriptional Activation; Virus Internalization; Virus Replication

The secreted frizzled-related protein 1 (SFRP1) gene, as a Wnt signaling modulator, is frequently inactivated by promoter methylation in many tumors including gastric cancer, breast cancer, oral squamous cell carcinoma, and esophageal adenocarcinoma. However, the role of SFRP1 in esophageal squamous cell carcinoma (ESCC) is not clear. In this study, we investigated the epigenetic inactivation of the SFRP1 gene in ESCC.. Nine ESCC cell lines, two immortalized human esophageal epithelial cell lines, twenty ESCC tissues, and paired adjacent nontumor tissues were analyzed in the study. Methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, reverse-transcription PCR, immunohistochemistry, and chromatin immunoprecipitation assay were used to detect SFRP1 promoter methylation, expression of the SFRP1 gene, and histone modification in the SFRP1 promoter region.. The SFRP1 promoter was found to be highly methylated in 95% (19/20) of the ESCC tissues and in nine ESCC cell lines, compared with 65% (13/20) of the paired nontumor tissues. Moreover, we confirmed that complete methylation of the SFRP1 gene promoter was correlated with its greatly reduced expression level. After individual treatment with 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA), the messenger RNA (mRNA) level of the SFRP1 gene was not obviously rescued in the EC9706 cell line. Combined incubation with DAC and TSA can, however, substantially increase the SFRP1 mRNA expression level in the EC9706 cell line. Chromatin immunoprecipitation assay showed that acetylated histone H3 and H4 were found in the SFRP1 promoter region.. Promoter hypermethylation of SFRP1 is a frequent event in ESCC. Promoter methylation and histone acetylation may cooperatively regulate expression of the SFRP1 gene.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Carcinoma, Squamous Cell; Cell Line, Tumor; Decitabine; DNA Methylation; Esophageal Neoplasms; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Membrane Proteins

The accumulation of reactive oxygen species and subsequent oxidative DNA damage underlie the development of Barrett's oesophagus (BO) and its progression to Barrett's dysplasia (BD) and adenocarcinoma (BAC).. The promoter regions of 23 genes of the glutathione S-transferase (GST) and glutathione peroxidase (GPX) families were systematically analysed. Quantitative bisulfite pyrosequencing, real-time RT-PCR, western blot and immunohistochemical (IHC) analysis methods were utilised in this study.. 14 genes were identified that have CpG islands around their transcription start sites: GSTs (GSTM2-M5, GSTA4, GSTP1, GSTZ1, GSTT2, GSTO1 and GSTO2) and GPXs (GPX1, GPX3, GPX4 and GPX7). Analysis of an initial set of 20 primary samples demonstrated promoter DNA hypermethylation and mRNA downregulation of GPX3, GPX7, GSTM2, GSTM3 and GSTM5 in more than half of the BAC samples. Further analysis of 159 primary human samples (37 normal, 11 BO, 11 BD and 100 BACs) indicated frequent hypermethylation (>or=10% methylation) of GPX3 (62%), GPX7 (67%), GSTM2 (69.1%) and GSTM3 (15%) in BACs. A significant inverse correlation between DNA methylation and mRNA expression level was shown for GPX3 (p<0.001), GPX7 (p = 0.002), GSTM2 (p<0.001) and GSTM5 (p = 0.01). Treatment of oesophageal cancer cell lines with 5-aza-2'-deoxycytidine and trichostatin-A led to reversal of the methylation pattern and re-expression of these genes at the mRNA and protein levels. The IHC analysis of GPX3, GPX7 and GSTM2 on a tissue microarray that contained 75 BACs with normal squamous oesophageal samples demonstrated an absent to weak staining in tumours (52% for GPX3, 57% for GPX7 and 45% for GSTM2) and a moderate to strong immunostaining in normal samples.. Epigenetic inactivation of members of the glutathione pathway can be an important mechanism in Barrett's tumourigenesis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Azacitidine; Barrett Esophagus; Cell Transformation, Neoplastic; CpG Islands; Decitabine; Disease Progression; DNA Methylation; DNA, Neoplasm; Down-Regulation; Epigenesis, Genetic; Esophageal Neoplasms; Gene Expression Regulation, Enzymologic; Glutathione Peroxidase; Glutathione Transferase; Humans; Hydroxamic Acids; Middle Aged; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Tumor suppressor genes (TSGs) are sometimes inactivated by transcriptional silencing through promoter hypermethylation. To identify novel methylated TSGs in melanoma, we carried out global mRNA expression profiling on a panel of 12 melanoma cell lines treated with a combination of 5-Aza-2-deoxycytidine (5AzadC) and an inhibitor of histone deacetylase, Trichostatin A. Reactivation of gene expression after drug treatment was assessed using Illumina whole-genome microarrays. After qRT-PCR confirmation, we followed up 8 genes (AKAP12, ARHGEF16, ARHGAP27, ENC1, PPP1R3C, PPP1R14C, RARRES1, and TP53INP1) by quantitative DNA methylation analysis using mass spectrometry of base-specific cleaved amplification products in panels of melanoma cell lines and fresh tumors. PPP1R3C, ENC1, RARRES1, and TP53INP1, showed reduced mRNA expression in 35-59% of the melanoma cell lines compared to melanocytes and which was correlated with a high proportion of promoter methylation (>40-60%). The same genes also showed extensive promoter methylation in 6-25% of the tumor samples, thus confirming them as novel candidate TSGs in melanoma.

Topics: Azacitidine; Carrier Proteins; Cell Line, Tumor; Colonic Neoplasms; CpG Islands; Decitabine; DNA Methylation; Esophageal Neoplasms; Gene Expression Profiling; Gene Silencing; Genes, Tumor Suppressor; Glioma; Heat-Shock Proteins; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Melanoma; Membrane Proteins; Microfilament Proteins; Neuropeptides; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG.

Topics: Alleles; Cell Line, Transformed; Cell Line, Tumor; Chromosome Mapping; Chromosome Segregation; Chromosomes, Human, Pair 13; Deoxycytidine; DNA Methylation; Epithelial Cells; Esophageal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genome, Human; Humans; Hydroxamic Acids; In Situ Hybridization, Fluorescence; Microarray Analysis; Microsatellite Repeats; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Thrombospondins; Transfection; Tumor Stem Cell Assay

Histone deacetylase inhibitors mediate a potent growth-inhibitory effect in cancer cells through induction of cell-cycle arrest and apoptosis. Moreover, these agents significantly induce transcriptional activation of nuclear factor kappaB, as well as p21 regulated by protein kinase C, and are thought to negatively influence the ability of histone deacetylase inhibitor to effectively mediate apoptosis. This study aimed to evaluate the effect of calphostin C (a protein kinase C inhibitor) on trichostatin A (a histone deacetylase inhibitor)-mediated upregulation of nuclear factor kappaB and p21 promotor transcriptional activity, as well as induction of apoptosis in lung and esophageal cancer cells.. Cultured lung and esophageal cancer cells were treated with calphostin C and trichostatin A. Nuclear factor kappaB transcriptional activity was quantitated by using the nuclear factor kappaB-luciferase assay. Transcription of p21 gene and p21 protein levels was evaluated by using the p21 promoter-luciferase assay and the p21 enzyme-linked immunoassay, respectively. Apoptosis was evaluated by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-based ApoBrdU assay. Levels of expression of nuclear factor kappaB-dependent antiapoptotic and proapoptotic proteins were evaluated by means of Western blotting.. Exposure of lung or esophageal cancer cells to trichostatin A resulted in a dose- and cell-dependent 2-fold to greater than 20-fold increase of nuclear factor kappaB and p21 transcriptional activity. Treatment with trichostatin A and calphostin C led to a 50% to 90% decrease of trichostatin A- mediated upregulation of nuclear factor kappaB and p21 activation. Inhibition of nuclear factor kappaB activity resulted in significant reduction (30% to >99%) of trichostatin A- mediated activation of not only nuclear factor kappaB transcription but also p21 promotor activity. Importantly, 90% to 96% of thoracic cancer cells under-went apoptosis after exposure to the combination of trichostatin A plus calphostin C.. Inhibition of protein kinase C abrogates trichostatin A-mediated upregulation of nuclear factor kappaB transcriptional activity and p21 expression that is associated with profound induction of apoptosis in lung or esophageal cancer cells. Protein kinase C might be a novel target for enhancing the efficacy of histone deacetylase inhibitor in cancer therapy.

Topics: Apoptosis; Blotting, Western; Cell Proliferation; Cell Survival; Drug Therapy, Combination; Esophageal Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Naphthalenes; NF-kappa B; Oncogene Protein p21(ras); Probability; Protein Kinase C; Risk Factors; Sensitivity and Specificity; Tumor Cells, Cultured; Up-Regulation

We have previously shown the reactivation of some methylation-silenced genes in cancer cells by (-)-epigallocatechin-3-gallate, the major polyphenol from green tea. To determine whether other polyphenolic compounds have similar activities, we studied the effects of soy isoflavones on DNA methylation.. Enzyme assay was used to determine the inhibitory effect of genistein on DNA methyltransferase activity in nuclear extracts and purified recombinant enzyme. Methylation-specific PCR and quantitative real-time PCR were employed to examine the DNA methylation and gene expression status of retinoic acid receptor beta (RARbeta), p16INK4a, and O6-methylguanine methyltransferase (MGMT) in KYSE 510 esophageal squamous cell carcinoma cells treated with genistein alone or in combination with trichostatin, sulforaphane, or 2'-deoxy-5-aza-cytidine (5-aza-dCyd).. Genistein (2-20 micromol/L) reversed DNA hypermethylation and reactivated RARbeta, p16INK4a, and MGMT in KYSE 510 cells. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARbeta by genistein were also observed in KYSE 150 cells and prostate cancer LNCaP and PC3 cells. Genistein (20-50 micromol/L) dose-dependently inhibited DNA methyltransferase activity, showing substrate- and methyl donor-dependent inhibition. Biochanin A and daidzein were less effective in inhibiting DNA methyltransferase activity, in reactivating RARbeta, and in inhibiting cancer cell growth. In combination with trichostatin, sulforaphane, or 5-aza-dCyd, genistein enhanced reactivation of these genes and inhibition of cell growth.. These results indicate that genistein and related soy isoflavones reactivate methylation-silenced genes, partially through a direct inhibition of DNA methyltransferase, which may contribute to the chemopreventive activity of dietary isoflavones.

Topics: Anticarcinogenic Agents; Antineoplastic Agents; Azacitidine; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p16; Decitabine; DNA Methylation; Dose-Response Relationship, Drug; Esophageal Neoplasms; Genistein; Glycine max; Humans; Hydroxamic Acids; Isoflavones; Isothiocyanates; O(6)-Methylguanine-DNA Methyltransferase; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfites; Sulfoxides; Tea; Thiocyanates

We performed a comprehensive survey of commonly inactivated tumor suppressor genes in esophageal squamous cell carcinoma (ESCC) based on functional reactivation of epigenetically silenced tumor suppressor genes by 5-aza-2'-deoxycytidine and trichostatin A using microarrays containing 12599 genes. Among 58 genes identified by this approach, 44 (76%) harbored dense CpG islands in the promoter regions. Thirteen of twenty-two tested gene promoters were methylated in cell lines, and ten in primary ESCC accompanied by silencing at the mRNA level. Potent growth suppressive activity of three genes including CRIP-1, Apolipoprotein D, and Neuromedin U in ESCC cells was demonstrated by colony focus assays. Pharmacologic reversal of epigenetic silencing is a powerful approach for comprehensive identification of tumor suppressor genes in human cancers.

Topics: Azacitidine; Base Sequence; Carcinoma, Squamous Cell; CpG Islands; Decitabine; DNA Methylation; Down-Regulation; Enzyme Inhibitors; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured