trichostatin-a and Colonic-Neoplasms

GLUT5, a key protein encoded by the SLC2A5 gene, is involved in the uptake of fructose from the intestine. Currently, with the increased consumption of this sugar and the associated increased incidence of obesity, diabetes and cancer, GLUT5 may represent an important molecular target in the prevention and treatment of these diseases. Here, we demonstrate that overexpression of the SNAI1 and SNAI2 transcription factors in cells expressing high levels of SLC2A5 mRNA reduced SLC2A5 gene expression. Furthermore, a histone deacetylase inhibitor, trichostatin A, which induces SNAI1 and SNAI2 expression, inhibits SLC2A5/GLUT5 expression and sensitizes colon cancer cells to cisplatin and oxaliplatin. This finding might have potential relevance for the development of therapeutic treatments aimed at modulating fructose transport or genes involved in this process for use with certain cancers.

Topics: Colonic Neoplasms; Fructose; Glucose Transporter Type 5; Humans; Platinum Compounds; Snail Family Transcription Factors; Transcription Factors

In differentiated Caco-2 cells, none of the genes had significant changes from D alone group. D + SFN (P = 0.99) demonstrated an opposing effect from D alone and decreased VDR expression. However, in proliferating Caco-2 cells, D + SFN (P < 0.04) increased VDR expression and decreased CYP27B1 (P < 0.01) more than D alone (P = 0.38 and 0.07, respectively). Although statistically significant, D + SFN (P = 0.01) effect on HDAC inhibitor activity was less than trichostatin A alone group (P < 0.0004) or SFN alone group (P < 0.0014).. The data suggest that colon cancer cells respond to dietary components differently under different conditions. The effect of vitamin D and SFN is selective and gene-specific in the complex multistep process of colorectal carcinogenesis in vitro. © 2020 Society of Chemical Industry.

Topics: Acetylation; Caco-2 Cells; Calcium Channels; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Hydroxamic Acids; Isothiocyanates; Receptors, Calcitriol; Sulfoxides; TRPV Cation Channels; Vitamin D; Vitamin D3 24-Hydroxylase

A pattern of epigenetic modifications and changes, DNA methylation and histone modification, is central to many human cancers. A variety of tumor suppressor genes (TSGs) have been demonstrated to be silenced because of histone deacetylation and DNA hypermethylation in several cancers. Recent in vitro studies have shown that two known mechanisms of epigenetic alteration consisting of methylation and histone deacetylation seem to be the best candidate mechanisms for inactivation of CIP/KIP family (p21Cip1/Waf1/Sdi1, and p27Kip1) in numerous cancers. Numerous investigations have indicated that DNA demethylating and histone deacetylase inhibitors (HDACIs) can restore the CIP/KIP family gene expression. Previously, we evaluated the effect of trichostatin A (TSA) and 5-aza-2'-deoxycytidine (5-AZA-CdR) on hepatocellular carcinoma (HCC). The present study was designed to investigate the effect of zebularine in comparison to and in combination with trichostatin A on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNMT1, DNMT3a and DNMT3b, Class I HDACs (HDACs 1, 2, 3) and Class II HDACs (HDACs 4, 5, 6) gene expression, cell growth inhibition and apoptosis induction in colon cancer LS 174T cell line.. The colon cancer LS 174T cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively.. Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively.. The zebularine and trichostatin A can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.

Topics: Apoptosis; Biomarkers, Tumor; Calcium-Binding Proteins; Cell Proliferation; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytidine; DNA (Cytosine-5-)-Methyltransferases; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Tumor Cells, Cultured

Tumor-associated fibroblasts (TAFs) contribute to transdifferentiation of stromal cells in tumor microenvironment. Epithelial-mesenchymal transition (EMT) is a procedure of phenotypic remodeling of epithelial cells and extensively exists in local tumoral stroma. Histone deacetylase (HDAC) inhibitor Tricostatin A (TSA) and sodium butyrate (SB) are reported to play important roles in the regulation of biological behaviour of cancer cells. However, whether TSA or SB is involved in control of EMT in colon epithelial cells induced by TAFs remains unidentified. In present study, we used conditioned medium (CM) form TAF-like CCD-18Co cells to stimulate 2D- and 3D-cultured colon epithelial HCoEpiC cells for 24 h and 4 d. We found that the CCD-18Co CM triggered multiple morphological changes in HCoEpiCs including prolonged cell diameters, down-regulation of E-cadherin and up-regulation of vimentin and α-SMA. Besides, ZEB1 and Snail expression and migration were also promoted by the CM. These phenomena were abolised by 5 μg/ml LY364947, a TGF-β receptor inhibitor. CCD-18Co induced up-regulation of HDAC1 and HDAC2 in the 2D and 3D models, while no change of HDAC4 exprerssion was found. Treatment of 2 μg/ml TSA reversed the CCD-18Co-induced morphological changes and migration of the HCoEpiCs, and suppressed the downregulation of E-cadherin and upregulation of vimentin, α-SMA, ZEB1 and Snail. However, the suppressive effect of 4 mg/ml SB on the EMT was not observed. TSA down-regulated the expressions of Smad2/3, p-Smad2/3 amd HDAC4. Besides, TSA promoted the apoptosis rate (36.84 ± 6.52%) comparing with the CCD-18Co-treated HCoEpiCs (3.52 ± 0.85%, P < 0.05), with promotion of Bax (0.5893±0.0498 in 2D and 0.8867±0.0916 in 3D) and reduction of Bcl-2 (0.0476±0.0053 in 2D and 0.0294±0.0075 in 3D). TSA stimulated expression of phosphorylated-p38 MAPK in 2D (0.3472±0.0249) and 3D (0.3188±0.0248). After pre-treatment with p38 MAPK inhibitor VX-702 (0.5 mg/ml), the apoptosis rate of TSA was decreased in 2D (10.32%) and 3D (5.26%). Our observations demonstrate that epigenetic treatment with HDAC inhibitor TSA may be a useful therapeutic tool for the reversion of TAF-induced EMT in colon epithelium through mediating canonical Smads pathway and non-canonical p38 MAPK signalling.

Topics: Apoptosis; Cancer-Associated Fibroblasts; Cell Line; Colon; Colonic Neoplasms; Epithelial Cells; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Neoplasm Proteins; Transforming Growth Factor beta

Ethanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied.. In this report, we mapped the important regulatory regions in the human ek1 promoter. 5' deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells.. In conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner.

Topics: Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Sp1 Transcription Factor; Sp3 Transcription Factor; Transcriptional Activation

Butyrate is a short-chain fatty acid produced by the intestinal microflora and it not only induces apoptosis but also inhibits the proliferation of cancer cells. Recently, it has been reported that butyrate may cause resistance in colon cancer cells. Therefore, we investigated the effects of increased resistance to butyrate in HCT116 colon cancer cells. We established HCT116 cells resistant to butyrate (HCT116/BR) by treating HCT116 parental cells (HCT116/PT) with increasing concentrations of butyrate to a maximum of 1.6 mM for 3 months. The butyrate concentrations that inhibited cell growth by 50% (IC50) were 0.508 and 5.50 mM in HCT116/PT and HCT116/BR cells. The values after treatment with paclitaxel, 5-fluorouracil (5-FU), doxorubicin and trichostatin A (TSA) were 2.42, 2.36, 4.31 and 11.3-fold higher, respectively, in HCT116/BR cells compared with HCT116/PT cells. The protein expression of drug efflux pumps, such as P-glycoprotein (P-gp), breast cancer-resistant protein (BCRP) and the multidrug resistance associated protein 1 (MRP1), did not differ between HCT116/PT and HCT116/BR cells. The expression level of the anti-apoptotic Bcl-xL protein was increased while those of pro-apoptotic Bax and Bim proteins were reduced in HCT116/BR cells. There were no significant differences in cell motility and invasion. This study suggests that exposure of colon cancer cells to butyrate results in development of resistance to butyrate, which may play a role in the acquisition of chemoresistance in colon cancer.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; bcl-X Protein; Butyrates; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Doxorubicin; Drug Resistance, Neoplasm; Fluorouracil; HCT116 Cells; Humans; Hydroxamic Acids; Multidrug Resistance-Associated Proteins; Neoplasm Invasiveness; Neoplasm Proteins; Paclitaxel

The effects of histone deacetylase (HDAC) inhibitors on epithelial-mesenchymal transition (EMT) differ in various types of cancers. We investigated the EMT phenotype in four colon cancer cell lines when challenged with HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA) with or without transforming growth factor-β1 (TGF-β1) treatment. Four colon cancer cell lines with different phenotypes in regards to tumorigenicity, microsatellite stability and DNA mutation were used. EMT phenotypes were assessed by the expression of E-cadherin and vimentin using western blot analysis, immunofluorescence, quantitative real-time RT-PCR following treatment with TSA (100 or 200 nM) or VPA (0.5 mM) with or without TGF-β1 (5 ng/ml) for 24 h. Biological EMT phenotypes were also evaluated by cell morphology, migration and invasion assays. TSA or VPA induced mesenchymal features in the colon carcinoma cells by a decrease in E-cadherin and an increase in vimentin expression at the mRNA and protein levels. Confocal microscopy revealed membranous attenuation or nuclear translocation of E-cadherin and enhanced expression of vimentin. These responses occurred after 6 h and increased until 24 h. Colon cancer cells changed from a round or rectangular shape to a spindle shape with increased migration and invasion ability following TSA or VPA treatment. The susceptibility to EMT changes induced by TSA or VPA was comparable in microsatellite stable (SW480 and HT29) and microsatellite unstable cells (DLD1 and HCT116). TSA or VPA induced a mesenchymal phenotype in the colon carcinoma cells and these effects were augmented in the presence of TGF-β1. HDAC inhibitors require careful caution before their application as new anticancer drugs for colon cancers.

Topics: Cadherins; Carcinoma; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; HCT116 Cells; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Microscopy, Confocal; Neoplasm Invasiveness; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta1; Valproic Acid; Vimentin

Radiotherapy is one of the main treatments for clinical cancer therapy. However, its application was limited due to lack of radiosensitivity in some cancers. Trichostatin A (TSA) is a classic histone deacetylases inhibitor (HDACi) that specifically inhibits the biochemical functions of HDAC and is demonstrated to be an active anticancer drug. However, whether it could sensitize colon cancer to radiation is not clear. Our results showed that TSA enhanced the radiosensitivity of colon cancer cells as determined by CCK-8 and clonogenic survival assay. Moreover, apoptotic cell death induced by radiation was enhanced by TSA treatment. Additionally, TSA also induced autophagic response in colon cancer cells, while autophagy inhibition led to cell apoptosis and enhanced the radiosensitivity of colon cancer cells. Our data suggested that inhibition of cytoprotective autophagy sensitizes cancer cell to radiation, which might be further investigated for clinical cancer radiotherapy.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Colonic Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Radiation Tolerance; Radiation-Sensitizing Agents

Cancer cells have extremely active metabolism, which supports high proliferation rates. Metabolic profiles of human colon cancer cells have been extensively studied, but comparison with non-tumour counterparts has been neglected.. Here we compared the metabolic flux redistribution in human colon adenocarcinoma cells (HT29) and the human colon healthy cell line NCM460 in order to identify the main pathways involved in metabolic reprogramming. Moreover, we explore if induction of differentiation in HT29 by trichostatin A (TSA) reverts the metabolic reprogramming to that of NCM460. Cells were incubated with [1,2-(13)C2]-d-glucose as a tracer, and Mass Isotopomer Distribution Analysis was applied to characterize the changes in the metabolic flux distribution profile of the central carbon metabolism.. We demonstrate that glycolytic rate and pentose phosphate synthesis are 25% lower in NCM460 with respect to HT29 cells. In contrast, Krebs cycle activity in the former was twice that recorded in the latter. Moreover, we show that TSA-induced HT29 cell differentiation reverts the metabolic phenotype to that of healthy NCM460 cells whereas TSA does not affect the metabolism of NCM460 cells.. We conclude that pentose phosphate pathway, glycolysis, and Krebs cycle are key players of colon adenocarcinoma cellular metabolic remodeling and that NCM460 is an appropriate model to evaluate the results of new therapeutic strategies aiming to selectively target metabolic reprogramming.. Our findings suggest that strategies to counteract robust metabolic adaptation in cancer cells might open up new avenues to design multiple hit and targeted therapies.

Topics: Adenocarcinoma; Antineoplastic Agents; Citric Acid Cycle; Colonic Neoplasms; Glucose; Glycolysis; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Lactic Acid; Pentose Phosphate Pathway

Histone deacetylase (HDAC) inhibitors have recently emerged as a new class of anti-cancer agents. Trichostatin A (TSA), a classical HDAC inhibitor, has been demonstrated to induce cell cycle arrest, promote cell apoptosis, and inhibit metastasis. However, the molecular mechanism underlying TSA function has not been fully elucidated. In the current study, we found that TSA treatment induced altered expression of cell cycle-associated genes in HCT116 cells by RT-PCR array. Among the 84 genes related to cell cycle control, 34 genes were significantly altered by TSA treatment, with 7 genes upregulated and 27 genes downregulated. Interestingly, gene expression of minichromosome maintenance protein-2 (MCM-2) was significantly downregulated by TSA treatment. This was confirmed by quantitative RT-PCR and Western blotting. Moreover, silencing of MCM-2 by siRNA led to cell cycle arrest and apoptosis in HCT116 cells. In addition, TSA caused an increase of phosphorylated JNK, which was involved in downregulation of MCM-2. Together, our results suggest that MCM-2 is a noval therapeutic target of TSA in colon cancer cells.

Topics: Adenocarcinoma; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Minichromosome Maintenance Complex Component 2; Molecular Targeted Therapy; Nuclear Proteins; RNA, Small Interfering; Up-Regulation

Expression of claudin-1, a tight junction protein, is highly upregulated in colon cancer. We have reported that claudin-1 expression in colon cancer cells is epigenetically regulated as histone deacetylase (HDAC) inhibitors decrease claudin-1 messenger RNA (mRNA) stability and thus expression. In this regard, our data suggested a role of the 3'-untranslated region (UTR) in the regulation of HDAC-dependent regulation of claudin-1 mRNA stability. In the current study, we demonstrate, based on our continued investigation, that the ELAV-like RNA-binding proteins (RBPs), human antigen R (HuR) and tristetraprolin (TTP) associate with the 3'-UTR of claudin-1 mRNA to modulate the latter's stability. Ribonomic and site-directed mutagenesis approaches were used to confirm the binding of HuR and TTP to the 3'-UTR of claudin-1. We further confirmed their roles in the stabilization of claudin-1 mRNA, under conditions of HDAC inhibition. In summary, we report that HuR and TTP are the critical regulators of the posttranscriptional regulation of claudin-1 expression in colon cancer cells. We also demonstrate that inhibition of HDACs by trichostatin treatment decreased the binding of HuR while increasing the binding of TTP to the 3'-UTR of claudin-1. Additionally, we provide data showing transcriptional regulation of claudin-1 expression, through the regulation of transcription factor Sp1. Taken together, we demonstrate epigenetic regulation of claudin-1 expression in colon cancer cells at the transcriptional and posttranscriptional levels.

Topics: 3' Untranslated Regions; Base Sequence; Blotting, Western; Breast Neoplasms; Cells, Cultured; Chromatin Immunoprecipitation; Claudin-1; Colonic Neoplasms; ELAV Proteins; Epigenesis, Genetic; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Kidney; Molecular Sequence Data; Mutagenesis, Site-Directed; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tristetraprolin

The role of the high mobility group box 1 (HMGB1) protein in chemotherapy-induced cell death was examined. CT26 mouse colon cancer cells were treated with trichostatin A (TSA; apoptosis inducer) or doxorubicin (DXR; necrosis inducer). DXR increased HMGB1 concentration in CT26 cell culture medium, whereas TSA did not. In a CT26 bilateral subcutaneous tumour model, DXR or TSA was injected in a single tumour. After injection, serum HMGB1 concentration in DXR-treated mice was 10 times higher than that in TSA-treated mice. After DXR treatment, the contralateral and remnant tumours showed more pronounced growth than did those treated with TSA. In mouse models, lung and liver metastasis was enhanced by DXR but not by TSA. DXR-enhanced metastasis was abrogated by anti-HMGB1 antibody treatment. In a cancer dormancy model, DXR induced regrowth of quiescent CT26 cells. HMGB1 induced tumour necrosis factor-α secretion via Toll-like receptor (TLR)4 in U937 monocytes; however, HMGB1 decreased the number of U937 cells, resulting in restriction of immune activation via receptor for advanced glycation endproducts (RAGE). RAGE showed a more pronounced effect on nuclear factor kappa B activation than did TLR4 in CT26 cells. These findings suggest that HMGB1 released from necrotic cancer cells treated with a necrosis inducer enhances regrowth and metastasis of remnant cancer cells via RAGE activation.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Doxorubicin; HMGB1 Protein; Humans; Hydroxamic Acids; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Necrosis; Neoplasm Metastasis; Receptor for Advanced Glycation End Products; Receptors, Immunologic; U937 Cells

GRP78, a major endoplasmic reticulum chaperone and signaling regulator, is commonly overexpressed in cancer. Moreover, induction of GRP78 by a variety of anti-cancer drugs, including histone deacetylase inhibitors, confers chemoresistance to cancer, thereby contributing to tumorigenesis. Thus, therapies aimed at decreasing GRP78 levels, which results in the inhibition of tumor cell proliferation and resensitization of tumor cells to chemotherapeutic drugs may hold promise for cancer treatment. Despite advances in our understanding of GRP78 actions, little is known about endogenous inhibitors controlling its expression. As endogenous regulators, microRNAs (miRNAs) play important roles in modulating gene expression; therefore, we sought to identify miRNA(s) that target GRP78, under the hypothesis that these miRNAs may serve as therapeutic agents. Here, we report that three miRNAs (miR-30d, miR-181a, miR-199a-5p) predicted to target GRP78 are down-regulated in prostate, colon and bladder tumors, and human cancer cell lines. We show that in C42B prostate cancer cells, these miRNAs down-regulate GRP78 and induce apoptosis by directly targeting its 3' untranslated region. Importantly, we demonstrate that the three miRNAs act cooperatively to decrease GRP78 levels, suggesting that multiple miRNAs may be required to efficiently control the expression of some genes. In addition, delivery of multiple miRNAs by either transient transfection or lentivirus transduction increased the sensitivity of cancer cells to the histone deacetylase inhibitor, trichostatin A, in C42B, HCT116 and HL-60 cells. Together, our results indicate that the delivery of co-transcribed miRNAs can efficiently suppress GRP78 levels and GRP78-mediated chemoresistance, and suggest that this strategy holds therapeutic potential.

Topics: 3' Untranslated Regions; Adenocarcinoma; Animals; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Heat-Shock Proteins; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Lentivirus; Male; Mice; Mice, Nude; MicroRNAs; Neovascularization, Pathologic; Prostatic Neoplasms; RNA; RNA, Messenger; Thapsigargin; Transcription, Genetic; Transfection; Tumor Stem Cell Assay; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

We previously found that enhanced expression of hepatocyte nuclear factor 4α (HNF-4α) is associated with hyper-proliferation of colon carcinoma cells. Here, the effect of histone deacetylase (HDAC) inhibitors on proliferation and the expression of HNF-4α and its downstream target genes were assessed in HM7, LS174T, HT29 and Caco-2 colon carcinoma cell lines.. HNF-4α expression was found to vary in the different colon carcinoma cell lines tested, being highest in HM7. Additionally, a direct correlation with proliferation was observed. In HM7 cells, the weak HDAC inhibitor butyrate significantly inhibited the transcription of HNF-4α, its downstream target gene MUC4, and genes associated with proliferation, including the proliferating cell nuclear antigen gene PCNA. siRNA-mediated silencing of HNF-4α exerted an effect similar to butyrate on HM7 cell proliferation. The stronger HDAC inhibitor trichostatin A (TSA) exerted an effect similar to that of siRNA-mediated HNF-4α silencing and, concomitantly, inhibited the expression of the transcription factor gene SP1. Also, siRNA-mediated silencing of HDAC3 and HDAC4 reduced HNF-4α expression. Chromatin immunoprecipitation (ChIP) assays revealed that TSA induces hyperacetylation of histones H3 and H4 and, concomitantly, inhibits SP1 binding to the HNF-4α promoter. Subsequent electromobility shift assays supported these latter findings.. HNF-4α transcriptional expression and activity are tightly controlled by epigenetic mechanisms. HDAC inhibitor targeting of HNF-4α may serve as an effective treatment for advanced colon carcinomas, since downstream cancer-associated target genes such as MUC4 are significantly down-regulated by this treatment.

Topics: Acetylation; Blotting, Western; Butyrates; Caco-2 Cells; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 4; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HT29 Cells; Humans; Hydroxamic Acids; Mucin-4; Promoter Regions, Genetic; Protein Binding; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sp1 Transcription Factor; Transcription, Genetic

New orally bioavailable 5-(thiophen-2-yl)-substituted 2-aminobenzamide-series histone deacetylase inhibitors were synthesized. These compounds possess a morpholine or piperadine-derived moiety as an aqueous soluble functional group. Among them, 8b, having a 4-ethyl-2,3-dioxopiperazine-1-carboxamide group as a surface recognition domain, showed promising inhibitory activities against HCT116 cell growth and HDAC1/2. Notably, unlike MS-275, this compound did not induce apoptosis in the cell cycle tests. We therefore conducted antitumor tests of 8b and MS-275 against HCT116 cell xenografts in nude mice. Compound 8b reduced the volume of tumor mass to T/C: 60% and 47% at 45 and 80mg/kg over 16days, respectively. These values were comparable to the rate (T/C: 51% at 45mg/kg) for MS-275. Furthermore, 8b, at neither 45 nor 80mg/kg, induced the weight loss which was observed in the mice given MS-275 at 45mg/kg.

Topics: Animals; Antineoplastic Agents; Benzamides; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Histone Deacetylase Inhibitors; Histone Deacetylases; Mice; Mice, Nude; Thiophenes; Xenograft Model Antitumor Assays

Elevated levels of survivin and histone deacetylases (HDACs) are often found over-expressed in human cancers, including colorectal cancer, and have been implicated in tumorigenesis. HDAC inhibition induces growth arrest and cell death in various transformed cell; however, the mechanisms by which this reduces cell viability in colorectal cancer cells remain unexplained.. We explored the actions of two HDAC inhibitors, trichostatin A (TSA) and sirtinol, in HT29 colon cancer cells.. TSA and sirtinol induced apoptosis and inhibited cell proliferation in HT29 cells. These results are associated with the modulation of survivin. Survivin promoter luciferase activity and Sp1, a transcription factor that contributes to survivin expression, were suppressed in cells exposed to TSA or sirtinol. TSA and sirtinol also activated p38 mitogen-activated protein kinase (p38MAPK) and AMP-activated protein kinase (AMPK). Inhibitors of p38MAPK or AMPK signaling abrogated TSA and sirtinol's effects of decreasing cell viability. Survivin promoter luciferase activity in the presence of TSA or sirtinol was restored by AMPK dominant negative mutant or p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p63 binding to the promoter region increased after TSA or sirtinol exposure.. We report a p38MAPK- and AMPK-mediated downregulation of survivin, and its functional correlation with decreased colon cancer cell viability in the presence of HDAC inhibitor. p63 and Sp1 may also contribute to TSA and sirtinol actions.. This study delineates, in part, the underlying mechanisms of TSA and sirtinol in decreasing survivin expression and subsequent colon cancer cell viability.

Topics: AMP-Activated Protein Kinases; Apoptosis; Benzamides; Cell Proliferation; Cell Survival; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Hydroxamic Acids; Inhibitor of Apoptosis Proteins; Luciferases; Naphthols; p38 Mitogen-Activated Protein Kinases; Plicamycin; Promoter Regions, Genetic; Protein Binding; Sp1 Transcription Factor; Survivin; Transcription Factors; Tumor Suppressor Proteins

MMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors.

Topics: Adenocarcinoma; Butyrates; Cell Line, Tumor; Colonic Neoplasms; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 11; Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Smad Proteins; Sp1 Transcription Factor

The purpose of this study on the involvement of epigenetic control of the expression of solute carrier (SLC) transporters by DNA methylation and histone deacetylation in 4 colon cancer cells is to find the epigenetic control mechanisms of drug transporters in colon cancers. Human colon cancer cell lines (HCT116, HT29, SW48, SW480) were treated with 5-aza-2'-deoxycytidine (DAC), as a DNA methyltransferase inhibitor, followed by trichostatin A (TSA), as a histone deacetylase inhibitor. The mRNA expression and DNA methylation of several SLC transporters were analyzed by real-time polymerase chain reaction (PCR) and methylation-specific PCR, respectively. Among 12 SLC transporters possessing cytosine-phosphate-guanine (CpG) islands, thiamine transporter 2 (THTR2) (SLC19A3) gene showed a correlation between its mRNA expression level and DNA methylation status. TSA treatment increased histone H3 acetylation of THTR2 promoter region in all 4 colon cancer cell lines examined. HCT116 and SW48 cells showed a lack of THTR2 mRNA expression and methylation of its promoter, and DAC treatment induced its re-expression. In addition, the co-treatment with DAC and TSA increased THTR2 mRNA expression more markedly than DAC treatment in HCT116 and SW48 cells. In HT29 and SW480 cells that showed little methylation of THTR2 promoter, TSA treatment induced THTR2 mRNA expression markedly, but DAC treatment did not. In the 4 colon cancer cells examined, THTR2 mRNA expression is down-regulated by DNA methylation and/or histone deacetylation.

Topics: Azacitidine; Cell Line, Tumor; Colonic Neoplasms; CpG Islands; Decitabine; DNA Methylation; DNA Modification Methylases; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Membrane Transport Proteins; Real-Time Polymerase Chain Reaction; RNA, Messenger

Colorectal cancer (CRC) is the third most common cancer in the United States. Approximately 90% of colon cancer deaths arise from the metastasis of primary tumors. Aberrant expression of Wnt5a, one of the WNT signaling factors, has been reported during colon cancer development and progression. We found that both mRNA and protein expression of Wnt5a were decreased in the highly metastatic human colon cancer cell line SW620 compared with the non-metastatic human colon cancer cell SW480. This study tested the hypothesis that the silencing of Wnt5a in metastatic human colon cancer cells is related to altered epigenetic modifications. Wnt5a expression was not responsive to DNA methyltransferase inhibitor 5-aza-cytidine treatment. However, histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate (NaBt) significantly increased Wnt5a mRNA expression in SW620. Importantly, lower transcription of Wnt5a in SW620 than SW480 corresponded to multiple histone modifications, including lower levels of acetylated histone H3, H4 and H3K4me2 and higher levels of H3K27me3 in the promoter region. The increase of H3Ac, H4Ac and H3K4me2 after NaBt treatment in SW620 confirmed the involvement of histone modifications in the transcriptional regulation of Wnt5a. Additionally, NaBt treatment increased β-catenin signaling and diminished the difference in cell adhesion ability between non-metastatic SW480 and metastatic SW620, suggesting that the HDAC inhibitor plays critical roles in the WNT signaling pathway and cell physiology that relate to metastasis. In conclusion, our study suggests the importance of Wnt5a in colon cancer metastasis and also indicates that Wnt5a silencing in the highly invasive human colon cancer cell line might result from transcriptional regulation of the gene by histone modifications.

Topics: Azacitidine; Cell Line, Tumor; Colonic Neoplasms; DNA Modification Methylases; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Neoplasm Metastasis; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Wnt Proteins; Wnt-5a Protein

Ca2+ is a chemopreventive agent for colon cancer. Ion transport systems are often altered in human cancer. The aim of this study was to clarify the alterations of calcium-sensing receptor (CASR), a member of the G protein-coupled receptor family, in colorectal carcinogenesis. We analyzed the expression of CASR in colorectal cancer cell lines and in cancer and adenoma tissues by RT-PCR and immunostaining. In addition, we analyzed methylation of the CASR promoter by using bisulfite sequence analysis and methylation-specific PCR. CASR mRNA and protein expression was significantly downregulated in most of the cancer cell lines. CpG islands were densely methylated in cancer cell lines with reduced CASR mRNA expression. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, and/or a histone deacetylase inhibitor, trichostatin A, restored CASR expression in the cancer cell lines. Disruption of CASR expression in CASR-unmethylated HCT-8 cells blocked the enhancing effect of Ca2+ on the cytotoxic response to 5-fluorouracil. CASR expression was observed in normal colonic epithelial cells and was retained in most adenoma tissues. CASR mRNA and protein expression was significantly downregulated in cancer tissues. There was an inverse relationship between CASR expression and degree of differentiation. Immunohistochemical CASR staining was reduced more predominantly in less-differentiated cancer tissues and/or in cancer cells at the invasive front, where nuclear/cytoplasmic β-catenin was often localized. CASR methylation was detected in 69% of colorectal cancer tissues and 90% of lymph node metastatic tissues and was significantly correlated with reduced CASR expression. CASR methylation was also detected in 32% of advanced adenoma tissues but was detected in only 9% of adenoma tissues and was not detected in hyperplastic polyp tissues. CASR methylation seems to occur at an early stage and progress in colorectal carcinogenesis. The results suggest that epigenetic inactivation of CASR has an important role in colorectal carcinogenesis.

Topics: Adenocarcinoma; Adenoma; Antineoplastic Agents, Alkylating; Azacitidine; Caco-2 Cells; Cell Survival; Colonic Neoplasms; CpG Islands; Decitabine; DNA Methylation; DNA, Neoplasm; Gene Expression; Gene Silencing; Humans; Hydroxamic Acids; Receptors, Calcium-Sensing; RNA, Messenger; Tissue Array Analysis

Our previous study demonstrated that, stanniocalcin-1 (STC1) was a target of histone deacetylase (HDAC) inhibitors and was involved in trichostatin A (TSA) induced apoptosis in the human colon cancer cells, HT29. In this study, we reported that the transcriptional factor, specificity protein 1 (Sp1) in association with retinoblastoma (Rb) repressed STC1 gene transcription in TSA-treated HT29 cells. Our data demonstrated that, a co-treatment of the cells with TSA and Sp1 inhibitor, mithramycin A (MTM) led to a marked synergistic induction of STC1 transcript levels, STC1 promoter (1 kb)-driven luciferase activity and an increase of apoptotic cell population. The knockdown of Sp1 gene expression in TSA treated cells, revealed the repressor role of Sp1 in STC1 transcription. Using a protein phosphatase inhibitor okadaic acid (OKA), an increase of Sp1 hyperphosphorylation and so a reduction of its transcriptional activity, led to a significant induction of STC1 gene expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binding on STC1 proximal promoter in TSA treated cells. The binding of Sp1 to STC1 promoter was abolished by the co-treatment of MTM or OKA in TSA-treated cells. Re-ChIP assay illustrated that Sp1-mediated inhibition of STC1 transcription was associated with the recruitment of another repressor molecule, Rb. Collectively our findings identify STC1 is a downstream target of Sp1.

Topics: Cell Line, Tumor; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Hydroxamic Acids; Okadaic Acid; Phosphorylation; Plicamycin; Protein Synthesis Inhibitors; Repressor Proteins; Retinoblastoma Protein; Sp1 Transcription Factor; Transcription, Genetic

Epigenetic deregulation revealed by altered profiles of DNA methylation and histone modifications is a frequent event in cancer cells and results in abnormal patterns of gene expression. Cancer silenced genes constitute prime therapeutic targets and considerable progress has been made in the epigenetic characterization of the chromatin scenarios associated with their inactivation and drug induced reactivation. Despite these advances, the mechanisms involved in the maintenance or resetting of epigenetic states in both physiological and pharmacological situations are poorly known. To get insights into the dynamics of chromatin regulation upon drug-induced reactivation, we have investigated the epigenetic profiles of two chromosomal regions undergoing long range epigenetic silencing in colon cancer cells in time-course settings after exposure of cells to chromatin reactivating agents. The DNA methylation states and the balance between histone H3K4 methylation and H3K27 methylation marks clearly define groups of genes with alternative responses to therapy. We show that the expected epigenetic remodeling induced by the reactivating drugs, just achieves a transient disruption of the bivalent states, which overcome the treatment and restore the transcriptional silencing approximately four weeks after drug exposure. The interplay between DNA methylation and bivalent histone marks appears to configure a plastic but stable chromatin scenario that is fully restored in silenced genes after drug withdrawal. These data suggest that improvement of epigenetic therapies may be achieved by designing strategies with long lasting effects.

Topics: Azacitidine; Chromatin; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 3; Colonic Neoplasms; CpG Islands; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Gene Silencing; HCT116 Cells; Histones; Humans; Hydroxamic Acids; Inhibin-beta Subunits; Jumonji Domain-Containing Histone Demethylases; Time Factors; Transcriptional Activation

As PTTG1 (pituitary tumor transforming gene) abundance correlates with adverse outcomes in cancer treatment, we determined mechanisms underlying this observation by assessing the role of PTTG1 in regulating cell response to anti-neoplastic drugs. HCT116 cells devoid of PTTG1 (PTTG1(-/-)) exhibited enhanced drug sensitivity as assessed by measuring BrdU incorporation in vitro. Apoptosis, mitosis catastrophe or DNA damage were not detected, but features of senescence were observed using low doses of doxorubicin and TSA. The number of drug-induced PTTG1(-/-) senescent cells increased ∼4 fold as compared to WT PTTG1-replete cells (p<0.001). p21, an important regulator of cell senescence, was induced ∼3 fold in HCT116 PTTG1(-/-) cells upon doxorubicin or Trichostatin A treatment. Binding of Sp1, p53 and p300 to the p21 promoter was enhanced in PTTG1(-/-) cells after treatment, suggesting transcriptional regulation of p21. p21 knock down abrogated the observed senescent effects of these drugs, indicating that PTTG1 likely suppresses p21 to regulate drug-induced senescence. PTTG1 also regulated SW620 colon cancer cells response to doxorubicin and TSA mediated by p21. Subcutaneously xenografted PTTG1(-/-) HCT116 cells developed smaller tumors and exhibited enhanced responses to doxorubicin. PTTG1(-/-) tumor tissue derived from excised tumors exhibited increased doxorubicin-induced senescence. As senescence is a determinant of cell responses to anti-neoplastic treatments, these findings suggest PTTG1 as a tumor cell marker to predict anti-neoplastic treatment outcomes.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cellular Senescence; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Doxorubicin; Female; Gene Knockdown Techniques; HCT116 Cells; Humans; Hydroxamic Acids; Immunohistochemistry; Mice; Mice, Nude; Mutation; Neoplasm Proteins; Promoter Regions, Genetic; Protein Binding; RNA Interference; Securin; Sp1 Transcription Factor; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety-a surface recognition domain introduced to increase in cellular uptake-and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.

Topics: Administration, Oral; Aminobenzoates; Animals; Benzamides; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Histone Deacetylase Inhibitors; Humans; Mice; Mice, Nude; Solubility; Structure-Activity Relationship; Transplantation, Heterologous; Tumor Burden

Expression and cellular distribution of claudin-1, a tight junction protein, is dysregulated in colon cancer and its overexpression in colon cancer cells induced dedifferentiation and increased invasion. However, the molecular mechanism(s) underlying dysregulated claudin-1 expression in colon cancer remains poorly understood. Histone deacetylase (HDAC)-dependent histone acetylation is an important mechanism of the regulation of cancer-related genes and inhibition of HDACs induces epithelial differentiation and decreased invasion. Therefore, in this study, we examined the role of HDAC-dependent epigenetic regulation of claudin-1 in colon cancer. In this study, we show that sodium butyrate and Trichostatin A (TSA), two structurally different and widely used HDAC inhibitors, inhibited claudin-1 expression in multiple colon cancer cell lines. Further studies revealed modulation of claudin-1 mRNA stability by its 3'-UTR as the major mechanism underlying HDAC-dependent claudin-1 expression. In addition, overexpression of claudin-1 abrogated the TSA-induced inhibition of invasion in colon cancer cells suggesting functional crosstalk. Analysis of mRNA expression in colon cancer patients, showed a similar pattern of increase in claudin-1 and HDAC-2 mRNA expression throughout all stages of colon cancer. Inhibition of claudin-1 expression by HDAC-2-specific small interfering RNA further supported the role of HDAC-2 in this regulation. Taken together, we report a novel post-transcriptional regulation of claudin-1 expression in colon cancer cells and further show a functional correlation between claudin-1 expression and TSA-mediated regulation of invasion. As HDAC inhibitors are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.

Topics: 3' Untranslated Regions; Butyrates; Cell Line, Tumor; Cell Movement; Claudin-1; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoblotting; Membrane Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA Stability; RNA, Messenger; Transcription, Genetic

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

Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa [29.5% (18/61)], adenomas [81.0% (47/58)] and carcinomas [82.7% (62/75)] (P = 8.6 x 10(-9))} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2'-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.

Topics: Azacitidine; Cell Line, Tumor; Chromosomes, Human, Pair 18; Colonic Neoplasms; CpG Islands; DCC Receptor; Decitabine; DNA Methylation; Epigenesis, Genetic; Histones; Humans; Hydroxamic Acids; Oxidoreductases, N-Demethylating; Promoter Regions, Genetic; Receptors, Cell Surface; Tumor Suppressor Proteins

Transcriptional activity of NF-kappaB is inhibited by the liganded glucocorticoid receptor (GR), which exists mainly in two splice variants as functional GRalpha and nonfunctional GRbeta. We investigated the effect of 5-aza-2'-deoxycytidine (5-dAzaC), trichostatin A (TSA), and sodium butyrate (NaBu) on GRalpha,GRbeta and ASF/SF2 splicing factor expression in HT-29 colon and MCF-7 breast carcinoma cells.. HT-29 and MCF-7 cells were cultured in the absence or in the presence of 5-dAzaC, TSA, and NaBu, followed by RNA and protein isolation. The transcript and protein levels of GRalpha, GRbeta ASF/SF2 were determined by reverse transcription, real-time quantitative PCR and Western blot analysis.. We found that 5-dAzaC, TSA, and NaBu lead to an increase in GRalpha and ASF/SF2 transcript levels and a decrease in GRbeta transcript levels in HT-29 and MCF-7 cells. The 5-dAzaC, TSA, and NaBu resulted in increased GRalpha and ASF/SF2 protein levels and GRbeta protein downregulation in HT-29 cells. The most increased GRalpha protein expression in MCF-7 cells was observed with NaBu. However, all of these compounds inhibited GRbeta protein expression in MCF-7 cells. The MCF-7 cells treated with NaBu demonstrated a remarkable increase in ASF/SF2 protein expression.. Because NF-kappaB is considered to be a factor in the augmentation of malignant properties of cells, treatment of tumors with 5-dAzaC, TSA, and NaBu may provide a novel approach to the enhancement of therapeutic effects of glucocorticoids in epithelial carcinomas.

Topics: Azacitidine; Breast Neoplasms; Butyrates; Colonic Neoplasms; Decitabine; Enzyme Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Nuclear Proteins; Receptors, Glucocorticoid; RNA-Binding Proteins; Serine-Arginine Splicing Factors

Drosophila Groucho and its human Transducin-like-Enhancer of Split orthologs (TLEs) function as transcription co-repressors within the context of Wnt signaling, a pathway with strong links to cancer. The current model for how Groucho/TLE's modify Wnt signaling is by direct competition with beta-catenin for LEF/TCF binding. The molecular events involved in this competitive interaction are not defined and the actions of Groucho/TLEs within the context of Wnt-linked cancer are unknown.. We used in vitro protein interaction assays with the LEF/TCF family member LEF-1, and in vivo assays with Wnt reporter plasmids to define Groucho/TLE interaction and repressor function.. Mapping studies reveal that Groucho/TLE binds two regions in LEF-1. The primary site of recognition is a 20 amino acid region in the Context Dependent Regulatory domain. An auxiliary site is in the High Mobility Group DNA binding domain. Mutation of an eight amino acid sequence within the primary region (RFSHHMIP) results in a loss of Groucho action in a transient reporter assay. Drosophila Groucho, human TLE-1, and a truncated human TLE isoform Amino-enhancer-of-split (AES), work equivalently to repress LEF-1*beta-catenin transcription in transient reporter assays, and these actions are sensitive to the HDAC inhibitor Trichostatin A. A survey of Groucho/TLE action in a panel of six colon cancer cell lines with elevated beta-catenin shows that Groucho is not able to repress transcription in a subset of these cell lines.. Our data shows that Groucho/TLE repression requires two sites of interaction in LEF-1 and that a central, conserved amino acid sequence within the primary region (F S/T/P/xx y I/L/V) is critical. Our data also reveals that AES opposes LEF-1 transcription activation and that both Groucho and AES repression require histone deacetylase activity suggesting multiple steps in Groucho competition with beta-catenin. The variable ability of Groucho/TLE to oppose Wnt signaling in colon cancer cells suggests there may be defects in one or more of these steps.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Cell Line, Tumor; Chlorocebus aethiops; Colonic Neoplasms; COS Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Lymphoid Enhancer-Binding Factor 1; Mutation; Protein Binding; Protein Structure, Tertiary; Repressor Proteins; Transfection

Muscle wasting, as occurring in cancer cachexia, is primarily characterized by protein hypercatabolism and increased expression of ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1. Myostatin, a member of the TGFbeta superfamily, negatively regulates skeletal muscle mass and we showed that increased myostatin signaling occurs in experimental cancer cachexia. On the other hand, enhanced expression of follistatin, an antagonist of myostatin, by inhibitors of histone deacetylases, such as valproic acid or trichostatin-A, has been shown to increase myogenesis and myofiber size in mdx mice. For this reason, in the present study we evaluated whether valproic acid or trichostatin-A can restore muscle mass in C26 tumor-bearing mice. Tumor growth induces a marked and progressive loss of body and muscle weight, associated with increased expression of myostatin and ubiquitin ligases. Treatment with valproic acid decreases muscle myostatin levels and enhances both follistatin expression and the inactivating phosphorylation of GSK-3beta, while these parameters are not affected by trichostatin-A. Neither agent, however, counteracts muscle atrophy or ubiquitin ligase hyperexpression. Therefore, modulation of the myostatin/follistatin axis in itself does not appear sufficient to correct muscle atrophy in cancer cachexia.

Topics: Animals; Cachexia; Colonic Neoplasms; Disease Models, Animal; Drug Evaluation, Preclinical; Enzyme Inhibitors; Follistatin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Hydroxamic Acids; Mice; Mice, Inbred BALB C; Muscles; Muscular Atrophy; Myostatin; Neoplasm Transplantation; Ubiquitin-Protein Ligases; Valproic Acid

The antineoplastic activity of HDAC inhibitors is an unquestionable property of these compounds, but recent studies in tumor cells have revealed the potential of HDAC inhibitors (e.g., suberoylanilide hydroxamic acid SAHA, valproic acid VPA) to cause acquisition of HDAC inhibitor resistance. We report that trichostatin A (TSA), an HDAC inhibitor structurally related to SAHA, causes the acquisition of multidrug resistance transporter-independent and irreversible 3-fold resistance to TSA in MLH1-deficient (absent MLH1 protein expression) but not in MLH1-proficient (expressing MLH1 protein) HCT116 colon tumor cells. This MLH1-deficient subline selected for TSA resistance by stepwise exposures to increasing TSA concentrations exhibited failure in the accumulation of acetylated histones, in p21 induction, and in apoptosis activation. These are cellular responses normally seen in tumor cells treated with HDAC inhibitors. Whereas the absence of acetyl-histone accumulation did not correlate with altered HDAC activity, the absence of apoptosis correlated with reduced expression of (pro-apoptotic) Bax. This TSA-resistant subline was cross-resistant to SAHA and VPA but not to 'classic' non-HDAC inhibitor-type anticancer agents such as docetaxel and doxorubicin. These herein presented results expand on a previous study reporting HDAC inhibitor resistance acquisition by SAHA which was independent of the MLH1 expression status. Taken together, the present study identifies TSA, besides SAHA and VPA, as another potential causative of HDAC inhibitor resistance acquisition specifically in MLH1-deficient HCT116 colon tumor cells, and it reveals a possible function of MLH1 protein in protecting colon tumor cells from resistance acquisition by TSA.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoprecipitation; Valproic Acid; Vorinostat

Growth arrest and DNA damage inducible 45 alpha (GADD45alpha) is a central player in mediating apoptosis induced by a variety of stress stimuli and genotoxic agents. Regular usage of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and sulindac is associated with reduced risk for various cancers, including colon cancer. The role of GADD45alpha in NSAID-induced colon cancer cell cytotoxicity is unknown. In this study, we report that indomethacin and sulindac sulfide treatments up-regulate GADD45alpha mRNA expression and protein levels in colon cancer HT-29, RKO and Caco-2 cells. This up-regulation of GADD45alpha is accompanied by necrotic cell death and apoptosis. Anti-sense suppression of GADD45alpha expression inhibited indomethacin and sulindac sulfide-induced necrotic cell death and apoptosis. These findings confirm a role for GADD45alpha in NSAID-induced cytotoxicity, a mechanism for the anti-neoplastic effect of NSAIDs in colon tumorigenesis and cancer growth.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caco-2 Cells; Cell Cycle Proteins; Colonic Neoplasms; Humans; Hydroxamic Acids; Indomethacin; Necrosis; Nuclear Proteins; Oligonucleotides, Antisense; RNA, Messenger; Sulindac; Up-Regulation

Annexin A1 is a member of a phospholipid and calcium binding family of proteins; it is involved in anti-inflammation and in the regulation of differentiation, proliferation, and apoptosis. Here, we show the existence of a functional binding site for the tumor suppressor p53 near the proximal CCAAT box and the fact that the basal expression of annexin A1 in human colon adenocarcinoma cells is driven by p53 at the transcriptional level. Posttranscriptional mechanisms may also play an important role in maintaining constitutive annexin A1 expression. In addition, a p53/NF-Y complex is detected bound to the p53 binding site on its promoter. Butyrate is a natural product of fiber degradation in the colon and a key regulator of colonic epithelium homeostasis. We show that butyrate, a class I and II histone deacetylase inhibitor, induces transcriptional activation of annexin A1 expression correlated with differentiation. The effect of butyrate is mediated through a release of NF-Y from the proximal CCAAT box and an enhancement of p53 binding. The interaction of p53 with the promoter is dependent on p38 MAPK activity either in the absence or in the presence of butyrate. Further, activation of p38 MAPK by this agent is required to increase annexin A1 promoter activity and to increase protein expression.

Topics: Adenocarcinoma; Annexin A1; Base Sequence; Binding Sites; Biomarkers, Tumor; Blotting, Western; Butyrates; CCAAT-Binding Factor; Cell Differentiation; Cell Line, Tumor; Chromatin Immunoprecipitation; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Molecular Sequence Data; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53; Up-Regulation

The membrane transporters such as P-glycoprotein (Pgp), the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines.. The MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses.. The MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression) but not SNU-668 (gastric, highest) and SNU-C5 (gastric, no expression) to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor) increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor) increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells.. These results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly lower than those in colon cancer cells, which is at least in part due to different epigenetic regulations such as DNA methylation and/or histone deacetylation. These results can provide a better understanding of the efficacy of combined chemotherapy as well as their oral bioavailability.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azacitidine; Cell Line, Tumor; Colonic Neoplasms; Decitabine; DNA Methylation; DNA, Neoplasm; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Profiling; Histones; Humans; Hydroxamic Acids; RNA, Messenger; Sequence Analysis, DNA; Stomach Neoplasms

The objective of this study was to investigate combinations of two chemopreventive dietary factors: EGCG 20 microM (or 100 microM) and SFN (25 microM) in HT-29 AP-1 human colon carcinoma cells.. After exposure of HT-29 AP-1 cells to SFN and EGCG, individually or in combination, we performed AP-1 luciferase reporter assays, cell viability assays, isobologram analyses, senescence staining, quantitative real-time PCR (qRT-PCR) assays, Western blotting, and assays for HDAC activity and hydrogen peroxide. In some experiments, we exposed cells to superoxide dismutase (SOD) or Trichostatin A (TSA) in addition to the treatment with dietary factors.. The combinations of SFN and EGCG dramatically enhanced transcriptional activation of AP-1 reporter in HT-29 cells (46-fold with 25 microM SFN and 20 microM EGCG; and 175-fold with 25 microM SFN and 100 microM EGCG). Isobologram analysis showed synergistic activation for the combinations with combination index, CI < 1. Interestingly, co-treatment with 20units/ml of SOD, a free radical scavenger, attenuated the synergism elicited by the combinations (2-fold with 25 muM SFN and 20 muM EGCG; and 15-fold with 25 microM SFN and 100 microM EGCG). Cell viability assays showed that the low-dose combination decreased cell viability to 70% whereas the high-dose combination decreased cell viability to 40% at 48 h, with no significant change in cell viability at 24 h as compared to control cells. In addition, 20 microM and 100 microM EGCG, but not 25 microM SFN, showed induction of senescence in the HT-29 AP-1 cells subjected to senescence staining. However, both low- and high-dose combinations of SFN and EGCG attenuated the cellular senescence induced by EGCG alone. There was no significant change in the protein levels of phosphorylated forms of ERK, JNK, p38, and Akt-Ser473 or Akt-Thr308. Besides, qRT-PCR assays corroborated the induction of the luciferase gene seen with the combinations in the reporter assay. Relative expression levels of transcripts of many other genes known to be either under the control of the AP-1 promoter or involved in cell cycle regulation or cellular influx-efflux such as cyclin D1, cMyc, ATF-2, Elk-1, SRF, CREB5, SLCO1B3, MRP1, MRP2 and MRP3 were also quantified by qRT-PCR in the presence and absence of SOD at both 6 and 10 h. In addition, pre-treatment with 100 ng/ml TSA, a potent HDAC inhibitor, potentiated (88-fold) the synergism seen with the low-dose combination on the AP-1 reporter transcriptional activation. Cytoplasmic and nuclear fractions of treated cells were tested for HDAC activity at 2 and 12 h both in the presence and absence of TSA, however, there was no significant change in their HDAC activity. In addition, the H2O2 produced in the cell system was about 2 microM for the low-dose combination which was scavenged to about 1 microM in the presence of SOD.. Taken together, the synergistic activation of AP-1 by the combination of SFN and EGCG that was potentiated by HDAC inhibitor TSA and attenuated by free radical scavenger SOD point to a possible multifactorial control of colon carcinoma that may involve a role for HDACs, inhibition of cellular senescence, and SOD signaling.

Topics: Catechin; Cell Survival; Cellular Senescence; Colonic Neoplasms; Drug Synergism; HT29 Cells; Humans; Hydroxamic Acids; Isothiocyanates; Reactive Oxygen Species; Sulfoxides; Superoxide Dismutase; Thiocyanates; Transcription Factor AP-1; Transcriptional Activation

Many studies aim at improving therapeutic efficacy by combining strategies with oxidative stress-inducing drugs and histone deacetylase (HDAC) inhibitors in colorectal cancer. As p53 and p21(WAF1) are essential in oxidative stress-induced DNA damage, we investigated epigenetic regulation of p21(WAF1) promoter. Firstly, HCT116 p53(+)/(+) and p53(-)/(-) colorectal cancer cells were treated with H(2)O(2) for 6 hrs and 24 hrs (early/late response). Chromatin immunoprecipitation revealed transcriptional transactivation of p21(WAF1) in HCT116 p53(+)/(+) cells as shown by increased binding of p53 and acetylated H4 around two p21(WAF1) promoter sites, the responsible element (RE) and the Sp1 site, while both proteins bound preferentially on the RE. Interestingly, H3 was not involved, suggesting H4-specific transactivation of the p21(WAF1) promoter. H(2)O(2) addition resulted in G(2)/M arrest of both HCT116 cell lines without significant cell death. To investigate whether a HDAC inhibitor strengthens G(2)/M arrest, we pretreated cells with Trichostatin A (TSA). In HCT116 p53(+)/(+) cells, we found (i) remarkably increased acetylated H4 around both p21(WAF1) promoter regions, especially at the Sp1 site; (ii) increased acetylation of p53 at lysines 320 and 382;(iii) displacement of HDAC1 from the Sp1 site, thus inhibiting its repression effect and increasing p53 binding.p53 seems to trigger H4-acetylation around the p21(WAF1) promoter because there was nearly no H4 acetylation in HCT116 p53(-)/(-) cells. For the first time we show that there is a time-dependent TSA mode of action with increased p53-dependent histone H4 acetylation at the p21(WAF1) promoter in early response, and decreased acetylation in late response. Reduced p53-triggered transactivation of p21(WAF1) in late response allows cells to re-enter cell cycle, and TSA causes p53 to simultaneously induce apoptosis.

Topics: Acetylation; Annexin A5; Apoptosis; Cell Cycle; Cell Line, Tumor; Chromatin Immunoprecipitation; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; G2 Phase; HCT116 Cells; Histones; Humans; Hydrogen Peroxide; Hydroxamic Acids; Lysine; Mitosis; Models, Biological; Promoter Regions, Genetic; Time Factors; Transcriptional Activation; Tumor Suppressor Protein p53

Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

Topics: Calcium; Cell Cycle; Cell Differentiation; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation, Neoplastic; Histone Deacetylases; HT29 Cells; Humans; Hydroxamic Acids; Intestines; Mucus; Promoter Regions, Genetic; Transcription, Genetic

Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDAC are involved in regulation of endothelial cell gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes down-regulated in tumor-conditioned versus quiescent endothelial cells, and reexpressed by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA). Among the 81 genes identified, 77% harbored a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant down-regulation in tumor-conditioned endothelial cells and reactivation by treatment with a combination of DAC and TSA, as well as by both compounds separately. Silencing of these genes in tumor-conditioned endothelial cells correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, but did not involve DNA methylation of promoter CpG islands. For six genes, down-regulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1, and quiescin Q6 are negative regulators of endothelial cell growth and angiogenesis. In summary, our data identify novel angiogenesis-suppressing genes that become silenced in tumor-conditioned endothelial cells in association with promoter histone modifications and reactivated by DNMT and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis.

Topics: Azacitidine; Caco-2 Cells; Clusterin; Colonic Neoplasms; Decitabine; DNA Methylation; DNA Modification Methylases; Endothelial Cells; Epigenesis, Genetic; Fibrillin-1; Fibrillins; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Microfilament Proteins; Neovascularization, Pathologic; Oxidoreductases Acting on Sulfur Group Donors; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Thioredoxins

To investigate the functions of promoter hypermethylation of secreted frizzled-related protein (sFRP) genes in colorectal tumorigenesis and progression.. Three colorectal cancer cell lines, RKO, HCTll6 and SW480, were treated hy 5-aza-2'-deoxycytidine and trichostatin A for demethylation. The promoter hypermethylation and expression of sFRP genes in colorectal tumor tissue and colorectal cancer cell lines were detected hy methylation-specific PCR and reverse transcription PCR, respectively.. None of the normal colorectal mucosa tissues showed methylation of sFRP genes. sFRP1, 2, 4 and 5 were frequently methylated in colorectal adenocarcinoma, adenoma and aberrant crypt foci (ACF) (sFRP1 > 85%, sFRP2 > 75%, sFRP5 > 50%), the differences between any two of them were not significant (P >0.05). Methylation was more frequent in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor. Hypermethylation of sFRP genes was present in three colorectal cancer cell lines. When sFRP genes were methylated, their corresponding mRNA expression was absent. After cells were treated by DAC/TSA combination, the silenced sFRP expression could be effectively re-expressed.. Hypermethylation of sFRP genes is a common early event in the evolution of colorectal tumors that occurs frequently in ACF. Methylation of sFRP1, 2 and 5 genes might serve as biomarkers for the early detection of colorectal tumors. Demethylation can effectively reverse gene expression that appears possibly to be an effective way for tumor therapy.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenoma; Adult; Aged; Azacitidine; Biomarkers, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Decitabine; DNA Methylation; DNA Modification Methylases; Eye Proteins; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; HCT116 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; RNA, Messenger

Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation and/or apoptosis in numerous cancer cell types and have shown promise in clinical trials. These agents are particularly novel, given their ability to selectively influence gene expression. Previously, we demonstrated that the HDIs butyrate and trichostatin A (TSA) directly repress c-Src proto-oncogene expression in many cancer cell lines. Activation and/or overexpression of c-Src have been frequently observed in numerous malignancies, especially of the colon. Therefore, our observation was particularly interesting since butyrate is a naturally abundant component of the large intestine and has been suggested to be a cancer-preventive agent. However, c-Src is not the only Src family kinase (SFK) member to be implicated in the development of human cancers, including those of the colon. Therefore, the relative expression levels of known SFKs were examined in a panel of human colon cancer cell lines. We found a surprisingly diverse expression pattern but noted that most cell lines expressed relatively high levels of at least 2 SFKs. When the effects of butyrate and TSA were examined in representative cell lines, the expression of all SFKs was repressed in a dose- and time-dependent manner. Further, detailed examination of Lck, Yes and Lyn demonstrated that this repression had a direct effect on transcription and was independent of new protein synthesis. These results mirror our earlier data obtained with c-Src and suggest that SFKs are a major target of HDIs and likely account in part for the anticancer effects of these promising new drugs.

Topics: Butyrates; Chloramphenicol O-Acetyltransferase; Colonic Neoplasms; Dose-Response Relationship, Drug; Down-Regulation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Oncogene Protein pp60(v-src); Proto-Oncogene Mas; Proto-Oncogene Proteins c-yes; src-Family Kinases; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

This study explored the eyes absent 4 (EYA4) gene promoter methylation in noncolitic colorectal tissues and assessed its discrimination for neoplasia in chronic ulcerative colitis (CUC).. The methylation status of noncolitic specimens was confirmed by direct bisulfite sequencing. Methylation-specific polymerase chain reaction (MSP) primers were designed to evaluate colorectal tissues, including 50 noncolitic patients comprising 24 normal epithelia, 14 polyps, and 12 cancers. The assay was tested on tissues from 67 CUC patients including 31 surveillance neoplasia-positive patients and nonneoplastic controls including 22 CUC surveillance-negative and 14 CUC short-disease duration. Remote colonic tissue was included from each of 27 of the 31 CUC neoplasia cases. The expression of EYA4 was quantified in cell lines by use of reverse-transcription polymerase chain reaction.. Within noncolitic tissues, bisulfite sequencing showed EYA4 promoter hypermethylation in 80% (8 of 10) of colorectal cancers but in none (0 of 9) of the normal tissues. MSP was positive in 81% (21 of 26) of cancers and polyps and in only 4% (1 of 14) of normal mucosa. In CUC, MSP was positive in 81% (25 of 31) of neoplastic cases but in none (0 of 36) of the nonneoplastic controls. RNA expression was decreased in methylated compared with unmethylated cell lines (P < .001). Treatment with 5-Aza-2'-deoxycytidine (DAC)/Trichostatin (TSA) increased the overall messenger RNA expression (P = .005).. The EYA4 gene promoter is hypermethylated commonly in sporadic and colitic neoplasia and may be associated with gene silencing. EYA4 methylation represents a candidate marker for CUC surveillance.

Topics: Azacitidine; Biomarkers; Cell Line; Chronic Disease; Colitis, Ulcerative; Colonic Neoplasms; Colonic Polyps; Decitabine; Humans; Hydroxamic Acids; Methylation; Polymerase Chain Reaction; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators

X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) antagonizes the anti-caspase activity of XIAP. XAF1 messenger RNA is present in normal tissues but undetectable in various cancers and thus poses a potential tumor suppressor gene. The aim of this study was to examine the novel pattern of methylation of XAF1 in gastric and colon cancers and locate the important CpG sites for transcriptional regulation and tumor progression.. XAF1 expression was detected by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis. Four different fragments around the transcription start site of XAF1 were cloned and examined putative promoter activities by luciferase reporter assay. Each CpG site in fragment F291 was mutated by site-directed mutagenesis technique, and the change of promoter activity of this fragment was detected by luciferase reporter assay. Methylation status of XAF1 was determined by methylation-specific PCR (MSP) and bisulfite DNA sequencing PCR analysis.. Down-regulation of XAF1 in association with hypermethylation was detected in 3 of 4 human gastric cancer cell lines and 6 of 8 colon cancer cell lines. Of the 4 promoter fragments, F291 showed the highest promoter activity, which could be down-regulated obviously by the mutation of particular CpG sites. Moreover, aberrant hypermethylation of these important CpG sites was strongly associated with the development of gastric and colon cancers.. A cluster of methylated CpG sites instead of CpG islands located in the promoter area resulted in gene silencing of XAF1, and CpGs at -2nd, -1st, and +3rd positions are functionally more important in its transcriptional regulation.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Apoptosis Regulatory Proteins; Azacitidine; Cell Line, Tumor; Colonic Neoplasms; CpG Islands; Decitabine; DNA Methylation; DNA, Neoplasm; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Male; Middle Aged; Neoplasm Proteins; Promoter Regions, Genetic; Protein Synthesis Inhibitors; RNA, Messenger; Stomach Neoplasms

We are interested in the mechanism of cyclooxygenase-2 (Cox-2) regulation in colon cancer cells because this knowledge could provide insight into colon carcinogenesis and suggest ways to suppress Cox-2 expression in colon tumors. Studying the HT-29 colon cancer cell line as a model, we found that Cox-2 mRNA and protein levels were activated over 10-fold by the inflammatory cytokine tumor necrosis factor (TNF)-alpha. Moreover, we found that the histone deacetylase inhibitors butyrate and trichostatin A could block Cox-2 activation in a gene-specific manner. TNF-alpha and butyrate did not significantly affect Cox-2 promoter activity, mRNA stability, or negative regulation by the Cox-2 3'-untranslated RNA region. A nuclear run-on assay showed that TNF-alpha increased Cox-2 transcription, whereas butyrate was suppressive. Because butyrate has been reported to suppress polymerase elongation on the c-myc gene, we employed the chromatin immunoprecipitation assay to determine the influence of butyrate and trichostatin A on polymerase distribution on the Cox-2 gene. These data indicated that butyrate restricted polymerase elongation from exon 1 to 2 on both the c-myc and Cox-2 genes. We propose that histone deacetylases regulate a transcriptional block on the Cox-2 and c-myc genes and that this block may be a potential target for pharmacological intervention.

Topics: Butyrates; Colonic Neoplasms; Cyclooxygenase 2; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; RNA Polymerase II; RNA, Messenger; Tumor Necrosis Factor-alpha

The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

Topics: Apoptosis; Butyric Acid; Carcinoma, Small Cell; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Nucleus; Chromatin; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Colonic Neoplasms; Enzyme Inhibitors; Fetus; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HT29 Cells; Humans; Hydroxamic Acids; Interphase; Lung Neoplasms

To investigate the effects of histone acetylation on the expression of p21(WAF1) and p16(INK4A) genes in two human colon cancer cell lines.. Two colon cancer cell lines (SW1116 and Colo-320) were treated with the DNA methyltransferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC) and/or the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) or sodium butyrate (NaBu). The cell cycle distribution was studied by flow cytometry (FCM). The expression of p21(WAF1) and p16(INK4A) genes mRNA was detected by real-time RT-PCR. The level of acetylated histones in chromatin associated with the p21(WAF1) and p16(INK4A) genes was examined by chromatin immunoprecipitation (ChIP) assay.. TSA or NaBu blocked cells mainly in the G(1) phase, whereas 5-aza-dC treatment failed to affect cell cycle distribution. Expression of p16(INK4A) was detected slightly and p21(WAF1) was not expression in SW1116 and Colo-320 cells before treatment. In SW1116 and Colo-320 cells, the expression of p16(INK4A) gene was markedly increased after treatment of 5-aza-dC, although 5-aza-dC treatment did not activate the expression of p21(WAF1) gene. Treatment of TSA and NaBu resulted in the significant over-expression of p21(WAF1) in these two cell lines and induced an accumulation of acetylate histones H3 and H4 in chromatin associated with p21(WAF1) gene.. In these two human colon cancer cell lines, HDAC inhibitors stimulate the p21(WAF1) gene expression by selectively increasing the degree of acetylation of the gene-associated histones, and induce a G(1) cell cycle arrest. The expression of the p16(INK4A) gene is regulated by DNA methylation.

Topics: Acetylation; Butyrates; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Cox-2 plays an important role in colon carcinogenesis and inflammation. Studying the HT-29 colon cancer cell line as a model, we found that Cox-2 expression and activity is increased approximately 25-fold by TNF-alpha. As previously reported for other Cox-2 inducers, this activation appears to result from a p38-mediated mRNA stabilization rather than an increase in promoter activity. The HDAC inhibitors butyrate and TSA blocked the TNF-alpha activation of Cox-2 protein and mRNA synthesis, and dramatically suppressed Cox-2 activity in HT-29 cells. The suppression of Cox-2 synthesis did not involve promoter inactivation and could be achieved even when applied after the TNF-alpha stimulus. The effect of the HDAC inhibitors was observed prior to the activation of p21 expression and did not require new protein synthesis. Finally, butyrate did not prevent p38 phosphorylation, so the block is likely to occur at a later step in the activation pathway. We propose that a component of the cytokine-induced Cox-2 mRNA stabilization pathway is sensitive to acetylation.

Topics: Butyrates; Cell Line, Tumor; Colonic Neoplasms; Cyclooxygenase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Isoenzymes; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Tumor Necrosis Factor-alpha

Sodium butyrate, a short-chain fatty acid naturally present in the human colon, is able to induce cell cycle arrest, differentiation and apoptosis in various cancer cells. Sodium butyrate is most probably related to the inhibition of deacetylases leading to hyperacetylation of chromatin components such as histones and non-histone proteins and to alterations in gene expression. In this study, we demonstrate for the first time that sodium butyrate selectively up-regulated DR5 but had no effect on the expression of the other TNF-alpha-related apoptosis-inducing ligand (TRAIL) receptor, DR4. Sodium butyrate-induced expression of DR5 involves the putative Sp1 site within the DR5 promoter region. Using a combination of the electrophoretic mobility shift assay and the luciferase reporter assay, we found that a specific Sp1 site (located at -195 bp relative to the transcription start site) is required for sodium butyrate-mediated activation of the DR5 promoter. When HCT116 cells were incubated with sodium butyrate and TRAIL, enhanced TRAIL-mediated apoptosis was observed. The enhanced apoptosis was measured by fluorescent activated cell sorting analysis, DNA fragmentation, poly (ADP-ribose) polymerase cleavage, down-regulation of XIAP and caspase activity. Taken together, the present studies suggest that sodium butyrate may be an effective sensitizer of TRAIL-induced apoptosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Butyrates; Caspases; Colonic Neoplasms; Down-Regulation; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Luciferases; Membrane Glycoproteins; Poly(ADP-ribose) Polymerases; Promoter Regions, Genetic; Proteins; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Sp1 Transcription Factor; TNF-Related Apoptosis-Inducing Ligand; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

The aim of this study is to assess the effects of DNA methylation and histone acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methylation-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Topics: Acetylation; Azacitidine; Butyrates; Cell Cycle; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Decitabine; DNA; DNA Methylation; Gene Expression Regulation, Neoplastic; Genes, APC; Genes, p16; Histones; Humans; Hydroxamic Acids

To investigate the effect of histone acetylation on regulation of p21WAF1 gene expression in human colon cancer cell lines.. Two cell lines, Colo-320 and SW1116 were treated with either trichostatin or sodium butyrate. Expressions of p21WAF1 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Acetylation of two regions of p21WAF1 gene-associated histones and total cellular histones were examined by chromatin immunoprecipitation assay and Western blotting.. Trichostatin or sodium butyrate re-activated p21WAF1 transcription resulted in up-regulated p21WAF1 protein level in colon cancer cell lines. Those effects were accompanied by an accumulation of acetylated histones in total cellular chromatin and p21WAF1 gene-associated region of chromatin.. Histone acetylation regulates p21WAF1 expression in human colon cancer cell lines, Colo-320 and SW1116.

Topics: Acetylation; Butyrates; Cell Cycle Proteins; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Protein Synthesis Inhibitors; RNA, Messenger

Activin A has been reported to play a role in the progression of colorectal cancer. Because dietary fiber protects against colorectal cancer, we hypothesized that butyrate, a fermentation product of dietary fiber, may affect the expression of activin A in colon cancer cells. Semiquantitative RT-PCR demonstrated that the activin A gene was upregulated by sodium butyrate in the human colon cancer cell lines HT-29 and Caco-2 in a concentration- and time-dependent manner. However, the activin A gene did not respond to sodium butyrate in the human normal colonic cell line FHC, rat normal intestinal epithelial cell (IEC) line IEC-6, and the explant of rat colon. Flow cytometry and agarose gel electrophoresis of genomic DNA revealed that cell cycle arrest and apoptosis were induced by sodium butyrate but not exogenous activin A in HT-29 cells, indicating that activin A could not act as an autocrine factor in colon cancer cells. By assuming that activin A promotes colorectal cancer spread as a paracrine factor, our findings suggest that butyrate could act as a tumor promoter in some circumstances.

Topics: Activins; Animals; Butyrates; Cell Cycle; Colonic Neoplasms; DNA Fragmentation; Humans; Hydroxamic Acids; Inhibin-beta Subunits; Male; Rats; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation

The inhibition of histone deacetylase activity is known to induce morphological changes of transformed cells. In this study, we investigated the effect of the specific HDAC inhibitor, trichostatin A (TSA), on colon carcinoma cell lines. Treatment of human colorectal carcinoma cells, KM1214 and KM12SM, with TSA induced distinct morphological changes. Both cell lines, which normally piled up in layers without clear boundary, became more flattened, and formed monolayers with evident boundaries between cells, with concomitant increased actin filament organization. Cell-cell interaction was not affected much, based on expression level, membrane localization, and interaction of E-cadherin with beta-catenin. In contrast, syndecan-2 expression was dramatically reduced and it was correlated with the morphological changes of colon carcinoma cells. Consistently, downregulation of syndecan-2 expression by antisense cDNA clearly mimicked the morphological changes in KM12SM and reduced anchorage-independent growth of colon cancer cells. All these results indicate that reduced syndecan-2 expression correlates with TSA-induced morphological changes and reduced tumorigenic activity in colon carcinoma cells.

Topics: beta Catenin; Cadherins; Cell Adhesion; Cell Communication; Colonic Neoplasms; Cytoskeletal Proteins; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Membrane Glycoproteins; Neoplasm Metastasis; Proteoglycans; Syndecan-2; Trans-Activators; Tumor Cells, Cultured

CDX2 is a caudal-related homeobox transcription factor whose expression in the adult is normally restricted to intestinal epithelium. Mice heterozygous for germ line Cdx2 inactivation develop intestinal polyps, and the lesions lack Cdx2 expression. Prior studies indicate some human colon carcinomas also lack CDX2 expression. To address the role of CDX2 defects in colon cancer development, we analyzed CDX2 expression in 45 primary colorectal carcinomas. Four carcinomas lacked CDX2 expression, and three others showed aberrant cytoplasmic localization of CDX2, although no significant CDX2 gene defects were seen in the seven tumors. Marked reductions in CDX2 transcript and protein levels were seen in five of 13 colorectal cell lines, and nuclear run-off data indicated reduced transcription was a major factor in CDX2 silencing. Treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A did not restore CDX2 expression in CDX2-negative lines. However, consistent with a role for dominant repression mechanisms in CDX2 silencing, all somatic cell hybrids resulting from pairwise fusions between colon cancer lines with intact CDX2 expression and lines lacking CDX2 had reduced CDX2 transcripts and protein. A roughly 9.5-kb 5'-flanking region from the human CDX2 gene contained key cis elements for regulating transcription in colon cancer cells. Restoration of CDX2 expression suppressed proliferation and soft agar growth in the CDX2-negative HT-29 colon cancer cell line. Our findings suggest CDX2 inactivation in colon cancer results from defects in trans-acting pathways regulating CDX2 transcription, and CDX2 silencing contributes to the altered phenotype of some colorectal cancers.

Topics: Adenoma; Azacitidine; Blotting, Western; CDX2 Transcription Factor; Cell Division; Colonic Neoplasms; Cytoplasm; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Immunohistochemistry; Methyltransferases; Mutation; Mutation, Missense; Phenotype; Phosphatidylinositol 3-Kinases; Rectal Neoplasms; Trans-Activators; Transcription, Genetic

Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC50 value of approximately 0.1-0.3 microM), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC50 value of approximately 0.3 microM) versus HDAC3 (IC50 value of approximately 8 microM) and had no inhibitory activity toward HDAC8 (IC50 value >100 microM). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.

Topics: Benzamides; Cloning, Molecular; Colonic Neoplasms; Drug Interactions; Enzyme Inhibitors; Gene Expression; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Protein Isoforms; Pyridines; Recombinant Proteins; Tumor Cells, Cultured

Utilizing tranexamic acid as a starting material, a series of N-hydroxycarboxamides were synthesized in order to seek new histone deacetylase (HDAC) inhibitors. Further structure optimization involving the replacement of the 1,4-cyclohexylene group with the 1,4-phenylene group yielded the promising HDAC inhibitors which possess a terminal bicyclic aryl amide.

Topics: Amides; Colonic Neoplasms; Drug Design; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Kinetics; Models, Molecular; Molecular Conformation; Structure-Activity Relationship; Tumor Cells, Cultured

The single-copy gene for rat gamma-glutamyltransferase (GGT) encodes at least seven distinct mRNAs that differ in their 5'-untranslated regions only. Tissue- and developmental-specific expression of GGT is partly achieved by the presence of many transcription factor-binding sites in the promoters of this gene. In an earlier study we found that GGT mRNAs II and IV levels were increased upon butyrate-induced differentiation of the rat colon carcinoma cell line CC531. The mechanism for this butyrate-induced upregulation remains unknown, but may result from altered promoter activity as butyrate is a known histone deacetylase inhibitor. In the present study, we show by transient transfection studies that butyrate enhanced the expression of the luciferase reporter gene driven by the rat GGT promoter 2 (P2). Trichostatine A (TSA), another histone deacetylase inhibitor, also enhanced transcription from this promoter. The role of the transcription factor site Sp1 in butyrate- or TSA-induced activation of the GGT P2 was examined as Sp1 has been previously shown to play a central role in the transcriptional activation of other genes during butyrate and TSA stimulation. A triple sequence-motif of this isolated Sp1 site linked to a minimal promoter was able to mediate butyrate- and TSA-induced expression of the luciferase reporter gene, while no effect was measured using the minimal promoter alone. Deleting the Sp1 site in the context of the rat GGT P2 strongly reduced the basal transcription activity and abrogated butyrate- and TSA-induced activation of the mutated promoter. These results suggest that butyrate- or TSA-induced activation of the rat GGT P2 can be mediated by a Sp1 binding motif.

Topics: Animals; Base Sequence; Butyrates; Colonic Neoplasms; DNA; Enzyme Activation; Enzyme Inhibitors; gamma-Glutamyltransferase; Histone Deacetylase Inhibitors; Hydroxamic Acids; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Rats; RNA, Messenger; Sp1 Transcription Factor; Tumor Cells, Cultured

Histone deacetylase (HDAC) inhibitors such as trichostatin (TSA) and butyrate have been shown to inhibit cancer cell proliferation, induce apoptosis and regulate the expression of genes involved in cell cycle. Although the precise mechanism underlying HDAC inhibitor-induced cell growth arrest is not fully understood, induction of cell cycle related genes such as p21(cip/waf), is thought to be important. Here we showed that in the SW620 human colon cancer cell line, TSA and butyrate induced the growth arrest and DNA damage gene 45alpha (GADD45alpha) and GADD45beta. Furthermore, GADD45beta and p21(cip/waf) messenger RNA were induced in the absence of protein synthesis, indicating that both genes were immediate target genes for TSA. Cyclohexamide and TSA super-induced the expression of GADD45alpha and beta, but not p21(cip/waf). Interestingly while mitogen-activated kinase (MEK) inhibitor PD98059 and p38 kinase inhibitor SB242235 were unable to affect GADD45 induction, two serine/threonine protein kinase inhibitors (H7 and H8) as well as curcumin completely blocked the super-induction. Concomitant to the inhibition of GADD45 induction, H7 and H8 also blocked TSA-induced apoptosis. Taken together, these results suggest that GADD45 induction may play important role in TSA-induced cellular effects.

Topics: Antigens, Differentiation; Blotting, Northern; Blotting, Western; Butyrates; Cell Cycle Proteins; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cycloheximide; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Nuclear Proteins; Protein Synthesis Inhibitors; RNA, Messenger; Tumor Cells, Cultured

Radioactive iodine is used to identify and treat recurrent and metastatic thyroid cancer of follicular cell origin. Between 30% and 40% of thyroid cancers are either resistant or become resistant to radioactive iodine. Increased sodium-iodide symporter (NIS) and decreased Pendrin (PDS) activity may be associated with increased radioactive iodine effectiveness. In this investigation the effects of Trichostatin A (TSA), a histone deacetylating inhibitor, on human thyroid NIS and PDS gene expression was investigated.. Cell lines from papillary, Hürthle, and follicular cell carcinomas were treated with TSA for 72 hours at concentrations up to 100 ng/mL. NIS and PDS gene expression was determined using quantitative RT-polymerase chain reaction.. . NIS messenger RNA expression in cell carcinomas was increased 107- (1.8-307) and 217- (5.7-408) fold in papillary, 39- (20-63) and 58- (37-80) fold in Hürthle, and 459- (178-810) and 781- (412-1229) fold in follicular after treatment with 50 and 100 ng/mL of TSA, respectively. PDS messenger RNA expression in cell carcinomas was decreased 0.22- (0.05-0.45) and 0.27- (0.09-0.47) fold in papillary, 0.53- (0.46-0.60) and 0.54- (0.44-0.64) fold in Hürthle, and 0.32- (0.26-0.39) and 0.56- (0.47-0.64) fold in follicular, after the same treatment.. In thyroid cancer cell lines, TSA dramatically increased NIS gene expression and reduced PDS expression. The increased NIS expression and reduced PDS expression may make radioiodine therapy more effective in patients with thyroid cancer, especially when the tumors have no or low uptake of radioiodine.

Topics: Carcinoma, Papillary; Carrier Proteins; Cell Survival; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; In Vitro Techniques; Iodine Radioisotopes; Membrane Transport Proteins; Radiopharmaceuticals; Sulfate Transporters; Symporters; Thyroid Neoplasms; Tumor Cells, Cultured

The Wnt signaling pathway modulates the transcription of genes linked to proliferation, differentiation and tumor progression. beta-Catenin-Tcf (BCT)-dependent Wnt signaling is influenced by the short-chain fatty acid sodium butyrate, which induces growth arrest and/or maturation of colonic carcinoma cells. We have compared the effects of sodium butyrate on BCT-dependent signaling in 2 colon carcinoma cell lines that differ in their physiologic response to butyrate, with SW620 cells responding to butyrate by undergoing terminal differentiation and apoptosis, and HCT-116 cells undergoing reversible growth arrest, but no significant apoptotic cell death. Furthermore, these colon carcinoma cell lines differ in their mechanism of Wnt pathway activation, with adenomatous polyposis coli (APC) mutant SW620 cells having high levels of BCT complexes and APC wild-type HCT-116 cells having mutant beta-catenin, low levels of BCT complexes and correspondingly higher levels of free Tcf. We have demonstrated that in SW620 cells, butyrate downregulates BCT-dependent expression of the Tcf-TK, matrilysin and cyclin D1 promoters, whereas in HCT-116 cells, butyrate upregulates expression of these promoters. Cotransfection with expression vectors that interfere with the Wnt pathway suggests that butyrate enhances BCT complex-DNA binding. Butyrate reduces the expression of Tcf4 in HCT-116 cells, consistent with the induction by butyrate of Tcf-repressible promoters in these cells. These findings indicate that sodium butyrate modulates the Wnt pathway in SW620 and HCT-116 cells in a different manner and that these differences have consequences for promoter activity that may influence the physiologic response to butyrate.

Topics: beta Catenin; Binding Sites; Butyrates; Colonic Neoplasms; Cyclin D1; Cytoskeletal Proteins; Enzyme Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 7; Promoter Regions, Genetic; Proto-Oncogene Proteins; Signal Transduction; TCF Transcription Factors; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors; Tumor Cells, Cultured; Wnt Proteins; Zebrafish Proteins

DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2'-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase-PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.

Topics: Acetylation; Antimetabolites, Antineoplastic; Azacitidine; Chromatin; Colonic Neoplasms; CpG Islands; Cross-Linking Reagents; DNA; DNA Methylation; DNA-Binding Proteins; Formaldehyde; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, p16; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Models, Genetic; Promoter Regions, Genetic; Protein Binding; Proteins; RNA, Messenger; Substrate Specificity; Transcriptional Activation; Tumor Cells, Cultured; Tumor Suppressor Protein p14ARF

A wealth of evidence correlates the chemopreventive activity of a fiber-rich diet with the production of butyrate. In order to identify the genes transcriptionally modulated by the molecule, we analyzed the expression profile of butyrate-treated colon cancer cells by means of cDNA expression arrays. Moreover, the effect of trichostatin A, a specific histone deacetylase inhibitor, was studied. A superimposable group of 23 genes out of 588 investigated is modulated by both butyrate and trichostatin A. Among them, a major target was tob-1, a gene involved in the control of cell cycle. tob-1 is also up-regulated by butyrate in a neuroblastoma-derived cell line, and its overexpression in the colon cells caused growth arrest. Our findings represent an extensive analysis of genes modulated by butyrate and identify completely new effectors of its biological activities.

Topics: Acetylation; Butyrates; Colonic Neoplasms; Cycloheximide; DNA-Binding Proteins; Enzyme Inhibitors; GATA2 Transcription Factor; Gene Expression; Gene Expression Profiling; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HT29 Cells; Humans; Hydroxamic Acids; Oligonucleotide Array Sequence Analysis; Protein Synthesis Inhibitors; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured

Butyrate has potent anti-tumorigenic effects on many colon cancer cell lines, including inhibition of growth and promotion of apoptosis in vitro. Nevertheless, despite the butyrate concentration in the colonic lumen being sufficient to result in the death of almost all cells in vitro, colon cancers still develop and grow in vivo, suggesting that cancer cells must develop mechanisms by which they escape the effects of butyrate observed in vitro. Insulin-like growth factor-II (IGF-II) is an autocrine growth factor in many colon cancer cells. The aim of this study was to determine whether IGF-II influences butyrate-mediated apoptosis in LIM 2405 human colon cancer cells. Butyrate and trichostatin A, both of which are histone deacetylase inhibitors although the latter is more specific, induced apoptosis as determined by floating cell counting, Hoechst 33258 staining, DNA laddering and a cell death detection ELISA. IGF-II inhibited the effects of both agents. Butyrate but not trichostatin A also induced LIM 2405 cell migration. In contrast to the above results, IGF-II enhanced butyrate-induced cell migration. Levels of IGF binding protein-3 (IGFBP-3), which may induce apoptosis by IGF-dependent or -independent mechanisms, were increased by butyrate and trichostatin A; IGF-II augmented this effect. It is therefore unlikely that IGFBP-3 mediates butyrate-induced apoptosis. We suggest that IGF-II inhibits the pro-apoptotic effect of butyrate downstream of histone deacetylase inhibition. In contrast, IGF-II promotes histone deacetylase-dependent IGFBP-3 expression and histone deacetylase-independent migration. IGF-II may promote tumour growth by mediating the development of resistance to the pro-apoptotic effects of butyrate.

Topics: Apoptosis; Bisbenzimidazole; Blotting, Western; Butyrates; Cell Movement; Cell Survival; Colonic Neoplasms; Drug Resistance, Neoplasm; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoblotting; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor II; RNA, Messenger; Tumor Cells, Cultured

The short-chain fatty acid butyrate, produced by microbial fermentation of dietary fiber in the large intestine, is a physiological regulator of major pathways of colonic epithelial cell maturation: cell cycle arrest, lineage-specific differentiation, and apoptosis. Microarray analysis of 8,063 sequences demonstrated a complex cascade of reprogramming of SW620 colonic epithelial cells upon treatment with butyrate characterized by the progressive recruitment of gene sets as a function of time. Comparison with the effects of trichostatin A, in conjunction with differences in the kinetics of alteration of histone acetylation induced by butyrate and trichostatin A, identified subsets of induced and repressed genes likely coordinately regulated by altered histone acetylation. The butyrate response was also compared in detail with that of sulindac, a nonsteroidal anti-inflammatory drug with significant chemopreventive activity for colon cancer, and curcumin, a component of mustard and curry structurally and functionally related to sulindac that also has chemopreventive activity. Although gene clusters were identified that showed similar responses to butyrate and sulindac, the data were characterized by the extensive differences in the effects of the two agents. This was striking for functional classes of genes involved in signaling pathways and in cell cycle progression, although butyrate and sulindac induce a similar G0-G1 arrest, elevation of beta-catenin-Tcf signaling, and apoptotic cascade. As regards cell cycle arrest, the underlying mechanism in response to butyrate was most similar to that of the Caco-2 cell line that had spontaneously undergone a G0-G1 arrest and least similar to the G2-M arrest stimulated by curcumin. Thus, high-throughput microarray analysis of gene expression profiles can be used to characterize and distinguish the mechanisms of response of colonic epithelial cells to physiological and pharmacological inducers of cell maturation. This has important implications for characterization of chemopreventive agents and recognition of potential toxicity and synergies. The data bases, gene clusters, and analyses are available at http:// sequence.aecom.yu.edu/genome/.

Topics: Acetylation; Anticarcinogenic Agents; Butyrates; Caco-2 Cells; Cell Cycle; Cell Differentiation; Colon; Colonic Neoplasms; Curcumin; Enzyme Inhibitors; Epithelial Cells; Gene Expression Profiling; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Kinetics; Multigene Family; Signal Transduction; Sulindac

Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.

Topics: Animals; Antineoplastic Agents; Benzamides; Butyrates; Cell Cycle; Cell Survival; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Female; Fluorouracil; Gelsolin; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; K562 Cells; KB Cells; Mice; Mice, Nude; Ovarian Neoplasms; Pancreatic Neoplasms; Pyridines; Transplantation, Heterologous; Tumor Cells, Cultured

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; beta Catenin; Butyrates; Colonic Neoplasms; Curcumin; Cytoskeletal Proteins; DNA-Binding Proteins; Enzyme Inhibitors; G2 Phase; Humans; Hydroxamic Acids; Lymphoid Enhancer-Binding Factor 1; Mice; Sulindac; Trans-Activators; Transcription Factors; Up-Regulation; Valinomycin

n-Butyrate inhibits the growth of colon cancer cell lines. In the HCT 116 cell line, butyrate-induced growth inhibition is almost fully reversible, whereas in the VACO 5 cell line, a subpopulation undergoes apoptosis within 30 hr of treatment with butyrate. Concurrent treatment of VACO 5 cells with butyrate and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) accelerates and increases the incidence of cell death to nearly 100% of the population, whereas HCT 116 cells largely remain alive during treatment with this combination. The action of butyrate as an inhibitor of histone deacetylase was assessed in these cell lines by examining extracted core histones for their electrophoretic mobility in Triton/acid/urea gels. The concentrations of butyrate that were effective for inducing apoptosis were similar to the concentrations that caused hyperacetylation of core histones in the VACO 5 cell line. Furthermore, an examination of other carboxylic acids for induction of apoptosis revealed a rank order that corresponded to the order of potency in causing hyperacetylation of core histones. Specifically, the active acids were 3-5 carbons in length and lacked substitution at the 2-position. Isovaleric and propionic acids, in particular, proved to be effective inducers of both hyperacetylation and apoptosis at 5 mM concentrations, a finding of potential relevance to the unusual pancytopenia occurring after acidotic episodes in isovaleric and propionic acidemias. The duration of butyrate treatment required for chromatin fragmentation (10-20 hr) corresponded to the time required for histone H4 to become predominantly tetraacetylated. Furthermore, trichostatin A, a structurally dissimilar inhibitor of histone deacetylase, mimicked butyrate-induced apoptosis of VACO 5 cells and growth inhibition of HCT 116 cells. The dramatic enhancement of VACO 5 cell death by TPA, and the high level resistance of HCT 116 cells to butyrate were not evident from histone acetylation determinations. Thus, applications of butyrate for cytoreduction therapy will benefit from pharmacodynamic assessment of histone acetylation, but will require additional work to predict susceptibility to butyrate-induced death.

Topics: Adenocarcinoma; Apoptosis; Butyrates; Butyric Acid; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fatty Acids, Volatile; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured