trichostatin-a and Colorectal-Neoplasms

Human intestinal peptide transporter PEPT1 is commonly repressed in human colorectal cancer (CRC), yet its relationship with sensitivity to the common CRC treatment ubenimex has not previously been elucidated. In this study, we confirmed PEPT1 suppression in CRC using real-time quantitative polymerase chain reaction and western blotting and then investigated the underlying epigenetic pathways involved using bisulfite sequencing, chromatin immunoprecipitation, siRNA knockdown, and reporter gene assays. We found that PEPT1 transcriptional repression was due to both DNMT1-mediated DNA methylation of the proximal promoter region and HDAC1-mediated histone deacetylation, which blocked P300-mediated H3K18/27Ac at the PEPT1 distal promoter. Finally, the effects of the epigenetic activation of PEPT1 on the CRC response to ubenimex were evaluated using sequential combination therapy of decitabine and ubenimex both in vitro and in xenografts. In conclusion, epigenetic silencing of PEPT1 due to increased DNMT1 and HDAC1 expression plays a vital role in the poor response of CRC to ubenimex.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Colorectal Neoplasms; DNA Methylation; Drug Resistance, Neoplasm; Drug Synergism; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intestinal Mucosa; Leucine; Mice; Mice, Inbred BALB C; Mice, Nude; Peptide Transporter 1; Vorinostat; Xenograft Model Antitumor Assays

Colorectal cancer (CRC) is known to be the third most common cancer disease and the fourth-leading cause of cancer-related deaths worldwide. Bile acid, especially deoxycholic acid and lithocholic acid, were revealed to play an important role during carcinogenesis of CRC. In this study, we found organic solute transporter

Topics: Acetylation; Carcinogenesis; Cholera Toxin; Colorectal Neoplasms; Down-Regulation; E1A-Associated p300 Protein; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histones; HT29 Cells; Humans; Hydroxamic Acids; Membrane Transport Proteins; Promoter Regions, Genetic; RNA, Small Interfering; Up-Regulation

Epithelial cancers are defined by a tumor-specific distribution of chromosomal aneuploidies that are maintained when cells metastasize and are conserved in cell lines derived from primary tumors. Correlations between genomic copy number and gene expression have been observed for different tumors including, colorectal (CRC), breast, and pancreatic cancer. These ploidy-driven transcriptional deregulations are characterized by low-level expression changes of most genes on the affected chromosomes. The emergence of these aberrations at an early stage of tumorigenesis and the strong selection for the maintenance of these aneuploidies suggest that aneuploidy-dependent transcriptional deregulations might contribute to cellular transformation and maintenance of the malignant phenotype. The histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) has anticancer effects and is well known to lead to large-scale gene-expression changes. Here we assessed if TSA could disrupt the aneuploidy-driven gene expression in the aneuploid colon cancer cell line SW480 and the artificially generated aneuploid cell line DLD-1 + 7. We found that TSA increases transcriptional activity throughout the genome, yet inhibits aneuploidy-induced gene-expression changes on chromosome 7. Among the TSA affected genes on chromosome 7, we identified potential CRC oncogenes. These experiments represent the first attempt to explain how histone acetylation affects aneuploidy-driven gene-expression changes.

Topics: Aneuploidy; Cell Line, Tumor; Chromosomes, Human, Pair 7; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Up-Regulation

In colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration.. CDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation.. CDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.

Topics: Benzamides; Caco-2 Cells; CDX2 Transcription Factor; Cell Line, Tumor; Colorectal Neoplasms; CpG Islands; Decitabine; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histones; HT29 Cells; Humans; Hydroxamic Acids; Male; Prognosis; Promoter Regions, Genetic; Retrospective Studies; Tissue Array Analysis

Recent studies from our group and many others have shown the ability of histone deacetylase (HDAC) inhibitors for retarding the growth of carcinomas of cervix, colon and rectum in vitro. A search for naturally occurring HDAC inhibitors continues due to the adverse effects associated with known HDAC inhibitors like SAHA and TSA. Therefore in the current study, naturally occurring cinnamic acids derivatives were screened for HDAC inhibitory effect using in silico docking method which identified cinnamic acids as potential candidates. Cinnamic acids (CA) are naturally occurring phenolic compounds known to exhibit anticancer properties. However, it is not clearly known whether the anticancer properties of CA derivatives are due to the inhibition of oncogenic HDACs, if so how the efficacy varies among various CA derivatives. Hence, the HDAC inhibitory potential of CA derivatives containing increasing number of hydroxylic groups or methoxy moieties was determined using Discovery Studio software and the most potent CA derivatives tested ex vivo (biochemical assay) as well as in vitro (using cell based assay). Among CA derivatives tested, dihydroxy cinnamic acid (DHCA, commonly known as caffeic acid) exhibited better interactions with HDAC2 (compared to other isoforms) in silico and inhibited its activity ex vivo as well as in vitro. Targeted reduction of HDAC activity using DHCA induced death of cancer cells by (a) generating reactive oxygen species, (b) arresting cells in S and G2/M phases; and (c) induction of caspase-3 mediated apoptosis. In conclusion, we demonstrated that DHCA inhibited cancer cell growth by binding to HDAC followed by the induction of apoptosis.

Topics: Apoptosis; Binding Sites; Butyric Acid; Cell Line, Tumor; Cinnamates; Colorectal Neoplasms; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Uterine Cervical Neoplasms

Trichostatin A (TSA) is a kind of classical histone deacetylase (HDAC) inhibitor. In this study, we reported the reversal effects of TSA on EMT and investigated the possible involved molecular mechanisms in SW480 and PC3 cells. Firstly, we observed that TSA induced the reversal process of epithelial-mesenchymal transition (EMT) in SW480 and PC3 cells, resulting in attenuated cell invasion and migration abilities. TSA-induced EMT reversal was characterized by up-regulation of E-cadherin and down-regulation of Vimentin. Then, treatment with TSA also decreased the expression of transcription factor Slug. Furthermore, over-expression of Slug significantly caused down-regulation of E-cadherin and up-regulation of Vimentin. Meanwhile, TSA treatment in Slug-expressing cells could prevent these changes. These findings suggested that Slug played a crucial role in TSA-induced EMT reversal. Additionally, the study showed that TSA could induce the increase of HDAC1 and HDAC2 on the Slug gene promoter, which might be responsible for the suppression of Slug. Overall, TSA could reverse EMT in SW480 and PC3 cells and TSA-mediated down-regulation of Slug was involved in the reversal process.

Topics: Antigens, CD; Cadherins; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Microscopy, Confocal; Neoplasm Invasiveness; Promoter Regions, Genetic; Prostatic Neoplasms; Snail Family Transcription Factors; Transcription Factors; Vimentin

It was previously reported that the histone deacetylase inhibitor (HDACI) trichostatin A (TSA) induced B cell lymphoma 2 (Bcl-2)-associated X protein (Bax)-dependent apoptosis in colorectal cancer (CRC) cells. In addition, Ku70 has been identified as a regulator of apoptosis, the mechanism of which proceeds via interacting with Bax. The aim of the present study was to investigate the role of Ku70 in TSA-induced apoptosis in the CRC cell lines HCT116 and HT29. The results showed that TSA induced the acetylation of Ku70, which was found to be associated with increased apoptosis. In addition, TSA treatment promoted the release of Bax from its complex with Ku70. Bax was then detected to have translocated from the cytoplasm into the mitochondria, while cytochrome c was detected to have translocated from the mitochondria into the cytoplasm. Furthermore, knockdown of Ku70 using small interfering RNA decreased TSA-induced apoptosis as well as downregulated the expression of Bax. These effects were rescued through pre-treatment of cells with the proteasome inhibitor MG132. In conclusion, the results of the present study suggested that Ku70 acetylation mediated TSA-induced apoptosis in CRC cells. In addition, Ku70 was found to be indispensable in TSA-induced apoptosis due to its role in protecting Bax from proteosomal degradation.

Topics: Acetylation; Antigens, Nuclear; Apoptosis; bcl-2-Associated X Protein; Colorectal Neoplasms; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Ku Autoantigen; Leupeptins

The RNA-binding protein tristetraprolin (TTP) promotes rapid decay of mRNAs bearing 3' UTR AU-rich elements (ARE). In many cancer types, loss of TTP expression is observed allowing for stabilization of ARE-mRNAs and their pathologic overexpression. Here we demonstrate that histone deacetylase (HDAC) inhibitors (Trichostatin A, SAHA and sodium butyrate) promote TTP expression in colorectal cancer cells (HCA-7, HCT-116, Moser and SW480 cells) and cervix carcinoma cells (HeLa). We found that HDAC inhibitors-induced TTP expression, promote the decay of COX-2 mRNA, and inhibit cancer cell proliferation. HDAC inhibitors were found to promote TTP transcription through activation of the transcription factor Early Growth Response protein 1 (EGR1). Altogether, our findings indicate that loss of TTP in tumors occurs through silencing of EGR1 and suggests a therapeutic approach to rescue TTP expression in colorectal cancer.

Topics: Butyric Acid; Cell Line, Tumor; Colorectal Neoplasms; Cyclooxygenase 2; Early Growth Response Protein 1; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; RNA, Messenger; Tristetraprolin; Vorinostat

The calcium-sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = -0.310, p < 0.05 and n = 45, ρ = -0.588, p < 0.001). Treatments with 5-aza-2'-deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine-specific demethylase 1 (LSD1) to prevent demethylation of mono- and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.

Topics: Acetylation; Aged; Apoptosis; Blotting, Western; Cell Proliferation; Chromatin Immunoprecipitation; Colorectal Neoplasms; DNA Methylation; Female; Fluorescent Antibody Technique; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Neoplasm Grading; Neoplasm Staging; Prognosis; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Receptors, Calcium-Sensing; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

In this study, we investigated the molecular mechanisms underlying the anti-proliferative effects of Compound K, with specific reference to histone modification. Exposure of HT-29 human colon cancer cells to Compound K resulted in time-dependent inhibition of histone deacetylase (HDAC) activity, mRNA and protein expression. Compound K treatment induced unmethylation of the RUNX3 promoter region such as TSA treatment and an accumulation of acetylated histones H3 and H4 within the total cellular chromatin, resulting in an enhanced ability of these histones to bind to the promoter sequences of the tumor suppressor gene Runt-related transcription factor 3 (RUNX3). Treatment of cells with Compound K increased the mRNA and protein expression of RUNX3, as well as p21, a downstream target of RUNX3. These alterations were consistent with cell cycle arrest at the G0/G1 phases and induction of apoptosis. Our results provide new insights into the mechanisms of Compound K action in human colorectal cancer cells and suggest that HDAC inhibition presents a novel approach to prevent or treat colorectal cancer.

Topics: Acetylation; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Core Binding Factor Alpha 3 Subunit; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Ginsenosides; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HT29 Cells; Humans; Hydroxamic Acids; Panax; Promoter Regions, Genetic; Protein Binding; RNA, Messenger

Diet-derived butyrate, a histone deacetylase inhibitor (HDI), decreases proliferation and increases apoptosis in colorectal cancer (CRC) cells via epigenetic changes in gene expression. Other HDIs such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) have similar effects. This study examined the role of microRNAs (miRNAs) in mediating the chemo-protective effects of HDIs, and explored functions of the oncogenic miR-17-92 cluster. The dysregulated miRNA expression observed in HT29 and HCT116 CRC cells could be epigenetically altered by butyrate, SAHA and TSA. These HDIs decreased expression of miR-17-92 cluster miRNAs (P < 0.05), with a corresponding increase in miR-17-92 target genes, including PTEN, BCL2L11, and CDKN1A (P < 0.05). The decrease in miR-17-92 expression may be partly responsible for the anti-proliferative effects of HDIs, with introduction of miR-17-92 cluster miRNA mimics reversing this effect and decreasing levels of PTEN, BCL2L11, and CDKN1A (P < 0.05). The growth effects of HDIs may be mediated by changes in miRNA activity, with down-regulation of the miR-17-92 cluster a plausible mechanism to explain some of the chemo-protective effects of HDIs. Of the miR-17-92 cluster miRNAs, miR-19a and miR-19b were primarily responsible for promoting proliferation, while miR-18a acted in opposition to other cluster members to decrease growth. NEDD9 and CDK19 were identified as novel miR-18a targets and were shown to be pro-proliferative genes, with RNA interference of their transcripts decreasing proliferation in CRC cells. This is the first study to identify competing roles for miR-17-92 cluster members, in the context of HDI-induced changes in CRC cells.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Butyric Acid; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Membrane Proteins; MicroRNAs; Phosphoproteins; Proto-Oncogene Proteins; PTEN Phosphohydrolase; RNA, Long Noncoding; RNA, Messenger; Transfection; Vorinostat

Aberrant janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is involved in the oncogenesis of several cancers. Suppressors of cytokine signaling (SOCS) genes and SH2-containing protein tyrosine phosphatase 1 (SHP1) proteins, which are negative regulators of JAK/STAT signaling, have been reported to have tumor suppressor functions. However, in colorectal cancer (CRC) cells, the mechanisms that regulate SOCS and SHP1 genes, and the cause of abnormalities in the JAK/STAT signaling pathway, remain largely unknown. The present study shows that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, leads to the hyperacetylation of histones associated with the SOCS1 and SOCS3 promoters, but not the SHP1 promoter in CRC cells. This indicates that histone modifications are involved in the regulation of SOCS1 and SOCS3. Moreover, upregulation of SOCS1 and SOCS3 expression was achieved using TSA, which also significantly downregulated JAK2/STAT3 signaling in CRC cells. We also demonstrate that TSA suppresses the growth of CRC cells, and induces G1 cell cycle arrest and apoptosis through the regulation of downstream targets of JAK2/STAT3 signaling, including Bcl-2, survivin and p16(ink4a) . Therefore, our data demonstrate that TSA may induce SOCS1 and SOCS3 expression by inducing histone modifications and consequently inhibits JAK2/STAT3 signaling in CRC cells. These results also establish a mechanistic link between the inhibition of JAK2/STAT3 signaling and the anticancer action of TSA in CRC cells.

Topics: Acetylation; Base Sequence; Cell Line, Tumor; Chromatin Immunoprecipitation; Colorectal Neoplasms; DNA Primers; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Janus Kinase 2; Promoter Regions, Genetic; STAT3 Transcription Factor; Suppressor of Cytokine Signaling Proteins

Many chemotherapeutic agents induce apoptosis via a p53-dependent pathway. However, up to 50% of human cancers have p53 mutation and loss of p53 function. Histone deacetylase inhibitors (HDACIs) are emerging as a potentially important new class of anticancer agents. Here, we report that, Trichostatin A (TSA), a pan-HDAC inhibitor, could induce G2/M cell cycle arrest and apoptosis in both colorectal cancer cell lines with wild-type p53 (HT116 cells) and mutant p53 (HT29 cells), although HCT116 cells had more apoptotic cells than HT29 cells. TSA induces apoptosis in both cell lines via the mitochondrial pathway as indicated by decrease of the mitochondrial membrane potential (MMP) and activation of caspase-3. Additionally, TSA induces expression of the pro-apoptotic protein Bax and decreases the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL in both cell lines. Bax knockdown by siRNA significantly impaired TSA-induced apoptosis in both cell lines. These data suggest that TSA induces G2/M cell cycle arrest and Bax-dependent apoptosis in colorectal cancer cells (HCT116 cells and HT29 cells) by both p53-dependent and -independent mechanisms. However, cells with normal p53 function are more sensitive to TSA-induced apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Colorectal Neoplasms; Enzyme Activation; G2 Phase Cell Cycle Checkpoints; HCT116 Cells; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Membrane Potential, Mitochondrial; Mutation; Signal Transduction; Tumor Suppressor Protein p53

Although growing evidence demonstrates that TWIST1 is an interesting tumor biomarker, little is known about the clinical significance of TWIST1 expression and TWIST1 methylation in human primary colorectal cancer. In this study, we examined the association of TWIST1 expression and TWIST1 methylation with clinicopathologic features in human primary colorectal tumors. Primary colorectal cancer (CRC) specimens from 319 patients, corresponding normal colorectal nontumorous mucosa from 251 patients with cancer, and colorectal adenomas from 189 patients were used. Methylation and expression levels of TWIST1 were compared with clinicopathologic features. The TWIST1 methylation level was higher in colorectal adenoma and cancer than in normal colorectal mucosa. Elevated TWIST1 mRNA expression in normal colorectal mucosa in patients with CRC as well as in primary CRC specimens was associated with unfavorable outcomes. There was no correlation between TWIST1 methylation and TWIST1 expression. Our results suggest that TWIST1 methylation may be a useful biomarker for screening colorectal tumors. In addition, TWIST1 mRNA expression is a possible molecular marker for predicting the outcome in patients with CRC. Confirmatory studies using independent data sets are needed to confirm our findings.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Azacitidine; Case-Control Studies; Cell Line, Tumor; Chromosome Mapping; Colorectal Neoplasms; Decitabine; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Intestinal Mucosa; Linear Models; Male; Middle Aged; Nuclear Proteins; Polymerase Chain Reaction; Prognosis; Sequence Analysis, DNA; Statistics, Nonparametric; Twist-Related Protein 1

DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes.. Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR.. Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated.. These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of gene expression level brought about by alterations in the epigenetic status following 5-aza-dC treatment.

Topics: Acetylation; Apoptosis; Azacitidine; Biomarkers, Tumor; Blotting, Western; Cell Proliferation; Cells, Cultured; Colorectal Neoplasms; CpG Islands; Decitabine; DNA Methylation; DNA Modification Methylases; DNA, Neoplasm; Enzyme Inhibitors; Epigenesis, Genetic; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome, Human; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Oncostatin M (OSM) is an interleukin-6 cytokine family member, which inhibits cell proliferation and induces cell differentiation and apoptosis in cancers. In melanoma cells, epigenetic silencing of OSM receptor (OSMR) by histone deacetylation contributes to escape of cell growth control by OSM. However, the silencing of OSMR by DNA methylation in any cancer has not been examined.. Methylation status of OSMR was determined by sequencing or methylation-specific PCR in primary tumors and cell lines. Cell lines were treated with DNA methyltransferase inhibitors 5-aza-2-deoxycytidine or DNA methyltransferase 1 small interfering RNA or a histone deacetylase inhibitor trichostatin A. OSMR mRNA level was determined by reverse transcription-PCR. The acetylation of histone H3 was analyzed by chromatin immunoprecipitation assay.. We observed methylation of OSMR in 88 of 98 (90%) colorectal cancers, 34 of 38 (89%) colorectal polyps, 17 of 31 (55%) normal-appearing mucosa adjacent to colorectal cancers, 13 of 40 (33%) gastric cancers, and 2 of 10 (20%) pancreatic cancers. OSMR methylation was absent or rarely detected in normal colonic mucosa from noncancer patients or in cancers of nondigestive organs, including breast, lung, liver, prostate, kidney, and melanoma. We observed a significant correlation between OSMR methylation and loss of mRNA expression in 39 cancer cell lines. Following the treatment of colorectal cancer cell lines with 5-aza-2-deoxycytidine, DNA methyltransferase 1 small interfering RNA, or trichostatin A, the induction of OSMR mRNA and the enrichment in the level of histone acetylation were observed.. The epigenetic silencing and DNA methylation of OSMR occur frequently in colorectal cancers and rarely in cancers of nondigestive organs. OSMR methylation is an early event in the colorectal carcinogenesis.

Topics: Acetylation; Azacitidine; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Epigenesis, Genetic; Gastrointestinal Tract; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Oncostatin M Receptor beta Subunit; Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Suppressor Protein p53

High-frequency microsatellite-instable (MSI-H) tumors account for approximately 15% of colorectal cancers. Therapeutic decisions for colorectal cancer are empirically based and currently do not emphasize molecular subclassification despite an increasing collection of gene expression information. Our objective was to identify low molecular weight compounds with preferential activity against MSI colorectal cancers using combined gene expression data sets.. Three expression/query signatures (discovery data set) characterizing MSI-H colorectal cancer were matched with information derived from changes induced in cell lines by 164 compounds using the systems biology tool "Connectivity Map." A series of sequential filtering and ranking algorithms were used to select the candidate compounds. Compounds were validated using two additional expression/query signatures (validation data set). Cytotoxic, cell cycle, and apoptosis effects of validated compounds were evaluated in a panel of cell lines.. Fourteen of the 164 compounds were validated as targeting MSI-H cell lines using the bioinformatics approach; rapamycin, LY-294002, 17-(allylamino)-17-demethoxygeldanamycin, and trichostatin A were the most robust candidate compounds. In vitro results showed that MSI-H cell lines due to hypermethylation of MLH1 are preferentially targeted by rapamycin (18.3 versus 4.4 mumol/L; P = 0.0824) and LY-294002 (15.02 versus 10.37 mumol/L; P = 0.0385) when compared with microsatellite-stable cells. Preferential activity was also observed in MSH2 and MSH6 mutant cells.. Our study shows that the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway is of special relevance in mismatch repair-deficient colorectal cancer. In addition, we show that amalgamation of gene expression information across studies provides a robust approach for selection of potential therapies corresponding to specific groups of patients.

Topics: Algorithms; Antineoplastic Agents; Benzoquinones; Cell Cycle; Cell Line, Tumor; Chromones; Colorectal Neoplasms; Computational Biology; DNA Mismatch Repair; Drug Evaluation, Preclinical; Enzyme Inhibitors; Gene Expression Profiling; Humans; Hydroxamic Acids; Immunosuppressive Agents; Lactams, Macrocyclic; Microsatellite Instability; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus

Deletion of 11q23-q24 is frequent in a diverse variety of malignancies, including breast and colorectal carcinoma, implicating the presence of a tumor suppressor gene at that chromosomal region. We examined a 6-Mb region on 11q23 by high-resolution deletion mapping, using both loss of heterozygosity analysis and customized microarray comparative genomic hybridization. LARG (leukemia-associated Rho guanine-nucleotide exchange factor) (also called ARHGEF12), identified from the analysed region, is frequently underexpressed in breast and colorectal carcinomas with a reduced expression observed in all breast cancer cell lines (n=11), in 12 of 38 (32%) primary breast cancers, 5 of 10 (50%) colorectal cell lines and in 20 of 37 (54%) primary colorectal cancers. Underexpression of the LARG transcript was significantly associated with genomic loss (P=0.00334). Hypermethylation of the LARG promoter was not detected in either breast or colorectal cancer, and treatment of four breast and four colorectal cancer cell lines with 5-aza-2'-deoxycytidine and/or trichostatin A did not result in a reactivation of LARG. Enforced expression of LARG in breast and colorectal cancer cells by stable transfection resulted in reduced cell proliferation and colony formation, as well as in a markedly slower cell migration rate in colorectal cancer cells, providing functional evidence for LARG as a candidate tumor suppressor gene.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 11; Colorectal Neoplasms; Decitabine; DNA Methylation; Female; Guanine Nucleotide Exchange Factors; Humans; Hydroxamic Acids; Male; Nucleic Acid Hybridization; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Rho Guanine Nucleotide Exchange Factors; Transfection; Tumor Suppressor Proteins

Genetic and epigenetic defects in Wnt/beta-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/beta-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly derepressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/beta-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/beta-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/beta-catenin signaling in colorectal cancer.

Topics: Adaptor Proteins, Signal Transducing; Adenosine; Adenosylhomocysteinase; Apoptosis; Azacitidine; beta Catenin; Colorectal Neoplasms; Decitabine; Dishevelled Proteins; DNA Methylation; DNA Modification Methylases; Down-Regulation; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Oligonucleotide Array Sequence Analysis; Phosphoproteins; Promoter Regions, Genetic; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection; Wnt Proteins

Topics: Acetylation; Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; HCT116 Cells; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HT29 Cells; Humans; Hydroxamic Acids; Repressor Proteins

Epigenetic parameters linked to E-cadherin gene were investigated in 5 human colorectal cancer cell lines. Treatment with trichostatin A led to enhanced acetylation of histone H3-K9 with concurrent induction of E-cadherin mRNA in 3 E-cadherin low/negative cell lines that are not DNA methylated. Co-treatment with 5-aza-2'-deoxycytidine and trichostatin A resulted in additive/synergic induction of E-cadherin mRNA in all 5 cell lines with concomitant enhancement of histone H3-K9 acetylation in 4 E-cadherin low/negative cell lines. Our results suggest that histone H3-K9 deacetylation appears to play a crucial role in transcriptional repression of E-cadherin in colorectal cancers.

Topics: Acetylation; Antigens, CD; Azacitidine; Cadherins; Cell Line, Tumor; Chromatin Immunoprecipitation; Colorectal Neoplasms; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; Transcription, Genetic

Dihydropyrimidine dehydrogenase (DPD) is one of the factors that determine the efficacy and toxicity of 5-fluorouracil. Variations in DPD activity may result from alterations at the transcriptional level of the DPYD gene. Heterogeneity in DPYD expression has been reported, but the molecular mechanisms responsible for this remain unclear. We investigated methylation of the DPYD promoter as a mechanism for transcriptional regulation of DPYD in the RKO colorectal cancer cell line. We demonstrate that the active transcription machinery for DPYD is present in RKO cells, but promoter binding of Sp1, a transactivator of DPYD, was inhibited, which on subsequent examination was shown to be associated with dense promoter methylation. Treatment with 5-aza-2'-deoxycytidine alone or the combination of 5-aza-2'-deoxycytidine and trichostatin A induced demethylation of the promoter and markedly increased the DPYD mRNA level in RKO cells but not in unmethylated WiDr cells. Furthermore, in vitro methylation of the DPYD promoter decreased promoter activity. These data suggest an important role for methylation in DPYD suppression. The transcriptional suppression of DPYD by methylation may be responsible for the increased 5-fluorouracil sensitivity observed in some patients. This may also provide insight into the mechanism underlying the downregulation of DPYD in some colorectal cancers.

Topics: Azacitidine; Base Sequence; Cell Line, Tumor; Colorectal Neoplasms; Decitabine; Dihydrouracil Dehydrogenase (NADP); DNA Methylation; DNA Primers; DNA, Neoplasm; Epigenesis, Genetic; Gene Expression Regulation, Enzymologic; Humans; Hydroxamic Acids; Promoter Regions, Genetic; RNA, Messenger; RNA, Neoplasm; Sp1 Transcription Factor; Suppression, Genetic

To investigate the functions of promoter hypermethylation of secreted Wnt-antagonist genes in colorectal tumorigenesis and progression.. Two colorectal cancer cell lines, HCT116 and SW480, were treated by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA) for demethylation. The promoter hypermethylation and expression of sFRP and WIF-1 genes in different stages of colorectal tumor and colorectal cancer cell lines were detected by methylation-specific PCR and reverse transcription PCR, respectively.. None of the normal colorectal mucosa samples showed methylated bands of any sFRP and WIF-1genes. Hypermethylation of sFRP1, 2, 4, 5 and WIF-1 was detected in 93.1% (67/72), 83.3% (60/72), 36.1% (26/72), 52.8% (38/72) and 84.7% (61/72) of adenocarcinomas, 87.9% (29/33), 81.8% (27/33), 24.2% (8/33), 57.6% (19/33) and 72.7% (24/33) of adenomas, 52.6%, 28.9%, 2.6%, 18.4%, 23.7% of the adjacent normal mucosa. Methylation was more frequently found in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor (P < 0.05). No significant association between Wnt-antagonist genes hypermethylation and clinicopathological characteristics was found (P > 0.05). SFRP1, 2, 4, 5 and WIF-1 genes were methylated in HCT116 cell line. SFRP1, 2 and WIF-1 were methylated in SW480 cell line. The mRNA expression of sFRPs and WIF-1 genes was absent or significantly downregulated (P < 0.01) when they were methylated in two colorectal cancer cell lines. SFRP3 was expressed in two colorectal carcinoma cell lines. DAC/TSA combination treatment re-expressed the silenced sFRPs and WIF-1 genes mRNA expressions effectively. A single application of TSA could not re-express sFRPs and WIF-1 genes mRNA expressions. The influence of demethylation treatment on sFRP3 expression was minimal.. Hypermethylation of Wnt-antagonist genes is a common early event in the evolution of colorectal tumor. Methylation of sFRP1, 2, 5 and WIF-1 genes might serve as biomarkers for the early detection of colorectal tumor.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenoma; Adult; Aged; Azacitidine; Cell Line, Tumor; Colorectal Neoplasms; Decitabine; DNA Methylation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; HCT116 Cells; Humans; Hydroxamic Acids; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Male; Middle Aged; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Carnitine palmitoyl transferase I (CPT1) catalyzes the transport of long-chain fatty acids into mitochondria for beta-oxidation. A link between CPT1 and apoptosis has been suggested on the basis of several experimental data. Nevertheless, results are contradictory about the effective role of CPT1 in cell survival control and cancer development. Conversely, Fatty acid synthase (FAS) enzyme, required for the synthesis of fatty acids, is found over-expressed in tumors and inhibition of FAS triggers apoptosis in human cancer cells. We have studied the tumor-specific modulation of CPT1 and FAS in human colorectal cancer (n = 11) and breast carcinomas (n = 24). CPT1 was significantly decreased in the cytoplasm of tumoral samples (p < or = 0.04), whereas FAS was increased (p < or = 0.04). A striking CPT1 nuclear localization was evident in the tumors (p < or = 0.04). In the nuclear environment the protein would modulate the levels of acetyl/acyl-CoA implicated in the regulation of gene transcription. At this purpose, we performed in vitro experiments using epithelial neoplastic (MCF-7, Caco-2, HepG2 cells) and non neoplastic cell lines (MCF-12F) confirming a nuclear localization of CPT1 protein exclusively in neoplastic cells. Moreover histone deacetylase (HDAC) activity showed significantly higher levels in nuclear extracts from neoplastic than from control cells. HDAC1 and CPT1 proteins coimmunoprecipitated in nuclear extracts from MCF-7 cells. The treatment with HDAC inhibitors such as trichostatin A and butyrate significantly decreased nuclear expression of CPT1 and its bond to HDAC1. We also identified the existence of CPT1A mRNA transcript variant 2 in MCF-7, beside to the classic isoform 1. The peculiar localization of CPT1 in the nuclei of human carcinomas and the disclosed functional link between nuclear CPT1 and HDAC1 propose a new role of CPT1 in the histonic acetylation level of tumors.

Topics: Acetylation; Adenocarcinoma; Aged; Breast Neoplasms; Carnitine O-Palmitoyltransferase; Cell Nucleus; Chromatin Immunoprecipitation; Colorectal Neoplasms; Fatty Acid Synthases; Female; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Lysine; Male; Transcription, Genetic

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

Regional DNA hypermethylation and global DNA hypomethylation are 2 epigenetic alterations associated with colorectal cancers. However, their correlation with microsatellite instability (MSI) and chromosomal instability (CIN) in colorectal cancer, and their relationship with chromatin conformation and histone modification are not clear. In this study, we analyzed regional and global methylation in 16 cell lines and 64 primary colorectal cancers. We found that MSI and CIN are 2 alternative events in most cell lines and tumors. Furthermore, regional hypermethylation and global hypomethylation are also alternative events in most cases. We also observed a strong correlation between MSI and regional hypermethylation and between CIN and global hypomethylation. We further analyzed chromatin conformation and histone acetylation in cell lines with CIN or MSI. CIN cancers had open chromatin conformation and enriched histone acetylation in repetitive as well as in gene-specific regions. MSI cancers, on the other hand, had closed chromatin conformation and low levels of histone acetylation. After a MSI cell line was treated with 5-aza-2'-deoxycytidine or trichostatin A, the closed chromatin conformation became open, and histone acetylation was enriched. These observations support our hypothesis that in colorectal cancer, regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation, which might have a causal correlation with MSI and CIN, respectively.

Topics: Acetylation; Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; Chromatin; Chromosomal Instability; Colorectal Neoplasms; DNA Methylation; DNA, Neoplasm; Epigenesis, Genetic; Histones; Humans; Hydroxamic Acids; Immunoprecipitation; Loss of Heterozygosity; Microsatellite Repeats; Protein Conformation

Colorectal cancer accounts for approximately 10% of all new cancer cases reported worldwide. High dietary fiber intake has been associated with a reduced risk for this type of neoplasia, and much of this effect is ascribed to the histone acetylase (HDAC) inhibitor n-butyrate produced in the gastrointestinal tract. Natural chemopreventive and several new synthetic HDAC inhibitors exert multiple effects on tumor cells including the induction of differentiation, cell cycle arrest and apoptosis. Since cancer cells undergo mutational changes, it will be important to understand precisely which pathway gains or losses modulate or compromise HDAC inhibitor efficacy. We have recently documented that n-butyrate can provoke apoptosis in human HCT116 colorectal carcinoma cells independently of the p53 tumor suppressor and p21Waf inhibitor. Here, we have developed cell lines on the basis of HCT116 p21-/- cells and HCT116 cells in which the retinoblastoma tumor suppressor protein Rb has been specifically knocked down by antisense expression. The cells were exposed to the DNA-damaging drugs adriamycin (ADR) and etoposide or the HDAC inhibitors n-butyrate and trichostatin A (TSA). While the maximal apoptotic response, observed in the absence of p21Waf, was unaffected by the additional knockdown of Rb when cells were treated with ADR or etoposide, the toxicity of the HDAC inhibitors was significantly reduced. This indicates that hyperphosphorylated Rb itself, dissociated from E2F1 transcription factor, can contribute - directly or indirectly - to tumor cell apoptosis provoked by HDAC inhibitors.

Topics: Adenocarcinoma; Apoptosis; Butyrates; Cell Cycle Proteins; Cell Line, Tumor; Colony-Forming Units Assay; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA, Neoplasm; Doxorubicin; Enzyme Inhibitors; Etoposide; Flow Cytometry; Gene Deletion; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kinetics; Retinoblastoma Protein; Transfection

The mechanism of DNA hypermethylation-associated tumor suppressor gene silencing in cancer remains incompletely understood. Here, we show by chromatin immunoprecipitation that for three genes (P16, MLH1, and the O(6)-methylguanine-DNA methyltransferase gene, MGMT), histone H3 Lys-9 methylation directly correlates and histone H3 Lys-9 acetylation inversely correlates with DNA methylation in three neoplastic cell lines. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in moderately increased Lys-9 acetylation at silenced loci with no effect on Lys-9 methylation and minimal effects on gene expression. By contrast, treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC) rapidly reduced Lys-9 methylation at silenced loci and resulted in reactivation for all three genes. Combined treatment with 5Aza-dC and TSA was synergistic in reactivating gene expression through simultaneous effects on Lys-9 methylation and acetylation, which resulted in a robust increase in the ratio of Lys-9 acetylated and methylated histones at loci showing dense DNA methylation. By contrast to Lys-9, histone H3 Lys-4 methylation inversely correlated with promoter DNA methylation, was not affected by TSA, and was increased moderately at silenced loci by 5Aza-dC. Our results suggest that reduced H3 Lys-4 methylation and increased H3 Lys-9 methylation play a critical role in the maintenance of promoter DNA methylation-associated gene silencing in colorectal cancer.

Topics: Adaptor Proteins, Signal Transducing; Azacitidine; Carrier Proteins; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p16; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lysine; Methylation; MutL Protein Homolog 1; Neoplasm Proteins; Nuclear Proteins; O(6)-Methylguanine-DNA Methyltransferase; Precipitin Tests; Promoter Regions, Genetic; Tumor Cells, Cultured

Aberrant methylation of CpG islands can be a good molecular marker for identifying genes inactivated in cancer. We found the proapoptotic gene HRK to be a target for hypermethylation in human cancers and examined the role of such methylation in silencing the gene's expression.. Methylation of HRK was evaluated by bisulfite-PCR and bisulfite sequencing in a group of colorectal and gastric cancer cell lines and primary cancers. Gene expression and histone acetylation were examined by reverse transcription-PCR and chromatin immunoprecipitation analyses, respectively. Apoptosis of cancer cells after treatment with a DNA methyltransferase inhibitor and/or histone deacetylase inhibitor was examined with fluorescence-activated cell-sorting analysis.. The region around the HRK transcription start site was methylated in 36% of colorectal and 32% of gastric cancer cell lines and was closely associated with loss of expression in those cell types. HRK expression was restored by treatment with a methyltransferase inhibitor, 5-aza-deoxycytidine, and enhanced further by addition of histone deacetylase inhibitor trichostatin A or depsipeptide. Such restoration of HRK expression was well correlated with induction of apoptosis and enhancement of Adriamycin-induced apoptosis. Expression of other proapoptotic genes, including BAX, BAD, BID, and PUMA, was unaffected by treatment with 5-aza-deoxycytidine. Aberrant methylation of HRK was also frequently detected in primary colorectal cancers that showed methylation of multiple genes, including p16INK4A and hMLH1, and was associated with wild-type p53.. HRK methylation can be a useful molecular target for cancer therapy in a subset of colorectal and gastric cancers.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Azacitidine; Biomarkers, Tumor; Cell Line, Tumor; Cell Separation; Chromatin; Colorectal Neoplasms; CpG Islands; Decitabine; Depsipeptides; DNA Methylation; Doxorubicin; Flow Cytometry; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Models, Genetic; Neuropeptides; Oligopeptides; Polymerase Chain Reaction; Precipitin Tests; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms; Sulfites

We examined the relationship between aberrant DNA hypermethylation and key histone code components at a hypermethylated, silenced tumor suppressor gene promoter in human cancer. In lower eukaryotes, methylated H3-lysine 9 (methyl-H3-K9) determines DNA methylation and correlates with repressed gene transcription. Here we show that a zone of deacetylated histone H3 plus methyl-H3-K9 surrounds a hypermethylated, silenced hMLH1 promoter, which, when unmethylated and active, is embedded in methyl-H3-K4 and acetylated H3. Inhibiting DNA methyltransferases, but not histone deacetylases, leads first to promoter demethylation, second to gene reexpression, and finally to complete histone code reversal. Our findings suggest a new paradigm-DNA methylation may directly, or indirectly by inhibiting transcription, maintain key repressive elements of the histone code at a hypermethylated gene promoter in cancer.

Topics: Acetylation; Adaptor Proteins, Signal Transducing; Azacitidine; Carrier Proteins; Colorectal Neoplasms; Decitabine; DNA Methylation; DNA Modification Methylases; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Methylation; MutL Protein Homolog 1; Neoplasm Proteins; Nuclear Proteins; Promoter Regions, Genetic; Transcription, Genetic; Tumor Cells, Cultured

15-Lipoxygenase-1 (15-LO-1) is expressed at higher levels in human colorectal tumors compared with normal tissue. 15-LO-1 is expressed in cultured human colorectal cells, but only after treatment with sodium butyrate (NaBT), which also stimulates apoptosis and cell differentiation. We examined the regulation of 15-LO-1 in human tissue and the colorectal carcinoma cell lines Caco-2 and SW-480 by treatment with histone deacetylase (HDAC) inhibitors: NaBT, trichostatin A (TSA) and HC toxin. Northern and western analysis showed that expression of 15-LO-1 was up-regulated by these HDAC inhibitors. Furthermore, HDAC inhibitors stimulated promoter activity of the 15-LO-1 gene approximately 12-to 21-fold using the -331/-23 region of the 15-LO-1 promoter, as measured with a luciferase-15-LO-1 promoter-reporter system, suggesting that 15-LO-1 is regulated at the transcriptional level by HDAC inhibitors. Histone proteins in colorectal cells were acetylated after treatment with HDAC inhibitors. Histone acetylation was also measured in human colorectal tissue and a correlation was observed between increased histone acetylation and 15-LO-1 expression. Thus, regulation of 15-LO-1 expression in colorectal tissues appears to occur by a novel and new mechanism associated with histone acetylation. Moreover, these results suggest that 15-LO-1 is a marker that reflects histone acetylation in colorectal carcinoma.

Topics: Acetylation; Arachidonate 15-Lipoxygenase; Blotting, Northern; Butyrates; Caco-2 Cells; Colorectal Neoplasms; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Peptides, Cyclic; Tumor Cells, Cultured

Inhibitors of histone deacetylase (HDAC) block cell cycle progression at G1 in many cell types. We investigated the mechanism by which trichostatin A (TSA), a specific inhibitor of HDAC, induces G1 arrest in human cervix carcinoma HeLa cells. TSA treatment induced histone hyperacetylation followed by growth arrest in G as well as hypophosphorylation of pRb. The Cdk4 kinase activity was essentially unchanged during the TSA-induced G1 arrest. On the other hand, the arrest was accompanied by down-regulation of kinase activity of Cdk2, although the total protein levels of Cdk2 and its activator Cdc25A were unaffected. Upon TSA treatment, amounts of cyclin E and the CDK inhibitor p21WAF1/Cip1 were markedly increased, while that of cyclin A was reduced. The induction of p21 and down-regulation of cyclin A correlated well with the decreased Cdk2 activity and cell cycle arrest. Furthermore, gel filtration chromatography showed the association of p21 with the cyclin E-Cdk2 complex, suggesting that the activation of Cdk2 by the enhanced expression of cyclin E is blocked by the increased p21. The elevated expression of p2 is also observed in cells treated with trapoxin and FR901228, structurally unrelated histone deacetylase inhibitors. A human colorectal carcinoma cell line lacking both alleles of the p21 gene (p21-/-) was resistant to TSA several times more than the parental line (p21+/+). These results suggest that the suppression of Cdk2 kinase activity due to p21 overexpression play a critical role in HDAC inhibitor-induced growth inhibition.

Topics: Antibiotics, Antineoplastic; CDC2-CDC28 Kinases; Cell Division; Colorectal Neoplasms; Cyclin A; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA-Binding Proteins; Enzyme Inhibitors; Female; G1 Phase; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Protein Serine-Threonine Kinases; Transcription Factors; Tumor Cells, Cultured