trichostatin-a and Adenocarcinoma

The aim of this study was to sensitize the resistant breast adenocarcinoma cells towards Tumour Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced apoptosis.. Breast cancer is a heterogeneous disease involving complex mechanisms. TRAIL is a potential anticancer candidate for targeted treatment due to its selective killing effects on neoplastic cells. Nonetheless, resistance occurs in many cancers either intrinsically or after multiple treatments.. Therefore, this research investigated whether the combination of Trichostatin A (TSA) and Zebularine (Zeb) (TZ) followed by TRAIL (TZT) could sensitize the human breast adenocarcinoma cells towards apoptosis.. The breast adenocarcinoma cells, MDA-MB-231, MCF-7 and E-MDA-MB-231 (E-cadherin re-expressed MDA-MB-231) were treated with TSA, Zeb, TZ, TRAIL and TZT. The cells were subjected to hematoxylin and eosin (H & E) staining and FITC-Annexin V/Propidium Iodide apoptosis detection prior to proteome profiling.. Based on morphological observation, apoptosis was induced in all cells treated with all treatment regimens though it was more evident for the TZT-treated cells. In the apoptosis detection analysis, TZ increased early apoptosis significantly in MDA-MB-231 and MCF-7 while TRAIL induced late apoptosis significantly in E-MDA-MB-231. Based on the proteome profiling on MDA-MB-231, TRAIL R2 and Fas expression was increased. For E-MDA-MB- 231, down-regulation of catalase, paraoxonase-2 (PON2), clusterin, an inhibitor of apoptosis proteins (IAPs) and cell stress proteins validated the notion that E-cadherin re-expression enhances TZT anti-cancer efficacy. Similar trend was observed in MCF-7 whereby TZT treatment down-regulated the anti-apoptotic catalase and PON2, increased the proapoptotic, B cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad) and Bcl-2-associated X (Bax), second mitochondria-derived activator of caspase (SMAC) and HtrA serine peptidase 2 (HTRA2) as well as TRAIL receptors (TRAIL R1 and TRAIL R2).. TZ treatment serves as an efficient treatment regimen for MDA-MB-231 and MCF-7, while TRAIL serves as a better treatment option for E-MDA-MB-231. Therefore, future studies on E-cadherin's positive regulatory role in TRAIL-induced apoptosis are warranted.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Cadherins; Catalase; Cell Line, Tumor; Female; Humans; Inhibitor of Apoptosis Proteins; Ligands; Proteome; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Insulin-like growth factor (IGF) signaling plays an important role in tumorigenesis and metastasis. Here, we analyzed insulin-like growth factor (IGF) binding protein-2 (IGFBP2) expression in 81 lung cancer patients and 36 controls consisting of healthy and benign pulmonary lesion participants for comparison, then validated the IGFBP2 expression in additional 84 lung cancer patients, and evaluated the prognostic and chemoresistant significance of IGFBP2 in two cohorts respectively. Next we detected the reversal effect of trichostatin A (TSA) on chemoresistance in cell lines with high IGFBP2 expression. As a result, the mean expression of IGFBP2 in lung cancer patients was significantly higher than that in controls and increased with lung cancer progressed to advanced stage. In addition, high IGFBP2 expression was independently predictive for chemoresistance; over-expressed IGFBP2 enhances cell activity and TSA can reverse the chemoresistance induced by high IGFBP2 expression through enhancing autophagy. Furthermore, multivariate analysis showed that lung cancer patients whose blood IGFBP2 was higher had a poor survival outcome, with a hazard ratio of 8.22 (95%CI 1.78-37.92, P = 0.007) after adjustment for stage, histopathology, EGFR mutation, age, smoking and surgery.

Topics: Adenocarcinoma; Autophagy; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Case-Control Studies; Drug Resistance, Neoplasm; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Insulin-Like Growth Factor Binding Protein 2; Lung Neoplasms; Male; Middle Aged; Prognosis; Retrospective Studies; Survival Rate; Tumor Cells, Cultured

Different from genetic alterations, the reversible nature of epigenetic modifications provides an interesting opportunity for the development of clinically relevant therapeutics in different tumors. In this study, we aimed to screen and validate candidate genes regulated by the epigenetic marker associated with transcriptional activation, histone acetylation, in gastric cancer (GC). We first compared gene expression profile of trichostatin A-treated and control GC cell lines using microarray assay. Among the 55 differentially expressed genes identified in this analysis, we chose the up-regulated genes BMP8B and BAMBI for further analyses, that included mRNA and histone acetylation quantification in paired GC and nontumor tissue samples. BMP8B expression was reduced in GC compared to nontumor samples (P < 0.01). In addition, reduced BMP8B expression was associated with poorly differentiated GC (P = 0.02). No differences or histopathological associations were identified concerning BAMBI expression. Furthermore, acetylated H3K9 and H4K16 levels at BMP8B were increased in GC compared to nontumors (P < 0.05). However, reduced levels of acetylated H3K9 and H4K16 were associated with poorly differentiated GC (P < 0.05). Reduced levels of acetylated H3K9 was also associated with diffuse-type histological GC (P < 0.05). Notably, reduced BMP8B mRNA and acetylated H4K16 levels were positively correlated in poorly differentiated GC (P < 0.05). Our study demonstrated that BMP8B seems to be a tumor suppressor gene regulated by H4K16 acetylation in poorly differentiated GC. Therefore, BMP8B may be a potential target for TSA-based therapies in this GC sample subset. J. Cell. Biochem. 118: 869-877, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Acetylation; Adenocarcinoma; Adult; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line, Tumor; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Histones; Humans; Hydroxamic Acids; Male; Middle Aged; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms

Claudin-2 is highly expressed in lung adenocarcinoma tissues and increases proliferation in adenocarcinoma cells. The chemicals that reduce claudin-2 expression may have anti-cancer effects, but such therapeutic medicines have not been developed. We found that azacitidine (AZA), a DNA methylation inhibitor, and trichostatin A (TSA) and sodium butyrate (NaB), histone deacetylase (HDAC) inhibitors, decrease claudin-2 levels. The effect of AZA was mediated by the inhibition of phosphorylated Akt and NF-κB. LY-294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and BAY 11-7082, an NF-κB inhibitor, decreased claudin-2 levels. The reporter activity of claudin-2 was decreased by AZA and LY-294002, which was blocked by the mutation in a putative NF-κB-binding site. NF-κB bound to the promoter region of claudin-2, which was inhibited by AZA and LY-294002. AZA is suggested to decrease the claudin-2 mRNA level mediated by the inhibition of a PI3K/Akt/NF-κB pathway. TSA and NaB did not change phosphorylated Akt and NF-κB levels. Furthermore, these inhibitors did not change the reporter activity of claudin-2 but decreased the stability of claudin-2 mRNA mediated by the elevation of miR-497 microRNA. The binding of histone H3 to the promoter region of miR-497 was inhibited by TSA and NaB, whereas that of claudin-2 was not. These results suggest that HDAC inhibitors decrease claudin-2 levels mediated by the elevation of miR-497 expression. Cell proliferation was additively decreased by AZA, TSA, and NaB, which was partially rescued by ectopic expression of claudin-2. We suggest that epigenetic inhibitors suppress the abnormal proliferation of lung adenocarcinoma cells highly expressing claudin-2.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Azacitidine; Butyric Acid; Cell Proliferation; Chromones; Claudin-2; Down-Regulation; Epigenesis, Genetic; Humans; Hydroxamic Acids; Lung Neoplasms; MicroRNAs; Morpholines; Nitriles; RNA, Messenger; Signal Transduction; Sulfones

The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Adenoviridae; Adenoviridae Infections; Cell Line, Tumor; Cisplatin; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Lung Neoplasms; Proteasome Inhibitors

Trichostatin A (TSA) is a histone deacetylase inhibitor and a potential therapeutic for various malignancies. The in vivo effect of TSA, however, has not been investigated in a transgenic lung cancer model. Previously, we generated transgenic mice with overexpression of Groucho-related-gene 1 (Grg1) and these mice all developed mucinous lung adenocarcinoma. Grg1 is a transcriptional co-repressor protein, the function of which is thought to depend on HDAC activity. However, functions outside the nucleus have also been proposed. We tested the supposition that Grg1-induced tumorigenesis is HDAC-dependent by assaying the therapeutic effect of TSA in the Grg1 transgenic mouse model. We found that TSA significantly inhibited lung tumorigenesis in Grg1 transgenic mice (p < 0.01). TSA did not affect overall Grg1 protein levels, but instead reduced ErbB1 and ErbB2 expression, which are upregulated by Grg1 in the absence of TSA. We confirmed this effect in A549 cells. Furthermore, lapatinib, an inhibitor of both ErbB1 and ErbB2, effectively masked the effect of TSA on the inhibition of A549 cell proliferation and migration, suggesting TSA does work, at least in part, by downregulating ErbB receptors. We additionally found that TSA reduced the expression of VEGF and VEGFR2, but not basic FGF and FGFR1. Our findings indicate that TSA effectively inhibits Grg1-induced lung tumorigenesis through the down-regulation of ErbB1 and ErbB2, as well as reduced VEGF signaling. This suggests TSA and other HDAC inhibitors could have therapeutic value in the treatment of lung cancers with Grg1 overexpression.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Co-Repressor Proteins; Genes, erbB-1; Genes, erbB-2; Humans; Hydroxamic Acids; Lung Neoplasms; Mice; Mice, Transgenic

To investigate the effect of trichostatin A (TSA) on gastric cancer cell line BGC-823, and identify the differentially expressed genes induced by TSA, which might participate in the progression of gastric cancer.. MTT, fluorescence microscopy, and flow cytometry were used to detect the effect of TSA on growth inhibition and apoptosis of BGC-823 cells. Using gene microarray, we analyzed the changes in gene expression. Change in growth differentiation factor-15 (GDF-15) was verified by qRT-PCR and Western blotting. The expression of GDF-15 in gastric cancer and adjacent normal tissues was detected by immunohistochemistry.. Apoptosis of BGC-823 cells induced by TSA (75 ng/mL for 48 h) was demonstrated by flow cytometry. There were significant variations between TSA treated groups and control groups (P = 0.02). Nuclear chromatin condensation and fluorescence intensity were observed by fluorescence microscopy. GDF-15 gene expression and protein level were significantly reduced in the TSA treated group (75 ng/mL for 48 h). Immunohistochemistry demonstrated that the expression of GDF-15 in gastric adenocarcinoma was significantly higher than in the surrounding normal tissues (P < 0.05).. Lower GDF-15 gene expression due to TSA-induced apoptosis was found in gastric cancer cell line BGC-823. Higher GDF-15 gene expression was seen in gastric adenocarcinoma tissues.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Disease Progression; Down-Regulation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 15; Humans; Hydroxamic Acids; Immunohistochemistry; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms

MicroRNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Increasing evidence suggests that miRNAs are functionally important in cancers. We demonstrated miR-204 exerts its function by targeting gene involved in tumor growth and chemotherapy drugs reactivity. Here, we show that Trichostatin A (TSA) could increase ERα expression in MCF-7 and MDA-MB-231 cells by reducing miR204. Analysis of tumors growth inhibition shows that TSA promotes ERα expression, which could be reversed by miR-204 mimic transfection. When miR-204 is down regulated, the inhibition of TAM on breast cancer cells is enhanced. Caspase 3 activity is also increased. TSA and TAM combination inhibits Mcl-1 expression by decreasing phosphorylation of AKT induced by ERα increase in vivo and in vitro.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line; Cell Proliferation; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; MCF-7 Cells; Mice, Nude; MicroRNAs; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Tamoxifen; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Commonly occurred in aged males, the incidence of prostate carcinoma is increasing by years. Histone deacetylase (HDACs) as one key enzyme in regulating gene transcription has been found to be related with cancer occurrence. Trichostatin A (TSA) is one HDAC inhibitor for suppressing tumor growth. This study thus treated prostate carcinoma cell line PC3 with TSA, to analyze the effect of HDAC on the occurrence and progression of HDAC. PC3 cells were treated with gradient concentrations of TSA. MTT assay was employed to detect the proliferation of PC3 cells, while flow cytometry was used to detect the cell apoptosis and cell cycle. Apoptotic proteins including caspase-3, caspase-9 and bcl-2 were further quantified by Western blotting. MTT assays showed a dose- and time-dependent manner of TSA in inhibiting PC3 cell proliferation. Most of PC3 cells were arrested at G1 phase after treating with TSA. The apoptotic ratio of cells was also elevated by higher concentrations of drugs. Apoptotic proteins including caspase-3, caspase-9 and bcl-2 were all up-regulated by TSA. HDAC inhibitor can effectively suppress the proliferation of prostate carcinoma cells, which can be arrested at G1 phase. The elevated apoptotic ratio was caused by up-regulation of apoptosis-related proteins caspase-3, caspase-9 and bcl-2, in both dose- and time-dependent manners.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Male; Prostatic Neoplasms

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

To understand the epigenetic mechanism underlying the PR-B gene silencing in endometrial cancer (EC) cells, we compared the chromatin composition between transcriptionally active and silenced PR-B genes in EC cell lines and cancer tissues.. Chromatin Immunoprecipitation (ChIP) assay was performed to measure MBD occupancy and histone acetylation/methylation in transcriptionally active and silenced PR-B genes. PR-B-positive/-negative, as well as epigenetic inhibitor-treated/-untreated EC cells were used as study models. Real-time polymerase chain reaction (PCR) and Western blot analysis were applied to measure the mRNA and protein levels of PR-B, MBD, and histones.. A close association among PR-B methylation, MBD binding and PR-B gene silencing was observed. Treatment with epigenetic inhibitors led to dynamic changes in the PR-B chromatin composition and gene expression. Increased H3/H4 acetylation and H3-K4 methylation, and decreased H3-K9 methylation were found to be associated with re-activation of silenced PR-B genes. MeCP2 knockdown resulted in a decreased MeCP2 binding to PR-B genes and an increased PR-B expression. ChIP analysis of MeCP2 binding to PR-B genes in the PR-B-positive/-negative EC samples confirmed the significant role of MeCP2 in PR-B silencing.. PR-B gene expression is regulated by a concerted action of epigenetic factors including DNA methylation, MBD binding, and histone modifications. MeCP2 occupancy of PR-B genes plays a critical role in PR-B gene silencing. These findings enriched our knowledge of the epigenetic regulation of PR-B expression in EC, and suggested that the epigenetic re-activation of PR-B could be explored as a potential strategy to sensitize the PR-B-negative endometrial cancers to progestational therapy.

Topics: Adenocarcinoma; Azacitidine; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; DNA Methylation; DNA-Binding Proteins; Endometrial Neoplasms; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Methyl-CpG-Binding Protein 2; Neoplasm Proteins; Protein Binding; Protein Interaction Mapping; Real-Time Polymerase Chain Reaction; Receptors, Progesterone; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Transcription Factors

Ultraconserved regions (UCR) are genomic segments of more than 200 base pairs that are evolutionarily conserved among mammalian species. They are thought to have functions as transcriptional enhancers and regulators of alternative splicing. Recently, it was shown that numerous RNAs are transcribed from these regions. These UCR-encoded transcripts (ucRNAs) were found to be expressed in a tissue- and disease-specific manner and may interfere with the function of other RNAs through RNA: RNA interactions. We hypothesized that ucRNAs have unidentified roles in the pathogenesis of human prostate cancer. In a pilot study, we examined ucRNA expression profiles in human prostate tumors.. Using a custom microarray with 962 probesets representing sense and antisense sequences for the 481 human UCRs, we examined ucRNA expression in resected, fresh-frozen human prostate tissues (57 tumors, 7 non-cancerous prostate tissues) and in cultured prostate cancer cells treated with either epigenetic drugs (the hypomethylating agent, 5-Aza 2'deoxycytidine, and the histone deacetylase inhibitor, trichostatin A) or a synthetic androgen, R1881. Expression of selected ucRNAs was also assessed by qRT-PCR and NanoString®-based assays. Because ucRNAs may function as RNAs that target protein-coding genes through direct and inhibitory RNA: RNA interactions, computational analyses were applied to identify candidate ucRNA:mRNA binding pairs.. We observed altered ucRNA expression in prostate cancer (e.g., uc.106+, uc.477+, uc.363 + A, uc.454 + A) and found that these ucRNAs were associated with cancer development, Gleason score, and extraprostatic extension after controlling for false discovery (false discovery rate < 5% for many of the transcripts). We also identified several ucRNAs that were responsive to treatment with either epigenetic drugs or androgen (R1881). For example, experiments with LNCaP human prostate cancer cells showed that uc.287+ is induced by R1881 (P < 0.05) whereas uc.283 + A was up-regulated following treatment with combined 5-Aza 2'deoxycytidine and trichostatin A (P < 0.05). Additional computational analyses predicted RNA loop-loop interactions of 302 different sense and antisense ucRNAs with 1058 different mRNAs, inferring possible functions of ucRNAs via direct interactions with mRNAs.. This first study of ucRNA expression in human prostate cancer indicates an altered transcript expression in the disease.

Topics: Adenocarcinoma; Aged; Azacitidine; Case-Control Studies; Cell Line, Tumor; Conserved Sequence; Decitabine; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Genome, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Metribolone; Middle Aged; Oligonucleotide Array Sequence Analysis; Prostate; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; RNA, Untranslated; Testosterone Congeners; Transcriptome

Cytoglobin (CYGB) is frequently downregulated in many types of human malignancies, and its exogenous overexpression reduces proliferation of cancer cells. Despite its implied tumour suppressor (TSG) functions, its exact role in carcinogenesis remains unclear as CYGB upregulation is also associated with tumour hypoxia and aggressiveness. In this study, we explore the TSG role of CYGB, its influence on the phenotype of cancerous cells under stress conditions and the clinical significance of CYGB expression and promoter methylation in non-small cell lung cancer (NSCLC). DNA methylation-dependent expression silencing of CYGB is demonstrated in both clinical samples and cell lines. CYGB promoter was more frequently methylated in lung adenocarcinomas (P = 1.4 × 10(-4)). Demethylation by 5'-azadeoxycytidine partially restored CYGB expression in cell lines. Interestingly, trichostatin A triggered upregulation of CYGB expression in cancer cell lines and downregulation in non-tumourigenic ones. CYGB mRNA expression in NSCLC surgical specimens correlated with that of HIF1α and VEGFa (P < 1 × 10(-4)). Overexpression of CYGB in cancer cell lines reduced cell migration, invasion and anchorage-independent growth. Moreover, CYGB impaired cell proliferation, but only in the lung adenocarcinoma cell line (H358). Upon hydrogen peroxide treatment, CYGB protected cell viability, migratory potential and anchorage independence by attenuating oxidative injury. In hypoxia, CYGB overexpression decreased cell viability, augmented migration and anchorage independence in a cell-type-specific manner. In conclusion, CYGB revealed TSG properties in normoxia but promoted tumourigenic potential of the cells exposed to stress, suggesting a bimodal function in lung tumourigenesis, depending on cell type and microenvironmental conditions.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Carcinoma, Non-Small-Cell Lung; Cell Hypoxia; Cell Line, Tumor; Cytoglobin; DNA Methylation; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Globins; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Oncogenes; Vascular Endothelial Growth Factor A

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

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

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

Histone deacetylase inhibitors can regulate gene expression through modulation of the degree of acetylation of histone and non-histone, thus affecting cell proliferation, survival and chemosensitivity. Histone deacetylase inhibitors combined with paclitaxel may enhance the inhibitory effect of drugs on lung cancer cells. This study aimed to observe the effect of trichostatin A (TSA)/paclitaxel on the proliferation and apoptosis in human A549 lung adenocarcinoma cells, and to investigate its mechanism.. A549 cells were cultured in Dulbecco modified Eagle's medium (DMEM) in the presence of paclitaxel and the histone deacetylase inhibitor TSA, and the growth curve was obtained by trypan-blue exclusion assay and cell count. Apoptosis was assessed using Hoechst 33258 staining and flow cytometry analysis, and cell cycle was detected by flow cytometry analysis. The proteins poly ADP-ribose polymerase (PARP), caspase-3, survivin, and tubulin acetylation were detected by Western blotting.. A significant reduction of proliferation was observed in A549 lung adenocarcinoma cells treated by paclitaxel or TSA. Combined treatment with TSA/paclitaxel caused the greatest inhibition of cell proliferation. The combined treatment with TSA and paclitaxel induced more severe apoptosis, and significantly more cells were arrested in G2/M phase (P < 0.05) then with a single drug. Using Western blotting, we demonstrated that treatment with TSA/paclitaxel led to synergistic increase in acetylated tubulin, PARP, caspase-3, and reduced the expression of survivin.. TSA and paclitaxel have a synergistic activity that can inhibit cell growth and induce apoptosis.

Topics: Acetylation; Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Paclitaxel; Tubulin

Src has a role in the anoikis resistance in lung adenocarcinomas. We focused on two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R+T790M), in suspension to elucidate whether suspended lung adenocarcinoma cells are eradicated by long-term treatment with Src tyrosine kinase inhibitors (TKIs). We also examined metastasis-positive lymph nodes from 16 EGFR-mutant lung adenocarcinoma patients for immunohistochemical expression of mutant-specific EGFR. Almost all suspended HCC827 cells underwent apoptosis after 144 h of combination treatment with AZD0530, trichostatin A (TSA), and ABT-263, whereas many suspended H1975 cells survived the treatment. AZD0530 is a Src TKI, TSA is a histone deacetylase inhibitor, and ABT-263 is a Bcl-2 inhibitor. During the therapy, the phosphorylation of EGFR decreased in HCC827 cells and remained stable in H1975 cells. The phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, was completely suppressed by the third generation EGFR TKI, WZ4002. Consequently, both the suspended cell lines were almost completely eradicated within 144 h, with the combined therapy of WZ4002, ABT-263, and TSA. Interestingly, treated suspended cells underwent apoptosis to a greater extent than did adherent cells. Intrasinus floating lung adenocarcinoma cells in the lymph nodes expressed a mutant-specific EGFR. These findings suggest that suspended EGFR-mutant lung adenocarcinoma cells depend significantly more on EGFR activation for survival than attached cells do. The tumor cells circulating in vessels, which express mutant-specific EGFR, would be highly susceptible to the combination therapy of WZ4002, ABT-263, and TSA.

Topics: Acrylamides; Adenocarcinoma; Aged; Aged, 80 and over; Aniline Compounds; Anoikis; Benzodioxoles; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Therapy, Combination; ErbB Receptors; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Male; Middle Aged; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Quinazolines; src-Family Kinases; Sulfonamides

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

AFP-producing adenocarcinoma is a variant of adenocarcinoma with high malignancy. Production of AFP suggests enteroblastic or hepatoid differentiation of cancer cells. GATA4 is a key molecule involved in the prenatal development of the stomach and liver. GATA4 is epigenetically silenced by hypermethylation of primer region in many types of cancers including gastric cancer. The aim of this study is to investigate the expression and epigenetic regulation of GATA4 in AFP-producing adenocarcinoma. Immunohistochemical analysis revealed that GATA4 was positive in 3/8 cases of AFP-producing gastric adenocarcinomas and in 28/30 cases of common type adenocarcinomas. Epigenetic modification of GATA4 promoter region was investigated with 3 AFP-producing and 4 common-type gastric cancer cell lines. GATA4 mRNA was detected in 1/3 of AFP-producing and 2/4 of common-type gastric cancer cell lines by RT-PCR. Methylation-specific PCR revealed no GATA4 methylation in any of the AFP-producing gastric cancers, whereas methylation was consistent with GATA4 expression in the common-type gastric cancers. Chromatin immunoprecipitation assay for AFP-producing gastric cancers revealed that histones H3 and H4 were hypoacetylated in the GATA4-negative cells, while they were hyperacetylated in the GATA4-positive cells. Treatment with trichostain A, an inhibitor for histone deacetylase, induced acetylation of histones H3 and H4, and tri-methylation of lysine 4 of histone H3, which was associated with the active transcription of GATA4 in GATA4-negative AFP-producing cells. These results indicated that histone deacetylation is a silencing mechanism for GATA4 expression in AFP-producing gastric cancer cells. Differences between AFP-producing gastric cancer and common-type gastric cancer in terms of the mechanism of GATA4 regulation may be reflected in the phenotypic deviation of AFP-producing gastric cancer from common-type gastric cancer.

Topics: Adenocarcinoma; alpha-Fetoproteins; Cell Line, Tumor; DNA Methylation; Epigenesis, Genetic; GATA4 Transcription Factor; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Lysine; Promoter Regions, Genetic; Protein Processing, Post-Translational; Stomach Neoplasms; Transcription, Genetic

Pancreatic cancer is the fourth-leading cause of death in the United States and one of the most aggressive known malignancies. New and innovative advances in treatment are desperately needed. One promising area of investigational treatment for pancreatic cancer involves the use of immunotherapy. The development of immunotherapy for pancreatic cancer has been hampered by difficulty in generating tumor-reactive lymphocytes from resected specimens and by a lack of appropriate target antigens expressed on tumor cells. Innovative strategies have been developed with the use of peripheral blood lymphocytes that are genetically engineered to express T-cell receptors targeting common tumor antigens, including cancer-testis antigens, such as the MAGE-A3 antigen. Cancer-testis antigens pose excellent targets for immunotherapy because they are expressed in cancer and in the testis, an immune-privileged site, but have limited expression in normal tissue. An additional advantage in targeting cancer-testis antigens for immunotherapy is that their expression can be selectively up-regulated in tumor cells via epigenetic regulation with chromatin remodeling agents. Current interest in targeting cancer-testis antigens in pancreatic cancer is well-founded because cancer-testis antigens have been shown to be expressed in pancreatic cancer as potential targets for therapy. In our studies, we validated the expression pattern of cancer-testis antigens in resected specimens of pancreatic cancer and tested the hypothesis that treatment of pancreatic cancer cells with chromatin remodeling agents would render them more sensitive to antigen-specific T lymphocytes. We focused predominately on the MAGE-A3 antigen because it is highly expressed in pancreatic cancer, and several immunotherapeutic strategies are in clinical trials targeting this specific antigen. The results of these studies have important translational implications and provide the rationale for combined treatment with chromatin remodeling agents and immunotherapeutic approaches for pancreatic cancer.

Topics: Adenocarcinoma; Antigens, Neoplasm; Azacitidine; Cell Line, Tumor; Chromatin Assembly and Disassembly; Cytokines; Decitabine; Deoxycytidine; DNA Methylation; Gemcitabine; Humans; Hydroxamic Acids; Immunotherapy; Male; Neoplasm Proteins; Pancreas; Pancreatic Neoplasms; Receptors, Antigen, T-Cell; T-Lymphocytes; Testis; Valproic Acid

To investigate the effect of trichostatin A (TSA)/paclitaxel on the growth and apoptosis in human lung adenocarcinoma cell line A549 cells.. Human lung adenocarcinoma A549 cells were cultured in DMEM in the presence of paclitaxel and the histone deacetylase inhibitor trichostatin A, and the growth curve was obtained by trypan-blue exclusion assay and cell count. Apoptosis was assessed using Hoechst 33258 staining and flow cytometry, and cell cycle was detected by flow cytometry analysis. The proteins of PARP, caspase-3, survivin and tubulin acetylation were detected by Western blotting.. Significant growth reduction was observed in the A549 cells following treatment with paclitaxel or the histone deacetylase inhibitor TSA. The combined treatment with TSA/paclitaxel caused the highest inhibition of cell growth. The apoptosis rate of A549 cells treated with TSA or paclitaxel for 24 hours was (17.6 ± 1.8)% and (39.2 ± 3.7)%, respectively, but a significantly higher apoptosis rate was (64.2 ± 4.2)% was induced by combined treatment with TSA and paclitaxel. In contrast with the control group, the cell cycle was markedly arrested at G2/M phase in the TSA and paclitaxel group (P < 0.05). The Western blot analysis demonstrated that treatment with TSA/paclitaxel led to a synergistic increase of acetylated tubulin, PARP and caspase-3, and reduced the expression of survivin.. TSA or paclitaxel alone can inhibit the cell growth and induce apoptosis, and the combination of TSA and paclitaxel exerts a synergistic effect on the growth and apoptosis in lung adenocarcinoma cells.

Topics: Acetylation; Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Paclitaxel; Poly(ADP-ribose) Polymerases; Survivin; Tubulin; Tubulin Modulators

Somatostatin (SST), a primary inhibitor of gastrin-stimulated gastric acid secretion, has potent antitumor and anti-secretory activity in several human cancers.. This study was performed to investigate the SST gene expression levels and possible epigenetic mechanisms that regulate expression of SST in gastric adenocarcinomas.. Quantitative real-time (RT)-PCR and quantitative bisulfite pyrosequencing were used to study primary gastric cancer tissue samples and cell lines.. Quantitative RT-PCR analysis revealed down-regulation of the SST transcript in 93% of gastric carcinoma samples (30/32), compared with 21 normal samples (P<0.001). Because of the presence of a large CpG island in the SST promoter, we next examined its promoter DNA methylation levels by use of quantitative bisulfite pyrosequencing. The results revealed a significant increase in SST promoter DNA methylation in tumor samples compared with normal samples (P<0.05). Promoter DNA hypermethylation and silencing of SST was also detected in seven gastric cancer cell lines that we tested. To confirm the role of promoter DNA methylation as an epigenetic mechanism regulating SST expression, AGS gastric cancer cells were treated with 5-Aza-dc. This treatment led to reduction of promoter DNA methylation levels of SST accompanied by restoration of its mRNA expression.. Our results indicate that promoter DNA methylation levels play a critical role in regulating SST expression in gastric cancer. This finding provides a foundation for further studies on the role of SST in gastric carcinogenesis and its potential as a biomarker for gastric cancers.

Topics: Adenocarcinoma; Antineoplastic Agents; Azacitidine; Biomarkers, Tumor; Cell Line, Tumor; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Methylation; Predictive Value of Tests; Reproducibility of Results; RNA, Messenger; Somatostatin; Stomach Neoplasms

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

Cisplatin-based chemotherapy is the standard therapy used to treat non-small-cell lung cancer. However, its efficacy is largely limited due to the development of drug resistance. The exact mechanism in which cancer cells develop resistance to the drug is not yet fully understood. The purpose of the present study is to test the role of volume-sensitive Cl(-) channels in cisplatin resistance in human lung adenocarcinoma cells (A549 cells) using patch-clamp recording, cell volume measurement and apoptosis assay. The results showed that cisplatin treatment induced an apoptotic volume decrease (AVD) and activated a Cl(-) current that showed properties similar to the volume-sensitive outward rectifying (VSOR) Cl(-) current in wild-type A549 cells. Both the AVD process and VSOR Cl(-) current were blocked by the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2' disulfonic acid. However, the A549/CDDP cells, a model of acquired cisplatin resistance cells, on the other hand, had almost no AVD process and VSOR Cl(-) current when treated with cisplatin. Treatment of A549/CDDP cells with trichostatin A (TSA), a drug that inhibits histone deacetylases, partially restored the VSOR Cl(-) current and increased cisplatin-induced cell apoptosis rate. These results suggest that impaired activity of VSOR Cl(-) channels contributes to the cisplatin resistance in A549/CDDP cells.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Base Sequence; Cell Line, Tumor; Chloride Channels; Cisplatin; DNA Primers; Drug Resistance, Neoplasm; Humans; Hydroxamic Acids; Lung Neoplasms; Patch-Clamp Techniques; Reverse Transcriptase Polymerase Chain Reaction

Lung cancer is the leading cause of cancer-related mortality and requires more effective molecular markers of prognosis and therapeutic responsiveness. Special AT-rich binding protein 1 (SATB1) is a global genome organizer that recruits chromatin remodeling proteins to epigenetically regulate hundreds of genes in a tissue-specific manner. Initial studies suggest that SATB1 overexpression is a predictor of poor prognosis in breast cancer, but the prognostic significance of SATB1 expression has not been evaluated in lung cancer.. A cohort of 257 lung cancers was evaluated by immunohistochemistry. Epigenetic silencing of SATB1 was examined in cell lines by 5-Aza 2-deoxycytidine and trichostatin A treatment, and chromatin immunoprecipitation.. Significant loss of SATB1 expression was found in squamous preinvasive lesions (p < 0.04) and in non-small cell lung cancers (p < 0.001) compared with matched normal bronchial epithelium. Loss of SATB1 independently predicted poor cancer-specific survival in squamous cell carcinomas (SCCs; hazard ratio: 2.06, 95% confidence interval: 1.2-3.7, p = 0.016). Treatment of lung cancer cell lines with the histone deacetylase inhibitor trichostatin A resulted in up-regulation of SATB1. SATB1 was associated with a decrease in the active chromatin mark acetylated histone H3K9 and an increase in the repressive polycomb mark trimethylated H3K27 in a SCC cell line relative to a normal bronchial epithelial cell line.. This is the first study showing that SATB1 expression is lost in early preinvasive squamous lesions and that loss of SATB1 is associated with poor prognosis in lung SCC. We hypothesize that the SATB1 gene is epigenetically silenced through histone modifications.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Azacitidine; Blotting, Western; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Chromatin Immunoprecipitation; Cohort Studies; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Lung Neoplasms; Male; Matrix Attachment Region Binding Proteins; Middle Aged; Neoplasm Staging; Prognosis; Tissue Array Analysis

To understand the mechanism of transcriptional down-regulation of BRCA1 by promoter methylation, we screened 51 breast cancer cell lines and identified HCC38 as another BRCA1 promoter-methylated cell line in addition to UACC3199. There was low expression of BRCA1 mRNA and BRCA1 protein in both cell lines as measured by quantitative RT-PCR and western blot analysis. After transient treatment with 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A (TSA), re-expression of BRCA1 mRNA and BRCA1 protein was detected in UACC3199 cells, but not in HCC38 cells. Another demethylating agent, zebularine, did not induce BRCA1 re-expression in either cell line. To test the hypothesis that methylation of CpG sites may affect accessibility of the BRCA1 promoter to transcription factors and consequently cause down-regulation of BRCA1, we analyzed the binding of four transcription factors (CTCF, Sp1, E2F1 and E2F6) to the BRCA1 promoter using chromatin immunoprecipitation assay (ChIP) and quantitative PCR. CTCF and E2F1 were enriched at the unmethylated BRCA1 promoter in MCF-7 cells. In contrast, these two transcription factors were not enriched at the methylated BRCA1 promoter in UACC3199 and HCC38 cells. Following demethylating drug treatment, E2F1 was enriched at the BRCA1 promoter in the demethylated UACC3199 cells. This indicates that reduced accessibility of transcription factors to the methylated promoter is one of the mechanisms for down-regulation of BRCA1 in heavily methylated cancer cells.

Topics: Adenocarcinoma; Azacitidine; BRCA1 Protein; Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Genes, BRCA1; Humans; Hydroxamic Acids; Neoplasm Proteins; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Transcription Factors; Transcription, Genetic

To investigate the apoptosis-inducing effect of trichostatin A (TSA) in the human lung adenocarcinoma cisplatin-resistant cell line (A549/CDDP) and to examine whether TSA can enhance sensitivity to cisplatin treatment and the underlying molecular mechanisms of such an enhancement.. Cell viability was evaluated using the Neutral Red assay. Apoptosis was assessed using Hoechst 33258 staining and flow cytometry analysis. Protein expression was detected by Western blotting. To determine the role of Death-associated protein kinase (DAPK) in TSA-induced apoptosis in the A549/CDDP cell line, cells were transfected with pcDNA3.1(+)-DAPK, which has a higher expression level of DAPK compared to endogenous expression, and DAPK activity was inhibited by both over-expression C-terminal fragment of DAPK which may competitive binding DAPK substrates to inhibit the function of DAPK and RNA interference.. TSA induced apoptosis in both A549 cells and A549/CDDP cells. TSA enhanced the sensitivity of A549/CDDP cells to cisplatin, along with concomitant DAPK up-regulation. When DAPK was over-expressed, A549/CDDP cells became sensitive to cisplatin and the cytotoxicity of TSA could be increased. Moreover, the cytotoxicity of TSA could be alleviated by inhibition of DAPK activity by the expression of a recombinant C-terminal fragment of DAPK or RNA interference.. TSA induced sensitivity to cisplatin treatment in cisplatin-resistant A549 cells. The up-regulation of DAPK is one of the mechanisms mediating sensitization to TSA-induced apoptosis in cisplatin-resistant cells.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cell Survival; Cisplatin; Death-Associated Protein Kinases; Drug Resistance, Neoplasm; Humans; Hydroxamic Acids; Lung Neoplasms; Protein Synthesis Inhibitors; Up-Regulation

To explore the effect of trichostatin A (TSA) on human lung cancer cell strains A549.. A549 cells were exposed to TSA at different concentrations, then the growth-inhibiting effects of the cell line were detected with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; After the cells were exposed to TSA for 48 and 96 hours at 300 nmol/L, the change of the cell cycle and apoptosis of A549 were analyzed with flow cytometry. p21 protein and extracellular signal regulated kinase (ERK) expression were detected by Western blot.. TSA inhibited the growth of A549 cells in time- and concentration-dependent manners. The proportion of apoptosis, G0/G1 and G2/M phase increased in accordance with raising of the TSA concentration. The expression of p21 protein was significantly up-regulated and the expression of phosphorylation ERK was significantly down-regulated after A549 cells were treated with TSA.. Histone deacetylase inhibitor TSA can inhibit the proliferation of human lung cancer cell strains A549 and induce the cell cycle arrest and apoptosis in the A549 cells. This may be related to up-regulation of p21 protein expression and the down-regulation of phosphorylation ERK.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Cell Cycle; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms

The licorice-derived compounds glycyrrhizin and 18β-glycyrrhetinic acid have been shown to induce apoptosis in various cancer cells. However, the effect of these licorice compounds on the apoptotic effect of histone deacetylase inhibitors in epithelial ovarian carcinoma cells has not been determined. We assessed the effect of 18β-glycyrrhetinic acid on trichostatin A-induced apoptosis in the human epithelial carcinoma cell lines OVCAR-3 and SK-OV-3. Trichostatin A induced nuclear damage, decreased Bid and Bcl-2 protein levels, increased in Bax levels, induced cytochrome c release, activated caspase-8, -9 and -3, and increased tumor suppressor p53 levels. 18β-Glycyrrhetinic acid potentiated the trichostatin A-induced apoptosis-related protein activation and cell death. Unlike 18β-glycyrrhetinic acid, up to 25 μM of the pro-compound glycyrrhizin did not induce cell death and did not affect trichostatin A-induced apoptosis. The results suggest that 18β-glycyrrhetinic acid may potentiate the apoptotic effects of trichostatin A against ovarian carcinoma cell lines by increasing the activation of the caspase-8-dependent pathway as well as the activation of the mitochondria-mediated cell death pathway, leading to activation of caspases. 18β-Glycyrrhetinic acid may enhance the therapeutic effect of trichostatin A against epithelial ovarian adenocarcinoma.

Topics: Adenocarcinoma; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Nucleus; Cell Survival; DNA Fragmentation; Drug Synergism; Enzyme Activation; Female; Glycyrrhetinic Acid; Glycyrrhizic Acid; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mitochondria; Ovarian Neoplasms; Prodrugs; Tumor Suppressor Protein p53

The imaging analytical capabilities of laser scanning cytometer (LSC) have been used to assess morphological features considered to be typical of the senescent phenotype. The characteristic "flattening" of senescent cells was reflected by the decline in the density of staining (intensity of maximal pixel) of DNA-associated fluorescence [4,6-diamidino-2-phenylindole (DAPI)] paralleled by an increase in nuclear size (area). The decrease in ratio of maximal pixel to nuclear area was even more sensitive senescence biomarker than the change in maximal pixel or nuclear area, each alone. The saturation cell density at plateau phase of growth recorded by LSC was found to be dramatically decreased in cultures of senescent cells, thereby also serving as an additional marker. The induction of cyclin dependent kinase inhibitors p21(WAF1) and p27(KIP1) and γH2AX and activation of ATM markers of DNA damage response were measured in parallel with DNA/DAPI maximal pixel and nuclear area. These biomarker indices were expressed in quantitative terms by reporting them as a fraction of the respective controls. The effect of treatment of A549 and WI-38 cells with different concentrations of mitoxantrone (Mxt) and trichostatin A for various time periods was studied to assess the degree (depth) of cell senescence. Also assessed was the effect of 2-deoxy-D-glucose, the agent attenuating metabolic cell activity, on the depth of senescence induced by Mxt. A relationship between the ability of cells to synthesize RNA (incorporate 5-ethynyluridine) that leads to growth imbalance and induction of cell senescence was also studied. The data show that morphometric analysis of cellular attributes by LSC offers an attractive tool to detect cell senescence and measure its degree particularly in assessing effects of the factors that enhance or attenuate this process. This methodology is of importance in light of the evidence that cellular senescence is not only a biological process that is fundamental for organismal aging but also impedes formation of induced-pluripotent stem cells providing the barrier for neoplastic transformation and is the major mechanism of induction of reproductive cell death during treatment of solid tumors.

Topics: Adenocarcinoma; Apoptosis; beta-Galactosidase; Biomarkers; Cell Count; Cell Line, Tumor; Cell Nucleus; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Fibroblasts; Fluorescent Dyes; Histones; Humans; Hydroxamic Acids; Indoles; Intracellular Signaling Peptides and Proteins; Laser Scanning Cytometry; Lung Neoplasms; Mitoxantrone

Epidemiological and experimental evidence implicates estrogens in the etiology and progression of breast cancer. The biosynthesis of estrogens from androgens is catalyzed by an enzymatic complex designated as aromatase (CYP19). Using quantitative real-time PCR and Western blot analysis, we demonstrated that trichostatin A (TSA) histone deacetylase inhibitor significantly reduced CYP19 transcript and protein contents in MCF-7 breast cancer cells. We also found that TSA lowered CYP19 transcript stability and significantly decreased the transcript's half-life from approximately 6h to 3.5h. Our results from experiments with a protein biosynthesis inhibitor suggest the involvement of an RNase and/or mRNA stabilization protein in CYP19 transcript stabilization. Since malignant tissue aromatase is a significant estrogen producer involved in breast tumor progression, our findings may have clinical implication.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; Aromatase; Aromatase Inhibitors; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Half-Life; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasm Proteins; RNA Stability; RNA, Messenger; RNA, Neoplasm

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

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

Glutathione-S-transferase (Gst) genes are downregulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors.. Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n = 15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5' CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured.. Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells.. Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression.

Topics: Adenocarcinoma; Animals; Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Drug Combinations; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Glutathione S-Transferase pi; Glutathione Transferase; Hydroxamic Acids; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Metastasis; Prostate; Prostatic Neoplasms; RNA, Messenger

The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-alpha, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2'-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions -666 to -426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2.

Topics: Adenocarcinoma; Azacitidine; CCAAT-Enhancer-Binding Protein-alpha; Cell Line, Tumor; Chromatin Immunoprecipitation; Decitabine; DNA; DNA Methylation; Gene Silencing; Humans; Hydroxamic Acids; Interleukin-6; Methyl-CpG-Binding Protein 2; NF-kappa B; Pancreatic Neoplasms

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 importance of alpha-2-glycoprotein 1, zinc (AZGP1) in lung adenocarcinoma (AD) remains largely unknown. Analysis of serum autoantibodies to tumor antigens combined with gene expression profiling of primary tumors may provide insight into the mechanisms underlying lung carcinogenesis and identify new AD biomarkers.. T7 phage cDNA libraries were used to identify AZGP1 autoantibodies in the serum of 473 patients (192 ADs, 192 matched controls, and 89 additional ADs for confirmation of findings). AZGP1 mRNA expression was examined in 86 ADs and 10 control lung tissue samples using oligonucleotide microarrays. AZGP1 protein expression was studied in 230 tissue samples (222 ADs; 8 controls) with immunohistochemistry. Kaplan-Meier analyses were used to correlate circulating autoantibody and tissue mRNA production with survival. AD cell lines A549 and SKLU1 were treated with 5-aza-2;-deoxycytidine (5-AZA) and trichostatin A (TSA) to examine the role of promoter methylation and histone deacetylation in the expression of AZGP1. Real-time polymerase chain reaction was used to quantify the effects of treatment.. In patients with AD, AZGP1 autoantibodies were observed in 40% of serum samples. Autoantibody production correlated with improved overall 5-year survival (p = 0.002) and improved survival in those with stage I to II disease (p = 0.008). A verification analysis was performed for the survival benefit and found similar results with p values of 0.02 and 0.036, respectively. Although abundant mRNA expression was found in a subset of tumors, mRNA expression did not correlate with prognosis. In normal lung, AZGP1 mRNA and protein expression were low or absent, whereas in AD they were highly expressed in 31.3% and 42.8% of samples, respectively. To determine whether AZGP1 expression in this subset of tumors might be affected by epigenetic mechanisms, low AZGP1-expressing A549 and SKLU1 AD cell lines were treated with TSA and 5-AZA. A 713-fold and 169-fold increase in mRNA expression were noted on treatment with TSA, respectively. Treatment with 5-AZA had minimal effect on AZGP1 mRNA expression.. The presence of AZGP1 serum autoantibody may be used as a prognostic marker in patients with AD. Furthermore, up-regulation of AZGP1 mRNA in AD may be affected by chromatin remodeling by means of histone acetylation.

Topics: Acetylation; Adenocarcinoma; Adipokines; Adult; Aged; Aged, 80 and over; Autoantibodies; Azacitidine; Biomarkers, Tumor; Carrier Proteins; Case-Control Studies; Enzyme Inhibitors; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycoproteins; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Tumor Cells, Cultured

The insulin-receptor substrate family plays important roles in cellular growth, signaling, and survival. Two new members of this family have recently been isolated: IRS5/Dok4 and IRS6/Dok5. This study examines the expression of IRS5/DOK4 in a panel of lung cancer cell lines and tumor specimens. The results demonstrate that expression of IRS5/DOK4 is frequently altered with both elevated and decreased expression in non-small-cell lung cancer (NSCLC) tumor specimens. The altered expression of IRS5/DOK4 observed in tumor samples is not due to aberrant methylation. In vitro cell culture studies demonstrate that treatment of NSCLC cell lines with the histone deacetylase inhibitor trichostatin A (TSA) upregulates IRS5/DOK4. This finding indicates that expression is regulated epigenetically at the level of chromatin remodeling. Chromatin immunoprecipitation experiments confirm that the IRS5/DOK4 promoter has enhanced histone hyperacetylation following treatments with TSA. Finally, hypoxia was demonstrated to downregulate IRS5/DOK4 expression. This expression was restored by TSA. The clinical relevance of altered IRS5/DOK4 expression in NSCLC requires further evaluation.

Topics: Acetylation; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Base Sequence; Bronchi; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Molecular Sequence Data; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.

Topics: Acetylation; Adenocarcinoma; Azacitidine; Biomarkers, Tumor; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Chromatin Immunoprecipitation; Colony-Forming Units Assay; DNA Methylation; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDACIs) have recently emerged as promising anticancer drugs to induce cell cycle arrest, cytodifferentiation, and apoptosis. It is suggested, however, that HDACIs promote cell migration and invasion depending on the cell type. We have reported previously that treatment with HDACIs, including trichostatin A and suberoylanilide hydroxamic acid (SAHA) or progesterone in combination with estrogen, can induce cytodifferentiation of endometrial adenocarcinoma Ishikawa cells through up-regulation of glycodelin, a progesterone-induced endometrial glycoprotein. Given the reported role of glycodelin in cell motility and the migration-modulating potential of HDACIs, we investigated using wound healing assay and transwell migration assay whether ovarian steroid hormones, trichostatin A, or SAHA affects cell migration in endometrial cancer cell lines, Ishikawa and RL95-2. Treatment with ovarian steroid hormones, trichostatin A, and SAHA enhanced cell migration together with up-regulation of glycodelin. SAHA-augmented cell migration was almost completely blocked by gene silencing of glycodelin. Furthermore, overexpression of gycodelin alone resulted in increased cell motility in Ishikawa cells. Our results collectively indicate that glycodelin positively regulates cell motility acting as a mediator of HDACI-enhanced endometrial cell migration, suggesting the involvement of glycodelin in the dynamic endometrial gland morphogenesis during menstrual cycle. Our results raise a possibility that the use of HDACIs in the therapy for glycodelin-inducible endometrial and presumably other gynecological cancers may enhance invasion in cases in which the HDACIs fail to exert differentiation-inducing and/or antiproliferative effects.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Endometrial Neoplasms; Enzyme Inhibitors; Female; Fibronectins; Glycodelin; Glycoproteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovary; Pregnancy Proteins; Up-Regulation; Vorinostat

We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.

Topics: 5'-Nucleotidase; Adenocarcinoma; Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Deoxycytidine; Enzyme Inhibitors; Gemcitabine; Glycoproteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Membrane Proteins; Mice; Pancreatic Neoplasms; Proto-Oncogene Proteins; Reactive Oxygen Species

HLA class I down-regulation in cancer cells confers immunological escape from cytotoxic T lymphocytes. We assessed the frequency of down-regulation of HLA class I antigens in a large series of prostate cancer tissues and determined the mechanism of up-regulation by investigating prostate cancer cell lines.. Immunohistochemical staining for HLA class I was done in specimens of 419 prostate cancers. We also investigated clinicopathological parameters, and the relationships between HLA class I down-regulation and the parameters. Furthermore, we examined whether HLA down-regulation was caused by epigenetic changes in vitro.. HLA class I was down-regulated in 311 prostate cancers (74.2%) and it significantly correlated with beta2-microglobulin down-regulation and a higher clinical stage. Flow cytometric analysis revealed a low level of HLA class I in LNCaP cells, which was up-regulated by the histone deacetylase inhibitor trichostatin A (Sigma). Trichostatin A up-regulated LNCaP beta2-microglobulin at the protein level. Furthermore, chromatin immunoprecipitation assay using an anti-acetylated histone H3 antibody provided direct evidence that trichostatin A up-regulated beta2-microglobulin by modulating the acetylation status of the promoter region in LNCaP cells.. The current study shows that the prevalence of HLA class I down-regulation is high in prostate cancer but histone deacetylase inhibitors can up-regulate HLA class I in LNCaP cells by up-regulating beta2-microglobulin. We suggest that the combination of an immunotherapeutic approach and histone deacetylase inhibition would accentuate the effects of current immunotherapies for prostate cancer.

Topics: Adenocarcinoma; beta 2-Microglobulin; Cell Line, Tumor; Down-Regulation; Enzyme Inhibitors; Flow Cytometry; Histocompatibility Antigens Class I; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Prostate; Prostatic Neoplasms; Up-Regulation

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

Pancreatic cancer is an aggressive neoplasia, and standard chemotherapies are by and large ineffective. The purpose of this work was to get a comprehensive preclinical study on the ability of anticancer drug combinations that best inhibit growth of pancreatic adenocarcinoma cells. We evaluated the in vitro growth inhibition of ten pancreatic cancer cell lines to gemcitabine and 5-fluorouracil, newer generation cytotoxic agents (oxaliplatin, irinotecan), targeted therapy (gefitinib) and a histone deacetylase (HDAC) inhibitor (trichostatin A). Cells were treated with the single drug alone and all pairwise drug association. Our results demonstrate that TSA can effectively increase the drug sensitivity of all the cell lines studied. The association of TSA and irinotecan determines an increase in growth inhibition on the highest percentage of cell lines (80%). Our findings may represent an experimental basis for potential clinical application of HDAC inhibitors, in particular in association with drugs used in cancer clinical treatment, supporting the idea that HDAC inhibitors could act as sensitizers for chemotherapy.

Topics: Adenocarcinoma; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Camptothecin; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Combinations; Drug Screening Assays, Antitumor; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Irinotecan; Pancreatic Neoplasms; Protein Synthesis Inhibitors

Using microarrays, we have screened for genes reactivated by drugs that modify epigenetic mechanisms in pancreatic cancer cells. One of the genes identified was tissue factor pathway inhibitor 2 (TFPI-2), which encodes for a broad-spectrum serine proteinase inhibitor that negatively regulates the extracellular matrix degradation, an essential step in tumor invasion and metastasis. We therefore investigated the expression and methylation patterns of the TFPI-2 gene in pancreatic adenocarcinoma, and determined its role in tumor growth and invasion. In contrast to its abundant expression in normal pancreas, TFPI-2 mRNA was undetectable in a high fraction of pancreatic cancer cell lines and in primary pancreatic ductal neoplasms (IPMNs). Loss of TFPI-2 expression was associated with aberrant hypermethylation of its promoter CpG island. Treatment with the phorbol ester (PMA), known to stimulate the TFPI-2 promoter activity, augmented the TFPI-2 expression in cell lines with unmethylated or partially methylated TFPI-2, but failed to induce the expression in cell lines that harbored fully methylated TFPI-2. Aberrant methylation of TFPI-2 was also detected in 73% (102/140) of pancreatic cancer xenografts and primary pancreatic adenocarcinomas, was more likely in older patients with pancreatic cancer, and significantly correlated with progression of IPMNs (P=0.0002). Restored expression of the TFPI-2 gene in nonexpressing pancreatic cancer cells resulted in marked suppression in their proliferation, migration, and invasive potential in vitro. We thus conclude that epigenetic inactivation of TFPI-2 is a common mechanism that contributes to the aggressive phenotype of pancreatic ductal adenocarcinoma.

Topics: Adenocarcinoma; Antimetabolites, Antineoplastic; Azacitidine; Base Sequence; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Decitabine; DNA Primers; Glycoproteins; Humans; Hydroxamic Acids; Loss of Heterozygosity; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous

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

Our previous study indicated that DUSP6/MKP-3/PYST1 could act as a tumor suppressor in human pancreatic cancer. DUSP6 was frequently underexpressed in primary pancreatic cancer tissues by an unknown mechanism. In this study, we demonstrated that hypermethylation of the expressional control region of DUSP6 could account for its abrogation in cultured human pancreatic cancer cells and in primary pancreatic cancer tissues. First, we checked intrinsic transcriptional expression levels of DUSP6 by a quantitative real time PCR assay in 16 cultured pancreatic cancer cell lines and found that the cells could be classified into four groups: very-low-level expression, low-level expression, high-level expression, and very-high-level expression. We observed restored expression of DUSP6 after treatment with 5-azacytidine and trichostatin A, a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, respectively, in cells with intrinsically very-low-level and low-level expression of DUSP6. Using a sodium-bisulfite-modification assay, we found that CpG sequences in intron 1 of DUSP6 were heavily methylated in MIA PaCa-2 and PAN07JCK, both showing the very low level of intrinsic expression of the gene. On the other hand, no methylation in this region was detected in 14 other cell lines. We checked the methylation state of this region by a methylation-specific PCR method in 12 primary pancreatic cancer tissues and compared it with the expression state of DUSP6 investigated by immunohistochemistry. Methylation was detected in five of eight cases with abolished expressions of DUSP6, four of which were poorly differentiated adenocarcinoma. On the other hand, none of the four cases with preserved expression of DUSP6 showed methylation. The methylation state significantly correlated with both the abolishment of protein expression (p = 0.038) and the histological subtype of adenocarcinoma (p = 0.023) by chi-square test. These results indicate that hypermethylation of the CpG islands in intron 1 may account for the strong suppression of DUSP6 expression. Other mechanism(s) and/or other CpG sites outside of our investigation may have some influence upon expressional suppression. Our combined results suggest that hypermethylation with modification of histone deacetylation play an important role in transcriptional suppression of DUSP6 in human pancreatic cancer.

Topics: Acetylation; Adenocarcinoma; Azacitidine; Base Sequence; Carcinoma, Pancreatic Ductal; Cell Differentiation; CpG Islands; DNA Methylation; Dual Specificity Phosphatase 6; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Molecular Sequence Data; Neoplasm Invasiveness; Pancreatic Neoplasms; Protein Tyrosine Phosphatases; Tumor Cells, Cultured

To determine if epigenetic interference can restore progesterone receptor-B (PR-B) expression in PR-B negative endometrial adenocarcinoma cell lines, and to characterize the kinetics of PR-B induction mediated by DNA methyltransferase and histone deacetylase inhibitors.. The PR-B negative endometrioid cancer cell lines KLE and HEC-1B were used as study models. PR-B mRNA and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. DNA methylation levels of the PR-B promoter were determined by methylation-specific PCR. Dose-response correlations and the duration of response to aza-deoxycytidine (ADC) and trichostatin A (TSA) were characterized. Cell responses to prolonged and repeated drug treatment were also examined.. Relatively low concentrations of ADC and TSA over a 24-h period induced PR-B expression. Furthermore, ADC and TSA acted synergistically to reactivate PR-B expression. Depending on the cell line used, PR-B mRNA was induced 10-110 fold. This elevated PR-B expression continued for 48 h after drug withdrawal. Sustained upregulation of PR-B mRNA and protein was observed during prolonged and repeated drug treatment.. The epigenetically silenced PR-B gene remains sensitive to changes in DNA demethylation and histone acetylation in uterine adenocarcinoma cell lines. Treatment with ADC and/or TSA results in a robust and sustainable PR-B upregulation. These small molecule epigenetic modifying agents may be used to sensitize poorly differentiated, PR-B negative endometrial cancers to progestational therapy.

Topics: Adenocarcinoma; Azacitidine; Cell Line, Tumor; Decitabine; DNA Methylation; Endometrial Neoplasms; Enzyme Inhibitors; Epigenesis, Genetic; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Polymerase Chain Reaction; Receptors, Progesterone; RNA, Messenger; Up-Regulation

The antitumor activity of the histone deacetylase inhibitors was tested in three well-characterized pancreatic adenocarcinoma cell lines, IMIM-PC-1, IMIM-PC-2, and RWP-1. These cell lines have been previously characterized in terms of their origin, the status of relevant molecular markers for this kind of tumor, resistance to other antineoplastic drugs, and expression of differentiation markers. In this study, we report that histone deacetylase inhibitors induce apoptosis in pancreatic cancer cell lines, independently of their intrinsic resistance to conventional antineoplastic agents. The histone deacetylase inhibitor-induced apoptosis is due to a serine protease-dependent and caspase-independent mechanism. Initially, histone deacetylase inhibitors increase Bax protein levels without affecting Bcl-2 levels. Consequently, the apoptosis-inducing factor (AIF) and Omi/HtrA2 are released from the mitochondria, with the subsequent induction of the apoptotic program. These phenomena require AIF relocalization into the nuclei to induce DNA fragmentation and a serine protease activity of Omi/HtrA2. These data, together with previous results from other cellular models bearing the multidrug resistance phenotype, suggest a possible role of the histone deacetylase inhibitors as antineoplastic agents for the treatment of human pancreatic adenocarcinoma.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Enzyme Inhibitors; Flavoproteins; High-Temperature Requirement A Serine Peptidase 2; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Pancreatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Serine Endopeptidases; Vorinostat

Histone reversible acetylation, which is controlled by histone acetyltransferases and deacetylases, plays a fundamental role in gene transcription. Histone deacetylase inhibitors (HDACIs), such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have been characterized not only as anticancer drugs, but also as cytodifferentiation-inducing agents. In human endometrium, postovulatory production of progesterone directs estrogen-primed endometrial glandular cells to differentiate and thereby produce a number of unique bioactive substances, including glycodelin, that are critical for implantation at the secretory phase of the menstrual cycle. In this study, we show that TSA and SAHA, belonging to the hydroxamic acid group of HDACIs, can induce the phenotype of a human endometrial adenocarcinoma cell line, Ishikawa (originally derived from the glandular component of the endometrium), to differentiate to closely resemble normal endometrial epithelium in a time- and dose-dependent manner, as determined by morphological changes, synthesis of glycogen, and expression of secretory phase-specific proteins, including glycodelin. The proliferation- and differentiation-modulating effects elicited by TSA and SAHA at their optimal concentrations were comparable or more potent than those exerted by combined treatment with progesterone and estradiol. Furthermore, the gene silencing of glycodelin by small interference RNA resulted in the blockade of HDACI-induced differentiation in Ishikawa cells, suggesting the requirement for glycodelin for endometrial epithelial differentiation. Our results collectively indicate that TSA and SAHA are potent differentiation inducers for endometrial glandular cells, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting glycodelin.

Topics: Adenocarcinoma; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Endometrial Neoplasms; Enzyme Inhibitors; Estradiol; Female; Gene Silencing; Glycodelin; Glycogen; Glycoproteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Interleukin-6; Leukemia Inhibitory Factor; Osmolar Concentration; Pregnancy Proteins; Progesterone; Promoter Regions, Genetic; RNA, Messenger; RNA, Small Interfering; Up-Regulation; Vorinostat

The histone deacetylase inhibitor trichostatin A (TSA) has been previously shown to block cellular growth in G2 and induce apoptosis in human pancreatic cancer cell lines. In order to better understand this phenomenon, we have analyzed the gene expression profiles in PaCa44 cells after treatment with TSA using microarrays containing 22,283 probesets. TSA was found to cause both the induction and repression of a large number of genes, although the number whose expression was up-regulated was greater than the number of genes that were down-regulated. When a threshold value of 3 was used as a cutoff level, a total of 306 (3.4%) of the detectable genes had altered expression. When categorized according to cellular function, the differentially expressed genes were found to be involved in a wide variety of cellular processes, including cell proliferation, signaling, regulation of transcription, and apoptosis. Moreover, Sp1/Sp3 transcription factor binding sites were significantly more abundant among TSA-induced genes. One prominent feature was the increased ratio between the levels of expression of pro-apoptotic (BIM) and anti-apoptotic (Bcl-XL and Bcl-W) genes. This result was confirmed in eight additional pancreatic cancer cell lines after treatment with TSA, suggesting that this event may be a strong determinant for the induction of apoptosis by TSA.

Topics: Adenocarcinoma; Apoptosis; Enzyme Inhibitors; Gene Expression Profiling; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Ras oncoproteins mediate multiple biological effects by activating multiple effectors. Classically, Ras activation has been associated with enhanced cellular growth and transformation. However, activated forms of Ras may also inhibit growth by inducing senescence, apoptosis, and differentiation. Induction of apoptosis by Ras may be mediated by its effector RASSF1, which appears to function as a tumor suppressor. We now show that the Ras effector Nore1, which is structurally related to RASSF1, can also mediate a Ras-dependent apoptosis. Moreover, an analysis of Nore1 protein expression showed that it is frequently down-regulated in lung tumor cell lines and primary lung tumors. Like RASSF1, this correlates with methylation of the Nore1 promoter rather than gene deletion. Finally, re-introduction of Nore1, driven by its own promoter, impairs the growth in soft agar of a human lung tumor cell line. Consequently, we propose that the Ras effector Nore1 is a member of a family of Ras effector/tumor suppressors that includes RASSF1.

Topics: 3T3 Cells; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Amino Acid Sequence; Animals; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Southern; Carrier Proteins; Cell Differentiation; Cell Division; Cell Line; Cellular Senescence; Cloning, Molecular; COS Cells; Down-Regulation; Gene Deletion; Humans; Hydroxamic Acids; Immunohistochemistry; Lung; Methylation; Mice; Molecular Sequence Data; Monomeric GTP-Binding Proteins; Promoter Regions, Genetic; ras Proteins; Rats; RNA, Messenger; Sequence Homology, Amino Acid; Time Factors; Tissue Distribution; Tumor Cells, Cultured

Recently, we used gene expression profiling of lung adenocarcinoma and paired normal tissue from smokers and nonsmokers to identify genes and molecular pathways associated with cigarette smoking and lung carcinogenesis. The gene encoding Glypican 3, a glycosylphosphatidylinositol-linked heparan sulfate proteoglycan, was decreased in lung adenocarcinoma. Within nonmalignant lung, GPC3 expression was decreased in smokers compared with nonsmokers; indicating that expression is associated with cigarette smoking. Microarray results were confirmed using an independent cohort of tumors and nonmalignant lung tissues. Immunohistochemical studies localized Glypican 3 protein expression to the apical surface of lung bronchiolar epithelial cells, potential cells of origin for adenocarcinoma. Northern blot analysis demonstrated expression was absent in all tested non-small cell lung carcinoma lines. Pharmacologic treatment of lung cell lines indicated that GPC3 expression was epigenetically silenced by promoter hypermethylation. Human lung carcinoma tumor cells ectopically expressing GPC3 demonstrated increased apoptosis response when exposed to etoposide and growth inhibition when implanted in nude mice. These findings suggest that GPC3 is a candidate lung tumor suppressor gene whose expression may be regulated by exposure to cigarette smoke and functions to modulate cellular response to exogenous damage.

Topics: Adenocarcinoma; Animals; Apoptosis; Azacitidine; Cell Line, Tumor; Decitabine; Enzyme Inhibitors; Gene Expression Profiling; Genes, Tumor Suppressor; Glypicans; Humans; Hydroxamic Acids; Lung Neoplasms; Membrane Proteins; Mice; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Respiratory Mucosa; Smoking; Tumor Suppressor Proteins

Increased histone acetylation has been associated with activated gene transcription and decreased acetylation with repression. However, there is a growing number of genes known, which are downregulated by histone deacetylase (HDAC) inhibitors through unknown mechanisms. This study examines the mechanism by which the mouse mammary tumor virus (MMTV) promoter is repressed by the HDAC inhibitor, trichostatin A (TSA). We find that this repression is transcriptional in nature and that it occurs in the presence and absence of glucocorticoids. TSA decreases MMTV transcription at a rapid rate, reaching maximum in 30-60 min. In contrast with previous reports, the repression does not correlate with an inhibition of glucocorticoid-induced nuclease hypersensitivity or NF1-binding at the MMTV promoter. Surprisingly, TSA does not induce sizable increases in histone acetylation at the MMTV promoter nor does it inhibit histone deacetylation, which accompanies deactivation of the glucocorticoid-activated MMTV promoter. Repression of MMTV transcription by TSA does not depend on the chromatin organization of the promoter because a transiently transfected MMTV promoter construct with a disorganized nucleoprotein structure was also repressed by TSA treatment. Mutational analysis of the MMTV promoter indicates that repression by TSA is mediated through the TATA box region. These results suggest a novel mechanism that involves acetylation of nonhistone proteins necessary for basal transcription.

Topics: Acetylation; Adenocarcinoma; Animals; Cell Transformation, Viral; Chromatin; Dexamethasone; Enzyme Inhibitors; Female; Genes, Reporter; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Nucleosomes; Promoter Regions, Genetic; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Sequence Deletion; TATA Box; Terminal Repeat Sequences; Transcription, Genetic; Transfection

In cells with an altered p53 gene, the expression of p21(WAF1/CIP1), a potent inhibitor of cyclin-dependent kinases, can be induced by histone deacetylase (HDAC) inhibitors via a p53-independent pathway, which may play a critical role in arrest of cell growth. Accordingly, HDAC inhibitors such as trichostatin A (TSA) have potential utility in pancreatic cancer, as most of these tumors possess mutations in p53, which in fact is the main cause of chemoresistance to 5-fluorouracil. We have analyzed the effect of TSA on the proliferation of nine pancreatic adenocarcinoma cell lines, all containing a mutated p53 gene. TSA strongly inhibited the cellular growth of all these cell lines at submicromolar concentrations. The cellular mechanisms underlying this effect consisted of cell cycle arrest at the G2 phase and apoptotic cell death. The expression of p21(WAF1/CIP1) normally induced at the transcriptional level by p53 was also strongly activated by TSA. These findings suggest that inhibitors of HDAC may represent a novel therapeutic strategy for treatment of pancreatic cancer.

Topics: Adenocarcinoma; Apoptosis; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Pancreatic Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Promoter hypermethylation has become apparent as a common mechanism of gene silencing in cancer. Based on our published microarray expression data, we noticed a prominent downregulation of ID4 in gastric adenocarcinoma. The dense 5' CpG island covering the previously mapped upstream promoter of ID4 has prompted us to relate its downregulation to promoter hypermethylation. ID proteins are distinct members in the helix-loop-helix family of transcriptional regulators, which modulate various key developmental processes. Emerging data have suggested the involvement of ID genes in tumorigenesis. In this study using bisulfite genomic sequencing, we have found hypermethylation of ID4 promoter in most gastric cancer cell lines and 30% of primary tumors. This correlated with decreased level of ID4 expression. Restoration of ID4 expression in various gastric cancer cell lines was achieved by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which at times required the synergistic action of the histone deacetylase inhibitor trichostatin A, but not with trichostatin A alone. Re-expression was accompanied by the corresponding ID4 promoter demethylation. Furthermore, we have found significant association of ID4 promoter methylation with hMLH1 promoter methylation (P=0.008) and microsatellite instability (P=0.006). Overall, our results have shown that transcriptional silencing of ID4 is related to the aberrant methylation of its promoter in gastric cancer. The significant association of ID4 and hMLH1 promoter hypermethylation suggested that ID4 may also be among the genes being targeted in the CpG island methylator phenotype tumorigenic pathway.

Topics: Adenocarcinoma; Azacitidine; Cell Line; Decitabine; DNA Methylation; DNA Modification Methylases; DNA-Binding Proteins; Down-Regulation; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Helix-Loop-Helix Motifs; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitor of Differentiation Proteins; Microsatellite Repeats; Promoter Regions, Genetic; Stomach Neoplasms; Transcription Factors

This paper was designed to investigate the expression of p53, p21 of A549 cell strains under hypoxic condition and the effect of trichostatin A (TSA), the inhibitor of histone deacetylasel (HDAC1) on their expression. The authors designed 1 normoxia group (control group) and 6 hypoxia groups (experimental group): hypoxia 6 h group (A), TSA+hypoxia 6 h (B), hypoxia 12 h group (C), hypoxia 24 h group (D), TSA+hypoxia 24 h (E), hypoxia 48 h group (F). The expression of HDAC1 in A549 cells was examined by using Western blot and the expression of p53, p21 in A549 cells and the effect of TSA on them were determined by using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The A value expressed by HDAC1 in A549 cell strains was 138+/-11 in the control group, 78+/-4, 86+/-5, 124+/-3, 120+/-9 in experimental groups A, C, D, F, respectively. The A value of the expression of the protein and mRNA of p53 in A549 cell strains were 0.12+/-0.02, 0.62+/-0.02 in the control group, 0.10+/-0.03, 0.32 +/-0.03; 0.11+/-0.01, 0.33+/-0.02; 0.13+/-0.03, 0.58+/-0.01; 0.12+/-0.02, 0.56+/-0.02 in experimental group A, B, D, E, respectively. The A value of the expression of the protein and mRNA of p21 in A549 cell strains were 0.17+/-0.03, 0.62+/-0.03 in the control group, 0.16+/-0.02, 0.50+/-0.02; 0.14+/-0.02, 0.36+/-0.02; 0.15+/-0.03, 0.49+/-0.03; 0.13+/-0.02, 0.33+/-0.02 in experimental groups A, B, D, E, respectively. These results indicate that the expression of HDAC1 is regulated by hypoxia and the effect of TSA is closely related to the expression of P21 under hypoxia condition.

Topics: Adenocarcinoma; Cell Hypoxia; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The FEZ1/LZTS1 (FEZ1) gene, located on chromosome 8p22 (8p22), was identified recently as a candidate tumor suppressor gene. Because loss of heterozygosity at 8p21-22 is a frequent event in lung cancers, we studied FEZ1 alteration in short-term cultures of resected lung cancer tumors and cell lines.. We examined FEZ1 expression in 17 non-small cell lung cancer (NSCLC), 19 small cell lung cancer (SCLC) cell lines, and 6 pairs of short-term cultures of resected NSCLCs and accompanying nonmalignant bronchial cells (NBECs) by reverse transcription-PCR and Western blotting. To investigate the mechanism for silencing, cells were cultured with 5-aza-2'-deoxycytidine or trichostatin A. We screened for genomic mutations by PCR-single-strand conformational polymorphism.. Thirteen of 17 NSCLC (76%) and 3 of 19 SCLC (16%) of cell lines showed absent expression (P = 0.001). Of the paired NSCLC-NBEC cultures, 3 of 6 showed loss of expression in tumor cell cultures. In the cell lines retaining expression, the amplicon products in SCLCs were more intense than those of NSCLCs and NBECs. Expression of FEZ1 was not restored by 5-aza-2'-deoxycytidine and trichostatin A. Although FEZ1 expression was moderately correlated with loss of heterozygosity of specific microsatellite makers at 8p21-22 in NSCLC cell lines, it was strongly correlated to D8S261 and LPL loci in SCLC cell lines. No mutation was found within cording region of FEZ1 by PCR-single-strand conformational polymorphism.. We found differential FEZ1 expression in NSCLC and SCLC cell lines, and the absent expression in 3 of 6 short-term cultures of NSCLC tumors. FEZ1 may be related to tumorigenesis of lung cancer.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Azacitidine; Blotting, Western; Carcinoma, Large Cell; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 8; CpG Islands; Decitabine; DNA Modification Methylases; DNA-Binding Proteins; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Loss of Heterozygosity; Lung Neoplasms; Nerve Tissue Proteins; Polymorphism, Single-Stranded Conformational; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Proteins

Although prostate-specific antigen (PSA) is considered a uniquely important tumor marker and is broadly used for early detection of prostate cancer, the molecular mechanisms underlying its elevated expression in tumors have been unknown. By using cDNA microarray gene expression profiling, we found a fourfold increase in the PSA mRNA level in prostatic carcinoma cell line LNCaP, in which the p53 pathway was suppressed by a dominant negative p53 mutant. Consistently, p53 suppression caused a 4-8-fold increase in secretion of PSA protein in culture medium, suggesting that PSA gene expression is under negative control of p53. While wild type p53 strongly repressed, dominant negative p53 mutants stimulated PSA promoter-driven transcription and secretion of PSA in transient transfection experiments. The inhibitory effect of wild type p53 was undetectable in the presence of trichostatin A, suggesting the involvement of histone deacetylation in negative regulation of PSA promoter activity. Thus, PSA is likely to be a tissue specific indicator of transformation-associated p53 suppression in prostate cells. This finding provides a plausible explanation for a frequent increase of PSA levels in advanced prostate cancer.

Topics: Acetylation; Adenocarcinoma; Chloramphenicol O-Acetyltransferase; Culture Media, Conditioned; DNA, Complementary; Gene Expression Regulation, Neoplastic; Genes, Dominant; Genes, p53; Genes, Reporter; Humans; Hydroxamic Acids; Male; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Processing, Post-Translational; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Histone deacetylase (HDAC) inhibitors and ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown previously to induce growth arrest and differentiation in a variety of cancer cell lines. The purpose of this study was to determine whether HDAC inhibitors function similarly in non-small cell lung cancer (NSCLC) and whether combination treatment with HDAC inhibitors and PPARgamma ligands is more efficacious than either agent alone.. Nanomolar concentrations of trichostatin A induced growth arrest in five of seven NSCLC cell lines, whereas sodium phenylbutyrate (PB) was markedly less potent. In adenocarcinomas, trichostatin A up-regulated general differentiation markers (gelsolin, Mad, and p21/WAF1) and down-regulated markers of the type II pneumocyte progenitor cell lineage (MUC1 and SP-A), indicative of a more mature phenotype. PB had a similar effect. Simultaneous treatment with a PPARgamma ligand and PB enhanced the growth inhibition in adenocarcinomas but not in nonadenocarcinomas. Growth arrest was accompanied by markedly decreased cyclin D1 expression but not enhanced differentiation.. The present study demonstrates potent growth-inhibitory and differentiation-inducing activity of HDAC inhibitors in NSCLC and suggests that combination differentiation therapy should be explored further for the treatment of lung adenocarcinomas.

Topics: Adenocarcinoma; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Synergism; Gelsolin; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Ligands; Lung Neoplasms; Phenylbutyrates; Receptors, Cytoplasmic and Nuclear; Repressor Proteins; RNA; Thiazoles; Thiazolidinediones; Transcription Factors; Tumor Cells, Cultured

Histone deacetylase inhibitor (HDAI) induces accumulation of highly acetylated histones by inhibiting the activity of histone deacetylase and inhibits cell proliferation, induces differentiation, and promotes apoptosis. TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis in various human cancer cells, a promising observation because it raises the possibility of a death ligand selectively for tumor cells. However, resistance to TRAIL-induced apoptosis was seen in colonic adenocarcinoma cell lines. So we investigated whether human colonic adenocarcinoma cell lines can be sensitized to TRAIL-induced apoptosis by the addition of HDAI. We investigated sensitivity to histone deacetylase inhibitor in colonic adenocarcinoma cell lines using the MTT assay. Cell viability decreased with sodium butyrate (SB) and trichostatinA (TSA) in a dose-dependent manner in LS 180 and HT-29 cells. Nuclear condensation and fragmentation were observed by DAPI staining after 24 h stimulation with SB or TSA in LS 180 cells. We also investigated the combination of HDAI and TNF family members (TRAIL, anti-Fas antibody or TNFalpha) in colonic adenocarcinoma cell lines. HDAI augmented TNF family-related apoptosis in LS 180 cells and HT-29 cells. HDAI sensitizes human colonic adenocarcinoma cell lines to TRAIL-mediated apoptosis. HDAI may be useful as an adjuvant agent for TRAIL in the treatment of human colonic adenocarcinomas that are resistant to TRAIL.

Topics: Adenocarcinoma; Apoptosis; Apoptosis Regulatory Proteins; Butyrates; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Membrane Glycoproteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Expression of the KAI1 gene, a metastasis-suppressor for prostate cancer, is reduced in all foci of prostatic metastasis. The altered regulatory mechanism is not strongly related to mutations or allelic losses of the KAI1 gene in prostate tumors. Since transcriptional silencing of genes has been found to be caused by epigenetic mechanisms, we have investigated the involvement of this epigenetic regulation of KAI1 expression in prostate cancers. The methylation status of the KAI1 promoter region was examined by restriction-enzyme digestion and sequencing, after amplifying a 331-bp fragment in the GC-rich promoter region from 4 human prostate cancer cell lines treated with bisulfite. The same 4 cell lines were also exposed to various concentrations of the demethylating agent, 5-aza-2'-deoxycytidine (5-AzaC) and / or the histone deacetylase inhibitor, trichostatin A (TSA). To clarify the influence of epigenetic modification on reduced KAI1 mRNA expression in the tumor cells, RT-PCR and northern-blot analyses were performed. Bisulfite-sequencing data showed a few methylated CpG islands in the promoter. RT-PCR analysis of 5-AzaC and / or TSA-treated cells indicated reversal of suppression of KAI1 transcription in two cell lines (PC-3 and DU-145), although the expression could not be detected by northern blots. From these results, it is suggested that epigenetic change is not the main mechanism of KAI1 down-regulation, though there remains a possibility that methylation in a more upstream region might be associated with this regulation.

Topics: Adenocarcinoma; Antigens, CD; Azacitidine; Base Sequence; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kangai-1 Protein; Male; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Proto-Oncogene Proteins; Sulfites; Tumor Cells, Cultured

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

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

Lung adenocarcinoma cells treated for 16 h with trichostatin A (TSA), an inhibitor of histone deacetylases, and untreated cells were analyzed with respect to differential gene expression. Complex hybridization of cDNA arrays revealed repression of Bcl-xL, CRAB2 and TFIID/TAFII31 as well as induction of p21waf1/cip1, GATA-2, hsp86, ID1, ID2 and ID3 mRNA expression, which could be verified by Northern blotting. ID2 induction was further confirmed by Taqman realtime quantitative RT-PCR. The described alterations of gene expression due to TSA renders the lung adenocarcinoma cells susceptible to induction of apoptosis.

Topics: Adenocarcinoma; bcl-X Protein; Blotting, Northern; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; DNA, Complementary; Enzyme Inhibitors; GATA2 Transcription Factor; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Inhibitor of Differentiation Protein 1; Inhibitor of Differentiation Protein 2; Inhibitor of Differentiation Proteins; Lung Neoplasms; Neoplasm Proteins; Nucleic Acid Hybridization; Proto-Oncogene Proteins c-bcl-2; Receptors, Retinoic Acid; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; TATA-Binding Protein Associated Factors; Trans-Activators; Transcription Factor TFIID; Transcription Factors; Transcription Factors, TFII; Tumor Cells, Cultured

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