trichostatin-a and Ovarian-Neoplasms

Topics: A549 Cells; Antineoplastic Agents; Cell Line, Tumor; Cell Size; Cell Survival; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms; Taurine

The influence of morin hydrate on changes of proliferative, metastatic, and adhesive potential of human ovarian cancer cells concerning the influence of decitabine, and decitabine with trichostatin A, and in comparison to untreated cells, were analyzed. The effect of morin hydrate, decitabine, and trichostatin A were examined in A2780 and SKOV-3 ovarian cancer cell lines using MTS assay, clonogenic assay, adhesion to endothelial HMEC-1 cells, transwell migration assay and cell cycle analysis. The expression level of epithelial to mesenchymal transition (EMT) markers was quantified using PCR Array in relation to the level of global methylation determined with Methylated DNA Quantification Kit. We observed statistically significant inhibition of adhesive and migratory potential of both cell lines and the accumulation of G0/G1 phase A2780 cells after treatment with morin hydrate. Our studies confirmed the influence of morin hydrate on down-regulation of genes considered as up-regulated during EMT, and up-regulation of some genes considered as down-regulated during EMT in A2780 and SKOV-3 cells. Phenotypic changes were associated with molecular changes in cells, eg. decrease of the expression level of genes associated with adhesion, and an increase of genes down-regulated during EMT, after morin hydrate treatment in comparison to untreated control cells in both cell lines, were observed.

Topics: Biomarkers, Tumor; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Decitabine; Epithelial-Mesenchymal Transition; Female; Flavonoids; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Ovarian Neoplasms

Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclin D1. Significantly, blockage of epidermal growth factor receptor (EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

Topics: Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Tyrphostins

Inhibition of histone deacetylase enzymes (HDACs) has been well documented as an attractive target for the development of chemotherapeutic drugs. The present study investigated the effects of two prototype hydroxamic acid HDAC inhibitors, namely Trichostatin A (TSA) and Belinostat (PXD‑101) and the benzamide Entinostat (MS‑275) in A2780 ovarian carcinoma and MCF7 breast adenocarcinoma cells. The three HDACi inhibited the proliferation of A2780 and MCF7 cells at comparable levels, below the µM range. Enzyme inhibition assays in a cell‑free system showed that TSA was the most potent inhibitor of total HDAC enzyme activity followed by PXD‑101 and MS‑275. Incubation of A2780 and MCF7 cells with the hydroxamates TSA and PXD‑101 for 24 h resulted in a dramatic increase of acetylated tubulin induction (up to 30‑fold for TSA). In contrast to acetylated tubulin, western blot analysis and flow cytometry indicated that the induction of acetylated histone H4 was considerably smaller. The benzamide MS‑275 exhibited nearly a 2‑fold induction of acetylated histone H4 and an even smaller induction of acetylated tubulin in A2780 and MCF7 cells. Taken together, these data suggest that although the three HDACi were equipotent in inhibiting proliferation of MCF7 and A2780 cells, only the benzamide MS‑275 did not induce acetylated tubulin expression, a marker of class IIb HDACs.

Topics: Acetylation; Benzamides; Breast Neoplasms; Cell Proliferation; Cell-Free System; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; MCF-7 Cells; Ovarian Neoplasms; Protein Processing, Post-Translational; Sulfonamides; Tubulin

Recently, there has been much interest in the field of nanomedicine to improve prevention, diagnosis, and treatment. Combination therapy seems to be most effective when two different molecules that work by different mechanisms are combined at low dose, thereby decreasing the possibility of drug resistance and occurrence of unbearable side effects. Based on this consideration, the study was designed to investigate the combination effect of reduced graphene oxide-silver nanoparticles (rGO-AgNPs) and trichostatin A (TSA) in human ovarian cancer cells (SKOV3).. The rGO-AgNPs were synthesized using a biomolecule called lycopene, and the resultant product was characterized by various analytical techniques. The combination effect of rGO-Ag and TSA was investigated in SKOV3 cells using various cellular assays such as cell viability, cytotoxicity, and immunofluorescence analysis.. AgNPs were uniformly distributed on the surface of graphene sheet with an average size between 10 and 50 nm. rGO-Ag and TSA were found to inhibit cell viability in a dose-dependent manner. The combination of rGO-Ag and TSA at low concentration showed a significant effect on cell viability, and increased cytotoxicity by increasing the level of malondialdehyde and decreasing the level of glutathione, and also causing mitochondrial dysfunction. Furthermore, the combination of rGO-Ag and TSA had a more pronounced effect on DNA fragmentation and double-strand breaks, and eventually induced apoptosis.. This study is the first to report that the combination of rGO-Ag and TSA can cause potential cytotoxicity and also induce significantly greater cell death compared to either rGO-Ag alone or TSA alone in SKOV3 cells by various mechanisms including reactive oxygen species generation, mitochondrial dysfunction, and DNA damage. Therefore, this combination chemotherapy could be possibly used in advanced cancers that are not suitable for radiation therapy or surgical treatment and facilitate overcoming tumor resistance and disease progression.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carotenoids; Cell Death; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Female; Graphite; Humans; Hydroxamic Acids; Lycopene; Metal Nanoparticles; Ovarian Neoplasms; Reactive Oxygen Species; Silver

In a previous study, we used a functional gene screen approach to identify the key genes responsible for the tumor-selective action of trichostatin A (TSA), of which LIV1, a novel zinc transporter, was isolated by its marked ability to confer resistance against TSA-induced apoptosis. The aim of the present study was to investigate the effect of LIV1 expression on the sensitivity of ovarian cancer cells to TSA. We tested the induction of LIV1 in ovarian cancer cells and clinical samples after TSA treatment by real-time PCR and western blot analysis. We investigated the effect of LIV1 expression on the sensitivity of ovarian cancer cells to TSA by MTT assay, flow cytometry and colony forming assays. Finally, we analyzed the mechanism of LIV1 in ovarian cancer cells by western blot analysis. We found that the levels of LIV1 mRNA and protein were significantly upregulated after TSA treatment. The viability and colony forming rates of the ovarian cancer cells transfected with AS-LIV1 (pCEP4 carrying antisense LIV1 cDNA) were obviously higher than the rates of the control as detected by MTT and colony forming assays, which could be reversed by FL-LIV1 (pCEP4 carrying full-length LIV1 cDNA). The apoptotic rate of the AS-LIV1 cells was markedly lower than the rate of the control as determined FACS. Using western blot analysis, we demostrated that the inhibition of TSA-induced apoptosis by knockdown of LIV1 might be associated with decreased endogenous levels of Bcl-2, enhanced levels of Bax and cleavage of procaspase-3. The present study suggests that the drug resistance of ovarian cancer cells to TSA may be related to expression of the LIV1 gene, and targeting LIV1 could be exploited as a novel strategy to more effectively kill ovarian cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Carrier Proteins; Cation Transport Proteins; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Neoplasm Proteins; Oligonucleotides, Antisense; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger

Resolution of aberrant epigenetic changes leading to altered gene expression during transformation and tumor progression is pertinent for mechanistic understanding of disrupted pathways in cancer. Such changes provide for biomarkers that can be applied in drug screening and improved disease management.. Genome-wide profiling and analyses of promoter DNA methylation, histone modifications, and gene expression of an in vitro progression model of serous ovarian adenocarcinoma were carried out. Similar in silico analyses and comparison of methylation and gene expression of early- and late-grade ovarian cancer samples in The Cancer Genome Atlas assigned a clinical relevance to our study. Candidate biomarkers were evaluated for epigenetic drug treatments in experimental animal models on a background of differing tumor cell responses arising from intratumor heterogeneity.. Differentially regulated genes during tumor progression were identified through the previously mentioned analyses as candidate biomarkers. In examining the tumor suppressor PTGIS as a potential biomarker for treatment with either 5-Aza-dC or TSA, 5-Aza-dC effectively stabilized cell cycling, restricted genetic instability, and derepressed PTGIS expression, while TSA led to emergence of drug-resistant progenitors lacking PTGIS expression. Profiling MEST and RXRγ for curcumin and CBB1007, respectively, indicated an inability of curcumin and CBB1007 in restricting residual tumor regenerative capabilities.. Our study provides novel insights into epigenetic regulation in ovarian cancer progression and potential biomarkers for evaluating efficacy of epigenetic drugs in restricting residual tumor regeneration. Such approaches may assign a new functional interpretation of drug efficacy and cell tumor responses in ovarian cancer.

Topics: Animals; Azacitidine; Benzamidines; Biomarkers, Tumor; Curcumin; Cytochrome P-450 Enzyme System; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Hydroxamic Acids; Molecular Targeted Therapy; Ovarian Neoplasms; Piperazines; Proteins; Retinoid X Receptor gamma

The istone deacetylase (HDAC) inhibitor trichostatin A (TSA) is known to mediate the regulation of gene expression and anti proliferation activity in cancer cells. Kruppel-like factor 4 (klf4) is a zinc finger- containing transcription factor of the SP/KLF family, that is expressed in a variety of tissues and regulates cell proliferation, differentiation, tumorigenesis, and apoptosis. It may either either function as a tumor suppressor or an oncogene depending on genetic context of tumors.. In this study, we tested the possibility that TSA may increase klf4 expression and cancer cell growth inhibition and apoptosis in SKOV-3 and A549 cells.. The cytotoxicity of TSA was determined using the MTT assay test, while klf4 gene expression was assessed by real time PCR and to ability of TSA to induce apoptosis using a Vybrant Apoptosis Assay kit.. Our results showed that TSA exerted dose and time dependent cytotoxicity effect on SKOV-3 and A549 cells. Moreover TSA up-regulated klf4 expression. Flow cytometric analysis demonstrated that apoptosis was increased after TSA treatment.. Taken together, this study showed that TSA increased klf4 expression in SKOV3 and A549 cell lines, consequently, klf4 may played a tumor-suppressor role by increasing both cell growth inhibition and apoptosis. This study sheds light on the details of molecular mechanisms of HDACI-induced cell cycle arrest and apoptosis.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lung Neoplasms; Ovarian Neoplasms; Up-Regulation

The aim of this study was to investigate the expression of Runt box 3 (RUNX3) and aplasia Ras homolog member I (ARHI) in ovarian tumors, and the effects of histone deacetylase inhibitors (HDACIs) on the expression of these genes and the growth of ovarian cancer cells. The mRNA expression of the RUNX3 and ARHI genes in normal ovaries and ovarian tumors was determined using reverse transcription polymerase chain reaction (RT-PCR). The effects of HDACIs on RUNX3 and ARHI expression in four ovarian cancer cell lines (SKOV3, A2780, COC1 and OC3) were determined using RT-PCR and the MTT assay. The expression of RUNX3 and ARHI in normal ovarian cells was 86 and 100%, respectively. Although the two genes were downregulated in ovarian tumors, the extent of downregulation differed. The expression of RUNX3 and ARHI was correlated with the degree of tumor malignancy (P<0.05). ARHI was expressed in all four ovarian cancer cell lines, whereas RUNX3 was expressed only in the OC3 cell line. Treatment with HDACIs upregulated the expression of ARHI and RUNX3 in the SKOV3 cell line only. In A2780 cells, HDACIs upregulated ARHI expression only in the presence of trichostatin A (TSA) plus cisplatin. HDACIs induced significant apoptosis in ovarian cancer cells, which was inversely correlated with the concentration and duration of treatment (P<0.05). In conclusion, RUNX3 and ARHI were shown to be expressed in normal ovarian cells; however, their expression was downregulated or lost in ovarian tumor cells. The combined detection of ARHI and RUNX3 expression may offer improved prediction and monitoring of ovarian malignancies. HDACIs were revealed to inhibit the growth of ovarian tumor cells and may constitute a novel therapeutic option for ovarian tumors.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; Aged; Aged, 80 and over; Apoptosis; Cell Proliferation; Cisplatin; Core Binding Factor Alpha 3 Subunit; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Middle Aged; Ovarian Neoplasms; RNA, Messenger; Tumor Suppressor Proteins; Young Adult

Ovarian cancer is one of the most lethal female malignancies and epigenetic abnormalities are thought to play a vital role in the pathogenesis, development and progression of ovarian cancer. Our goal was to investigate whether the combination of trichostatin A (TSA) and 5-aza-2'-deoxycytidine (decitabine) was superior to single agent on tumorigenicity of ovarian cancer cells. We found that tumorigenicity and metastasis of SKOV3 cells were significantly suppressed by the combination of TSA and decitabine in xenograft mouse models. Migration capacity was markedly suppressed through the induction of E-cadherin and suppression of N-cadherin when treated with TSA and decitabine. Invasion was also suppressed at least partially through inhibition of MMP-2 and MMP-9 with the combined treatment. The combination drugs markedly inhibited spheroid formation and significantly impaired migration and invasion capacity of spheroid derived cells through inhibition of Twist, N-cadherin, MMP-2, MMP-9 and induction of E-cadherin. Epigenetically, the activity of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) were markedly inhibited when TSA was used in combination with decitabine, especially the expression of DNMT3A/3B and HDAC1/2. Acetylation of histone H3 and H4 were more markedly stimulated with the combination than with either agent alone. The expression level of lysine-specific demethylase-1 (LSD1) was also suppressed. The transcription activity marker dimethylated-H3K4 was induced, but the dimethylated-H3K9 was suppressed by exposure to the combined drugs. These results suggest that the combination of TSA and decitabine significantly suppresses tumorigenicity by inhibiting migration and invasion of ovarian cancer cells via regulating the expression of the cadherins and MMPs, which may be epigenetically regulated by DNA methylation and histone modification.

Topics: Acetylation; Animals; Ascites; Azacitidine; Cadherins; Carcinogenesis; Cell Movement; Decitabine; DNA Methylation; DNA Modification Methylases; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Histone Demethylases; Histones; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C3H; Mice, SCID; Neoplasm Invasiveness; Neoplasm Metastasis; Nuclear Proteins; Ovarian Neoplasms; Spheroids, Cellular; Tumor Cells, Cultured; Twist-Related Protein 1

RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies.

Topics: Antineoplastic Agents; Azacitidine; Carcinogenesis; Case-Control Studies; Cystadenoma, Serous; DNA Methylation; DNA, Neoplasm; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Hydroxamic Acids; Methyltransferases; Neoplasm Staging; Ovarian Neoplasms; Ovary; rhoB GTP-Binding Protein

To evaluate the anticancer efficacy of the combination of epigenetic modifiers and cisplatin in human ovarian cancer.. The effect of trichostatin A (TSA) and 5-aza-2'-deoxycytidine alone or in combination with low-dose cisplatin was evaluated on human ovarian cancer cell lines in vitro. We measured drug interaction by MTS assay, migration by transwell assay, expression of epithelial to mesenchymal transition (EMT) markers (Twist, Snail, Slug, E-cadherin, and N-cadherin), pluripotency markers (Oct4, Sox2, and Nanog), and epigenetic markers (DNMT3A, LSD1 and H3K4me2, H3K4me3, H3K9me2, and H3K9me3) by western blot, and the impact on and characteristics of spheroid growth when exposed to these drugs. Mouse xenografts were used to evaluate the anticancer effect of sequential drug treatment.. Combination treatment had greater efficacy than single drugs and significantly suppressed cell viability, migration, and spheroid formation and growth. Sequential treatment of cisplatin (1 mg kg(-1)) followed by TSA (0.3 mg kg(-1)) significantly suppressed tumorigenicity of HEY xenografts through inhibition of EMT and decreased pluripotency of ovarian cancer cells.. Epigenetic modifiers potentiate the anticancer efficacy of low-dose cisplatin in ovarian cancer through regulation of EMT and pluripotency, and may provide a promising treatment for ovarian cancer patients.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Blotting, Western; Cell Adhesion; Cell Movement; Cell Proliferation; Cisplatin; Decitabine; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Mice, Inbred C3H; Mice, SCID; Ovarian Neoplasms; Spheroids, Cellular; Tumor Cells, Cultured

Previously we reported that liposomal cisplatin (CDDP) overcomes CDDP resistance of ovarian A2780cis cancer cells (Krieger et al., Int. J. Pharm. 389, 2010, 10-17). Here we find that the cytotoxic activity of liposomal CDDP is not associated with detectable DNA platination in resistant ovarian cancer cells. This suggests that the mode of action of liposomal CDDP is different from the free drug. To gain insight into mechanisms of liposomal CDDP activity, we performed a transcriptome analysis of untreated A2780cis cells, and A2780cis cells in response to exposure with IC50 values of free or liposomal CDDP. A process network analysis of upregulated genes showed that liposomal CDDP induced a highly different gene expression profile in comparison to the free drug. p53 was identified as a key player directing transcriptional responses to free or liposomal CDDP. The free drug induced expression of essential genes of the intrinsic (mitochondrial) apoptosis pathway (BAX, BID, CASP9) most likely through p38MAPK activation. In contrast, liposomal CDDP induced expression of genes from DNA damage pathways and several genes of the extrinsic pathway of apoptosis (TNFRSF10B-DR5, CD70-TNFSF7). It thus appears that liposomal CDDP overcomes CDDP resistance by inducing DNA damage and in consequence programmed cell death by the extrinsic pathway. Predictions from gene expression data with respect to apoptosis activation were confirmed at the protein level by an apoptosis antibody array. This sheds new light on liposomal drug carrier approaches in cancer and suggests liposomal CDDP as promising strategy for the treatment of CDDP resistant ovarian carcinomas.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Humans; Hydroxamic Acids; Liposomes; Ovarian Neoplasms; Tumor Suppressor Protein p53

Chemoresistance is the major factor limiting long-term treatment success in patients with epithelial ovarian cancers. Most cytotoxic drugs kill cells through apoptosis; therefore, defective execution of apoptotic pathways results in a drug-resistant phenotype in many tumor types.. A panel of ovarian cancer cell lines was screened for expression and function of the apoptosome components Apaf-1 and caspase-9. Expression levels were analyzed by immunohistochemistry and immunoblotting; Apaf-1 function was determined by assessing the ability of endogenous Apaf-1 to cleave caspase-9 in the presence or absence of cytochrome c. The effect of the histone deacetylase inhibitor trichostatin A on Apaf-1 expression and function was evaluated.. The authors report here that the resistance of ovarian cancer cells to the proapoptotic effects of chemotherapy is due in part to deficient Apaf-1 activity. Although Apaf-1 is expressed in most ovarian cancers, the functional activity is impaired, as Apaf-1 has a diminished ability to recruit and activate caspase-9. Treatment of ovarian cancer cells with trichostatin A results in restoration of Apaf-1 function independent of alterations in Apaf-1 expression. Furthermore, treating chemoresistant cells with sublethal doses of trichostatin A restores Apaf-1 function and sensitizes cells to cisplatin-induced apoptosis.. Targeting intrinsic pathway defects for therapeutic intervention may result in sensitizing tumors to standard chemotherapy or triggering apoptosis in the absence of other apoptotic signals. The identification of drugs that can use Apaf-1 when it is present, yet can overcome its functional inactivation, may be an important clinical advance.

Topics: Antineoplastic Agents; Apoptotic Protease-Activating Factor 1; Caspase 9; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms

Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Drug Resistance, Neoplasm; Drug Synergism; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; RNA Interference; Time Factors

Histone deacetylase inhibitor-induced apoptosis in cancer cells may be mediated by the Ras/Raf/MEK/ERK and protein kinase B/Akt signaling pathways. However, inhibition of ERK and Akt activity has different effects on proliferation and apoptosis in cancer cells. We assessed and compared the inhibitory effects of Akt and ERK pathways on the apoptotic effect of trichostatin A using the human epithelial carcinoma cell lines OVCAR-3 and SK-OV-3. Trichostatin A induced nuclear damage, decrease in Bid and Bcl-2 protein levels, increase in Bax levels, cytochrome c release, activation of caspases (8, 9, and 3) and increase in tumor suppressor p53 levels. Akt inhibitor potentiated trichostatin A-induced apoptosis-related protein activation and cell death, whereas ERK inhibitor exhibited an additive toxic effect. These results suggest that the Akt and ERK inhibitors may have a differential effect on trichostatin A-induced apoptosis in human epithelial ovarian carcinoma cell lines. Akt inhibitor may potentiate the apoptotic effect of trichostatin A on ovarian carcinoma cell lines by increasing the activation of the caspase-8-dependent pathway and the mitochondria-mediated cell death pathway, leading to caspase activation. In contrast, ERK inhibitor may exhibit an additive toxic effect on trichostatin A toxicity by increasing apoptosis-related protein activation.

Topics: Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carcinoma, Ovarian Epithelial; Caspase 8; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inositol Phosphates; Microscopy, Fluorescence; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up-regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re-expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts.. Ovarian cancer cells (Hey and SKOv3) were treated with demethylating agents (5-aza-20-deoxycytidine [DAC] or 5-azacitidine [AZA]) or with HDAC inhibitors (suberoylanilide hydroxamicacid [SAHA] or trichostatin A [TSA]) to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, autophagy, and re-expression of 2 growth inhibitory imprinted tumor suppressor genes: guanosine triphosphate-binding Di-RAS-like 3 (ARHI) and paternally expressed 3 (PEG3). The in vivo activities of DAC and SAHA were assessed in a Hey xenograft model.. The combination of DAC and SAHA produced synergistic inhibition of Hey and SKOv3 cell growth by apoptosis and cell cycle arrest. DAC induced autophagy in Hey cells that was enhanced by SAHA. Treatment with both agents induced re-expression of ARHI and PEG3 in cultured cells and in xenografts, correlating with growth inhibition. Knockdown of ARHI decreased DAC-induced autophagy. DAC and SAHA inhibited the growth of Hey xenografts and induced autophagy in vivo.. A combination of DAC and SAHA inhibited ovarian cancer growth while inducing apoptosis, G2/M arrest, autophagy, and re-expression of imprinted tumor suppressor genes.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Azacitidine; Cell Division; Cell Line, Tumor; Cell Proliferation; Decitabine; Drug Synergism; Drug Therapy, Combination; Epigenomics; Female; G2 Phase; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genomic Imprinting; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; Transplantation, Heterologous; Vorinostat

Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1alpha dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

Topics: Base Sequence; Breast Neoplasms; Cell Cycle Proteins; Cell Hypoxia; Cell Proliferation; Disulfides; E1A-Associated p300 Protein; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Histone Deacetylases; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Indole Alkaloids; Intercellular Signaling Peptides and Proteins; Luciferases; Molecular Sequence Data; Ovarian Neoplasms; Response Elements; Time Factors

Histone deacetylase inhibitors and casein kinase 2 inhibitors have been shown to induce apoptosis. However, the combined effect of casein kinase 2 inhibition on the apoptotic effect of histone deacetylase inhibitor is unknown. We assessed the effect of casein kinase 2 inhibition on the apoptotic effect of trichostatin A in human epithelial carcinoma cell lines with respect to cell death signaling pathways. At concentrations that did not induce cell death, the casein kinase 2 inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited activation of apoptotic proteins and changes in mitochondrial membrane permeability induced by the histone deacetylase inhibitor trichostatin A. These results suggest that casein kinase 2 inhibition may reduce trichostatin A-induced apoptosis in ovarian carcinoma cell lines by suppressing activation of apoptotic proteins and changes in mitochondrial membrane permeability, which both lead to caspase-3 activation. Casein kinase 2 inhibition, which does not induce a cytotoxic effect, may prevent histone deacetylase inhibitor-mediated apoptosis.

Topics: Animals; Apigenin; Apoptosis; Casein Kinase II; Cell Line, Tumor; Cell Nucleus; DNA Fragmentation; Epithelium; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms; Signal Transduction; Triazoles

Resistance to TGF-beta is frequently observed in ovarian cancer, and disrupted TGF-beta/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-beta/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-beta/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; P<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan-Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, P<0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-beta/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Cisplatin; Decitabine; DNA Methylation; Down-Regulation; Drug Resistance, Neoplasm; Epigenesis, Genetic; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Muscle Proteins; Ovarian Neoplasms; Prognosis; Promoter Regions, Genetic; Proportional Hazards Models; SKP Cullin F-Box Protein Ligases; Smad4 Protein; Taiwan; Transforming Growth Factor beta; Young Adult

Epothilone B (EpoB), like Taxol, stabilizes microtubules resulting in an inhibition of microtubule dynamic instability. The drug is being evaluated in phase III clinical trials. An EpoB analog, Ixabepilone, was approved by the FDA for the treatment of taxane-resistant metastatic breast cancer. Epithelial cell adhesion antigen (EpCAM) expression is significantly higher in epithelial ovarian cancer cells compared to normal cells. The effects of EpoB and other microtubule-interacting agents on surface EpCAM expression were studied.. Biochemical methods, immunofluorescence and flow cytometry were used to identify EpCAM expression on the surface of the ovarian cancer cell line, Hey, after exposure to EpoB. The relationship between EpoB-mediated surface EpCAM expression and EpoB-induced α-tubulin acetylation, a surrogate marker for stable microtubules, in Hey cells also was investigated.. Nanomolar concentrations of EpoB, Taxol, discodermolide or vinblastine caused a marked increase in surface EpCAM expression in Hey cells. Alpha-tubulin acetylation was increased following treatment with Taxol, EpoB and discodermolide, but not with vinblastine, indicating that drug-enhanced surface EpCAM expression does not correlate with tubulin acetylation or stabilization. Unexpectedly, EpoB did not have a significant effect on EpCAM mRNA expression, nor did it alter the level of total cellular EpCAM in Hey cells.. The results indicate that disruption of the microtubule cytoskeleton is associated with the redistribution of cell surface antigens in ovarian cancer cells. The increase in cell surface EpCAM antigen density may facilitate the antibody targeting of EpCAM-positive ovarian cancer cells.

Topics: Acetylation; Alkanes; Antigens, Neoplasm; Carbamates; Cell Adhesion Molecules; Cell Line, Tumor; Epithelial Cell Adhesion Molecule; Epothilones; Female; Humans; Hydroxamic Acids; Lactones; Ovarian Neoplasms; Paclitaxel; Pyrones; Tubulin; Tubulin Modulators; Vinblastine

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

Ovarian cancer is of worldwide importance, and has a significantly high mortality rate due to therapy failure. Drug resistance might be one of most importance factors. Histone deacetylase inhibitors (HDACi) have been reported to be a new class of promising anti-tumor agents, thus this study aimed to investigate the effect of HDAC on the chemo-resistance genes of human ovarian carcinoma cell lines.. The expressions of ATP binding cassette (ABC) transporter genes, multidrug-resistant protein (MDR1) and multidrug resistance-associated proteins (MRP1 and 2) of ovarian cancer cell lines OC-109 and SK-OV-3 after HDACi treatment were determined.. HDACi, including sodium butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) reduced ovarian cancer cell viability from 4.4% to 68.8%, in both dose- and time-dependent manners. The effect of HDACi on MDR1, MRP1, and MRP2 showed induced expression of MDR1 mRNA, but reduced mRNA expression of MRP1 and MRP2.. The effect of HDACi on the reduced viability of ovarian cancer cell lines, concomitant with the induced expression of MDR1 and reduced expression of MRP1 and 2, might provide additional benefits in the management of ovarian cancers in the future.

Topics: Antineoplastic Agents; ATP-Binding Cassette Transporters; Butyrates; Cell Line, Tumor; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms

The mechanisms of estrogen insensitivity or estrogen resistance in ovarian cancer cells are not known. Studies on regulation of the estrogen receptor (ER) gene have suggested a role for epigenetics in silencing ER expression.. Cells from insensitive ovarian cancer cells, SKOV3 and HEY, were cultured with and without the DNA methyltransferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine (AzaC) and the histone deacetylase (HDAC) inhibitor, trichostatin (TSA). ERbeta promoter methylation was examined using bisulfite sequencing. RNA was collected for oligonucleotide array studies.. Cell type-specific ERbeta promoter methylation was found as well as relative hypomethylation of the ERbeta promoter in SKOV3 compared to HEY cells. Preferential demethylation of specific CpGs by different treatments was found. AzaC and TSA resulted in significant tumor growth inhibition and alterations in expression of numerous genes.. The ERbeta promoter is differentially methylated in ovarian cancer cells. Moreover, AzaC and TSA can inhibit ovarian cancer cell growth.

Topics: Azacitidine; Cell Growth Processes; Cell Line, Tumor; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Estrogen Receptor beta; Female; Histone Acetyltransferases; Humans; Hydroxamic Acids; Ovarian Neoplasms; Promoter Regions, Genetic

TRAIL-resistant cancer cells can be sensitized to TRAIL by combination therapy. In this study, we investigated the effect of trichostatin A (TSA), a histone deacetylase inhibitor, to overcome the TRAIL resistance in human ovarian cancer cells. Co-treatment of human ovarian cancer cells with TSA and TRAIL synergistically inhibited cell proliferation and induced apoptosis. The combined treatment of ovarian cancer SKOV3 cells with TSA and TRAIL significantly activated caspase-8 and truncated Bid, resulting in the cytosolic accumulation of cytochrome c as well as the activation of caspase-9 and -3. Moreover, we found that down-regulation of c-FLIP(L) might contribute to TSA-mediated sensitization to TRAIL-induced apoptosis in SKOV3 cells, and this result was supported by showing that down- or up-regulation of c-FLIP(L) with transfection of siRNA or plasmid sensitized or made SKOV3 cells resistant to TRAIL-induced apoptosis, respectively. TSA or co-treatment with TSA alone and TRAIL also resulted in down-regulation of EGFR1/2 and dephosphorylation of its downstream targets, AKT and ERK. Treatment of SKOV3 cells with PKI-166 (EGFR1/2 inhibitor), LY294002 (AKT inhibitor), and PD98059 (ERK inhibitor) decreased c-FLIP(L) expression and co-treatment with TRAIL further reduced the level of c-FLIP(L,) respectively, as did TSA. Collectively, our data suggest that TSA-mediated sensitization of ovarian cancer cells to TRAIL is closely correlated with down-regulation of c-FLIP(L) via inhibition of EGFR pathway, involving caspase-dependent mitochondrial apoptosis, and combination of TSA and TRAIL may be an effective strategy for treating TRAIL-resistant human ovarian cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Caspase 8; Caspase Inhibitors; Cell Culture Techniques; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Female; Humans; Hydroxamic Acids; Ovarian Neoplasms; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand

To analyze the effect and mechanism of TSA on cell cycles in human ovarian cancer cells.. cells were cultured in RPMI 1640 supplemented. Flow cytometry analysis and RT-PCR were used to examine the distribution of cell cycles and the level of P21(WAF/CIPI) mRNA.. TSA induced increase of the G2/M phase accompanied by decrease of the S phase and enhanced level of P21(WAF/CIPI) mRNA in a concentration and time-dependent manner in A2780 cells.. Trichostatin A affects the activity of cyclin-dependent kinase through increased expression of P21(WAF/CIPI) mRNA. TSA causes A2780 cell blockage in the G2/M phase and inhibits cell proliferation of A2780 cells. The minimum level of active TSA is 100 nM and the minimum time is 12 hours. The effect relies on time and concentration.

Topics: Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction

Several studies in the last years evidenced that deregulation of proapoptotic and antiapoptotic pathways are key players in the onset and maintenance of chemoresistance in advanced ovarian cancers. To characterize the signaling events and molecules involved in the acquisition of cisplatin resistance, we used the human ovarian cancer cell line A2780 and its derivative cisplatin-resistant subline A2780 CIS. We found that the mitochondrial intrinsic apoptotic pathway, induced by cis-dichlorodiammineplatinum (CDDP) in A2780 wild-type cells, was compromised in the resistant subline CIS. The analysis of expression of proteins involved in mitochondria-dependent apoptosis revealed a role of Bax and p73 but not p53. Indeed, we found that CDDP treatment induced the up-regulation of p53 in both sensitive and resistant A2780 cell lines. By contrast, p73 and Bax expressions were compromised in resistant cells. Pretreatment of resistant A2780 CIS cells with the histone deacetylase inhibitor trichostatin A overcomes apoptosis resistance to CDDP by restoring both p73 and Bax but not p53 expression. Altogether, these data indicate that p73, but not p53, is involved in the regulation of apoptosis susceptibility to cisplatin in A2780 ovarian cancer cells and evidence a key contribution of histone deacetylase activation in the acquisition of chemotherapy resistance in human ovarian cancer cells.

Topics: Apoptosis; Base Sequence; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Humans; Hydroxamic Acids; Membrane Potential, Mitochondrial; Mitochondria; Molecular Sequence Data; Nuclear Proteins; Ovarian Neoplasms; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation

Cisplatin (DDP) can cause DNA damage in cells. DNA mismatch repair proteins serve to detect DDP-caused DNA damage and generate an injury signal that eventually contributes to the triggering of tumor cell apoptosis. As a member of the mismatch repair system, the absence of hMLH1 expression contributes to the resistance of tumor cells to DDP. This study was to explore the role of hMLH1 expression and DNA methylation in DDP-resistance of human ovarian cancer, and to evaluate the reversal effects of 5-aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) on DDP-resistance.. We cultured human ovarian cancer cell line COC1 and its DDP-resistant subline, COC1/DDP. We treated the two cell lines with 5-Aza-dC and TSA. DNA methylation at hMLH1 gene promoter was detected by methylation-specific polymerase chain reaction (MSP). The expression of hMLH1 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The inhibition rate of cell proliferation was detected by MTT assay.. In COC1 cells, both hMLH1 mRNA and protein were detected, while no DNA methylation of hMLH1 gene was detected. 5-Aza-dC and TSA used alone or in combination had no effects on DNA methylation, hMLH1 mRNA or protein expression (P>0.05), and cell proliferation. In COC1/DDP cells, DNA hypermethylation of hMLH1 gene was detected, while no hMLH1 mRNA or protein was detected. 5-Aza-dC resulted in DNA demethylation and restoration of hMLH1 expression. TSA had no effect on DNA demethylation or restoration of hMLH1 expression. 5-Aza-dC plus TSA also resulted in DNA demethylation, restored hMLH1 expression more obviously than 5-Aza-dC did (P<0.05), and restricted the proliferation of COC1/DDP cells.. Hypermethylation of DNA promoter is related to the silencing of hMLH1 in ovarian cancer COC1/DDP cells. 5-Aza-dC alone or in combination with TSA results in DNA demethylation of hMLH1 gene, restoration of hMLH1 expression and reversal of DDP-resistance of COC1/DDP cells.

Topics: Adaptor Proteins, Signal Transducing; Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; Cell Proliferation; Cisplatin; Decitabine; DNA Methylation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MutL Protein Homolog 1; Nuclear Proteins; Ovarian Neoplasms; Promoter Regions, Genetic; RNA, Messenger

Telomerase is composed primarily of catalytic subunit (hTERT) and RNA template (hTERC). Histone deacetylase (HDAC) inhibitors are known to modulate transcription and change the expression of hTERT and hTERC mRNA and telomerase activity in several types of cancer cells, but it is unclear if there is a similar effect in ovarian cancer cells.. The present study was designed to evaluate the effects of HDAC inhibitors on hTERT and hTERC mRNA expression in ovarian cancer cells. SK-V-3 cells were treated with the HDAC inhibitors, trichostatinA (TSA) and sodium butyrate (NaB); the expression of hTERC and hTERT mRNA and telomerase activity were evaluated by RT-PCR and TRAP assay, respectively.. In SK-OV-3 cells, TSA and NaB inhibited cell proliferation and induced apoptosis. The expression of hTERT and hTERT mRNA was not suppressed even after treatment with 1.0 microM TSA and 6 mM NaB, respectively. The telomerase activity was not changed by either TSA or NaB.. Histone deacetylase inhibitors inhibited cell proliferation and induced apoptosis, but had no effect on the expression of hTERC and hTERT mRNA and on telomerase activity.

Topics: Apoptosis; Butyrates; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Telomerase

ARHI is a maternally imprinted tumor suppressor gene that is expressed in normal breast and ovarian epithelial cells but not in most breast and ovarian cancers. Our earlier studies showed that histone deacetylases (HDACs) in complexes with transcription factors E2F1 and E2F4 play an important role in downregulating ARHI expression in breast cancer cells. To determine which HDAC or HDACs are responsible for repressing ARHI, we cotransfected vectors expressing HDACs 1-11 with an ARHI/luciferase reporter into SKBr3 and MCF-7 breast cancer cells. Expression of multiple HDACs consistently reduced ARHI promoter activity in a dose-dependent manner. We also found that the expression level of HDACs 1-3 was higher in breast cancer cell lines than in normal breast epithelial cells. In agreement with their repressive function, depletion of HDACs 1, 3 and 11 not only significantly increased the ARHI promoter activity of the transfected reporter but also activated the transcription of the endogenous ARHI gene. Furthermore, depletion or inhibition of HDACs by small interfering RNA of HDAC11 or by trichostatin A, respectively, increased E2F acetylation. Chromatin immunoprecipitation assays revealed that HDACs 1 and 3 are bound to the ARHI promoter. Taken together, our results suggest that the activity of multiple HDACs contributes to the repression of the ARHI tumor suppressor gene in breast cancer cells. Since HDAC inhibitors are now being used to treat breast cancer, the reactivation of ARHI in these cancer cells may serve as a new biomarker with which to monitor the treatment effects.

Topics: Acetylation; Breast; Breast Neoplasms; Cell Line; Cell Nucleus; Chromatin Immunoprecipitation; DNA Methylation; E2F1 Transcription Factor; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Luciferases; Ovarian Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA, Small Interfering; Transcription, Genetic

Gene silencing associated with aberrant DNA methylation of promoter CpG islands is one mechanism through which several genes may be inactivated in human cancers. Cyclin D2, a member of the D-type cyclins, implicated in cell cycle regulation, differentiation and malignant transformation, is inactivated due to aberrant DNA methylation in several human cancers. In the present study, we examined the promoter methylation status and expression of Cyclin D2 in human epithelial ovarian cancer, and then determined the relationship between methylation status and various clinicopathological variables. Twelve ovarian cancer cell lines and 71 surgical specimens were examined by methylation-specific polymerase chain reaction and quantitative reverse transcription-polymerase chain reaction to evaluate the methylation status and expression of the Cyclin D2 gene. The relationship between methylation status and various clinicopathological variables was evaluated using statistical analysis. Aberrant methylation of Cyclin D2 was present in five of 12 ovarian cancer cell lines and 16 of 71 primary ovarian cancer tissues. In five cell lines with methylation, expression of the Cyclin D2 gene tended to be lower than in cell lines without methylation. In ovarian cancer tissues, methylation bands were detected in 16 of 71 cases. The methylation status of Cyclin D2 was associated with advanced stage and a residual tumor size (>2 cm) (P = 0.027 and P = 0.031, respectively). Based on univariate analysis, patients with aberrant methylation of the Cyclin D2 promoter had a significantly worse chance of disease-free survival than those without methylation (P = 0.021). Our results suggest that aberrant promoter methylation of the Cyclin D2 gene is significantly associated with patient prognosis in epithelial ovarian cancer.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; Cyclin D2; Cyclins; Decitabine; DNA Methylation; Enzyme Inhibitors; Female; Humans; Hydroxamic Acids; Immunohistochemistry; Ovarian Neoplasms; Prognosis; Promoter Regions, Genetic; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.

Topics: Azacitidine; Binding Sites; Cell Line, Tumor; Claudin-3; Claudin-4; CpG Islands; Decitabine; DNA Methylation; Female; Humans; Hydroxamic Acids; Membrane Proteins; Mutagenesis, Site-Directed; Ovarian Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Sp1 Transcription Factor; Sp3 Transcription Factor

Trichostatin A (TSA), a hydroxamate-type inhibitor of mammalian histone deacetylases, is emerging as one of a potentially new class of anticancer agents. TSA is known to act by promoting the acetylation of histones, leading to uncoiling of chromatin and activation of a variety of genes implicated in the regulation of cell survival, proliferation, differentiation, and apoptosis. In addition, there is an increasing appreciation of the fact that TSA may act through mechanisms other than induction of histone acetylation. Accumulated experimental data indicate that TSA activates phosphatidyl inositol-3-kinase (PI3K)/AKT signaling. Using human ovarian cancer cell line Caov3 cells, we observed that TSA induced cell death in a time- and dose-dependent manner and also inhibited cell migration. TSA transiently activated EGFR tyrosine phosphorylation and AKT activation in a time- and dose-dependent manner, which had been inhibited by EGFR inhibitor PD153035 and PI3 kinase inhibitor LY294002. We also observed that TSA transiently induced survivin expression that had been inhibited by PD153035 and LY294002, suggesting that TSA-induced survivin expression is mediated by EGFR/PI3 kinase pathway. Combination of EGFR inhibitor 153035 or PI3 kinase inhibitor LY294002 with TSA enhanced TSA-induced cell death and TSA reduction of cell migration. Collectively, our data demonstrate that TSA transiently activated EGFR/PI3K/AKT cell survival pathway, leading to expression of survivin. Inhibition of this pathway enhanced TSA-induced cell death and inhibited cell migration. Our data suggest that combination of EGFR/PI3K/AKT cell survival pathway inhibitors with TSA be a better approach to ovarian cancer treatment.

Topics: Acetylation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chromones; Dose-Response Relationship, Drug; Enzyme Inhibitors; ErbB Receptors; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Morpholines; Neoplasm Proteins; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Survivin; Time Factors

Trichostatin A produces predominantly G(1) cell-cycle blockade and differentiation of the cisplatinum-sensitive A2780 ovarian cancer cell line. Given the propensity of ovarian tumors to become resistant to cisplatinum, often leading to cross-resistance to other agents, we have extended these observations by examining how the emergence of resistant phenotypes in A2780 cells affects the actions of histone deacetylase (HDAC) inhibitors. Trichostatin A exposure (100 ng/mL, 24 hours) induced ultrastructural differentiation of the "intrinsically" cisplatinum-resistant A2780-9M subline, with the reappearance of intercellular junctions and lumina containing primitive microvilli. Similar trichostatin A exposure in the acquired resistance A2780CP cells produced minimal differentiation consisting of occasional weak intercellular junctions. Independent of the differences in trichostatin A-induced differentiation, in both resistant sublines trichostatin A produced a similar reduction in cell viability, by >90%, within 5 days of treatment. Diminished viability in both A2780-9M and CP cells was associated with the absence of cell cycle arrest in G1, resulting in predominant G2-checkpoint arrest accompanied by a 10- to 20-fold increase in Annexin V binding and the reemergence of apoptosis. Similar cell cycle arrests and apoptosis were also observed using other HDAC inhibitors and in other resistant ovarian cancer cell lines (OVCAR-3 and SK-OV-3). Trichostatin A-induced apoptosis in resistant cells is in sharp contrast to its effects on the parental cisplatinum-sensitive A2780 and normal MRC-5 fibroblast cell lines (predominant cycle arrest in G1 with no detectable apoptosis). Western immunoblot analysis indicated trichostatin A triggers apoptosis in resistant ovarian cancer cells via p53-independent activation of the intrinsic "mitochondrial" pathway, commensurate with induction of the Bcl-2-related protein Bad. These results suggest cisplatinum resistance alters the effects of HDAC inhibition through a shift in cell cycle arrest from the G1 to the G2 checkpoint and reactivation of the intrinsic mitochondrial apoptotic cascade.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; bcl-Associated Death Protein; Blotting, Western; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Female; Fibroblasts; Flow Cytometry; G1 Phase; G2 Phase; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Microscopy, Electron; Mitochondria; Mitosis; Ovarian Neoplasms; Phenotype; Proto-Oncogene Proteins c-bcl-2; Time Factors

The presence of Coxsackie and adenovirus receptor (CAR) on target cell surface is required for efficient adenovirus transfection; lack or down-regulated expression of CAR on cancer cells is the main cause of inefficiency of adenovirus-based gene therapy. This study was to evaluate enhancive effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on the transfection efficiency of adenovirus in ovarian carcinoma cell line A2780, and explore its possible application to adenovirus-based gene therapy.. mRNA and protein levels of CAR on A2780 cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot before and after treatment of TSA. Transfection efficiency of adenovirus was valued by flow cytometry (FCM). In vitro antitumor effect of adenovirus/thymidine kinase (ADV/TK) was detected by MTT assay.. After treatment of TSA, mRNA and protein levels of CAR on A2780 cells were obviously increased. Transfection rates of adenovirus were (1.24+/-0.14)% in untreated group, (7.58+/-0.32)% in 5 nmol/L of TSA treated group, and (7.94+/-0.28)% in 100 nmol/L of TSA treated groups. In vitro antitumor effect of ADV/TK was 4-10 folds in TSA (5 or 100 nmol/L) treated groups compared with that in untreated group.. TSA could enhance transfection efficiency of adenovirus in ovarian carcinoma cells, and may be useful in gene therapy for ovarian carcinoma.

Topics: Adenoviridae; Cell Line, Tumor; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms; Receptors, Virus; RNA, Messenger; Thymidine Kinase; Transfection

Allelic imbalance on chromosome arm 22q has been detected in 50-70% of ovarian cancers, suggesting the presence of a tumor-suppressor gene on this chromosome arm that is involved in ovarian carcinogenesis. Recently, we isolated a candidate tumor-suppressor gene, MYO18B, at 22q12.1, which is deleted, mutated and hypermethylated in approximately 50% of lung cancers. In our study, we analyzed genetic and epigenetic alterations of the MYO18B gene in ovarian cancers. Missense MYO18B mutations were detected in 1 of 4 (25%) ovarian cancer cell lines and in 1 of 17 (5.9%) primary ovarian cancers. MYO18B expression was reduced in all 4 ovarian cancer cell lines and in 12 of 17 (71%) of primary ovarian cancers. MYO18B expression was restored by treatment with 5-aza-2'-deoxycytidine and/or trichostatin A in 3 of 4 cell lines with reduced MYO18B expression, and hypermethylation of the promoter CpG island for MYO18B was observed in 2 of these 3 cell lines. Its hypermethylation was also observed in 2 of 15 (13%) primary ovarian cancers. Thus, it was indicated that MYO18B expression is reduced in a considerable fraction of ovarian cancers by several mechanisms, including hypermethylation, while the MYO18B gene is mutated in a small subset of ovarian cancers. The present results suggest that MYO18B alterations, including both epigenetic and genetic alterations, play an important role in ovarian carcinogenesis.

Topics: Azacitidine; Carcinoma, Endometrioid; Case-Control Studies; Chromosomes, Human, Pair 22; Cystadenocarcinoma, Mucinous; Cystadenocarcinoma, Serous; Decitabine; DNA Methylation; DNA Mutational Analysis; Female; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Mutation, Missense; Myosin Heavy Chains; Ovarian Neoplasms; Ovary; Promoter Regions, Genetic; Tumor Cells, Cultured

Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In the current studies, exposing A2780 ovarian cancer cells to the histone deacetylase inhibitor trichostatin A (TSA) produced a marked change in cellular morphology, proliferation, and differentiation. Within 24 h of TSA treatment, there was a morphological transformation of the cells, with increased cytoplasm, a more epithelial-like columnar appearance, and the emergence of distinct cellular boundaries. Commensurate with the morphological transformation, TSA also inhibited cell proliferation; cells treated with TSA for 72 h increased to 110% of the initial cell numbers versus control cell numbers of 622%, with a corresponding reduction in mitotic activity and a flow cytometry S-phase fraction of 3.9% in TSA-treated cells versus 28.8% for control. TSA also induced epithelial-like differentiation with increased cytokeratin expression from 2% of controls to 22-25% of TSA-treated cells and the reappearance of intercellular plasma membrane junctions and primitive microvilli. Immunocytochemical analyses indicate the molecular mechanism underlying the actions of TSA on A2780 cell cycle progression and differentiation involves reexpression of the CDK inhibitor p21. Elevated levels of p21, in TSA-treated cells, were associated with a reduction in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). TSA also caused a decrease in the helix-loop-helix inhibitor of differentiation/DNA binding protein Id1, with no change in Id2 levels. In conclusion, the observed TSA-induced changes in p21, Rb, and Id1 are consistent with cell cycle senescence and differentiation of A2780 ovarian cancer cells.

Topics: Cell Cycle; Cell Differentiation; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Enzyme Inhibitors; Epithelial Cells; Female; Flow Cytometry; Helix-Loop-Helix Motifs; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Inhibitor of Differentiation Protein 1; Inhibitor of Differentiation Protein 2; Keratins; Ki-67 Antigen; Microfilament Proteins; Microscopy, Electron; Microvilli; Muscle Proteins; Ovarian Neoplasms; Phosphorylation; Repressor Proteins; Retinoblastoma Protein; Transcription Factors; Tumor Suppressor Protein p53

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

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