trichostatin-a and Carcinoma--Squamous-Cell

The present in vitro study aims to investigate the potential use of epigenetic inhibitors as treatment modalities in tongue squamous cell carcinoma.. The human tongue squamous cell carcinoma cell line (CAL-27) was cultured and exposed to varying concentrations of 5-Azacitidine (5-Aza) or Trichostatin A (TSA) in the culture medium. The cell apoptosis was evaluated using Annexin V/PI by flow cytometry. To evaluate DNA damage response, γH2AX foci analysis was performed using immunofluorescence. Single cell gel electrophoresis (SCGE) was applied to measure DNA strand breaks. Gene expression was assessed by quantitative real-time PCR.. The results showed that 5-Aza and TSA had apoptotic effects on the SCC cell line at concentrations of 50-200 µM and 0.5-5 µM, respectively. Immunofluorescence analysis showed increased expression of γH2AX, the marker of DNA damage response after treatment of 5-Aza and TSA that was associated with increased DNA strand breaks. The expressions of urokinase plasminogen activator, its receptor and matrix metalloproteinase-2, were significantly reduced in TSA- and 5-Aza-treated cells.. Our results showed that 5-Aza and TSA increase apoptotic and DNA damage response in squamous cell carcinoma cell line while reducing the expression of tumor invasion genes that further indicating the potential therapeutic value of two epigenetic modifiers in squamous cell carcinoma.

Topics: Azacitidine; Carcinoma, Squamous Cell; Decitabine; DNA Damage; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Tongue; Tongue Neoplasms

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

To investigate the apoptotic event of trichostatin A (TSA) and its associated mechanism in oral squamous cell carcinoma (OSCC) lines.. HSC-3 and Ca9.22 cell lines were evaluated using a trypan blue exclusion assay, histone isolation, soft agar assay, live/dead assay, 4%,6-diamidino-2-phenylindole staining, JC-1 mitochondrial membrane potential (MMP) assay, and Western blot analysis to demonstrate the anticancer activity of TSA.. TSA decreased OSCC cell viability and proliferation without affecting the histone acetylation. TSA-induced caspase-dependent or -independent apoptosis according to cell types, TSA enhanced the expression levels of Bim protein by dephosphorylating ERK1/2 pathway in HSC-3 cells. TSA also damaged MMP and increased cytosolic apoptosis-inducing factor (AIF) in Ca9.22 cells.. The present study suggests that TSA may be a potential anticancer drug candidate for the treatment of OSCC through the induction of apoptosis.

Topics: Acetylation; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Mouth Neoplasms

Histone deacetylase inhibitors (HDACis) play an important role as valuable drugs targeted to cancer therapy: several HDACis are currently being tested in clinical trials. Two new potential HDACis 1a and 1d, characterized by the presence of a biphenyl-4-sulfonamide group as a connection unit between the N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-hydroxyphenyl} and the 2-hydroxy-N-(trifluoroacetyl)benzamide moiety, respectively, as two zinc-binding group (ZBG), have been designed, synthesized and tested for their biological activity. Surprisingly, compounds 1a and 12, this last exclusively obtained in place of 1d, exhibited a very low HDAC inhibitory activity. A serendipitous assay of these two compounds, conducted on three chemoresistant cell lines of head and neck squamous cell carcinoma (HNSCC), showed their antiproliferative activity at low nanomolar concentrations, better than cisplatin. In vitro, biological assays indicated that compounds 1a and 12 are able to increase acetylation of histone H3 and to interfere with the PI3K/Akt/mTOR pathway by inducing the accumulation of PTEN protein.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Proliferation; Dose-Response Relationship, Drug; Drug Discovery; Drug Screening Assays, Antitumor; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Molecular Structure; Structure-Activity Relationship; Sulfonamides

Trichostatin A (TSA), a histone deacetylase inhibitor, is used as an anti-carcinogenic and radiosensitizing agent in various cancers. However, the role and mechanism underlying its radiosensitization of tongue squamous cell carcinoma (TSCC) remains unclear. Thus, in this study we aimed to confirm the promotion of miR‑375 expression by TSA, and to investigate the effects of TSA and miR‑375 in the radiosensitivity of TSCC cells. The results showed that TSA had significant radiosensitizing effects on TSCC cells and miR‑375 overexpression had effects similar to TSA in sensitizing these cells to radiotherapy. By contrast, miR‑375 knockdown attenuated apoptosis induced by radiation combined with TSA. Mechanistically, the histone acetylation status of the miR‑375 promoter region was increased by TSA, resulting in the upregulation of miR‑375, which led to a decline of PDK1 and phosphorylated AKT. Taken together, our data suggest that TSA increases the radiosensitization and apoptosis in TSCC cells at least partially via miR‑375, and TSA or miR‑375 in combination with radiotherapy may provide a valuable approach for the treatment of TSCC.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; MicroRNAs; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Radiation-Sensitizing Agents; Tongue Neoplasms

Chemoresistance is a major barrier to effective chemotherapy of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Recently, autophagy, a highly conservative intracellular recycling system, has shown to be associated with chemoresistance in cancer cells. However, little is known about how autophagy plays a role in the development of chemoresistance in HNSCC and how autophagy is initiated when HNSCC cells undergo cytotoxic stress. Here, we report that autophagy was activated when HNSCC cells are treated with the proteasome inhibitor bortezomib, proposed as an alternative chemotherapeutic agent for both primary and cisplatin-resistant HNSCC cells. Ablation of histone deacetylase 6 (HDAC6) expression and its activity in HNSCC cells significantly inhibited autophagy induction by altering the phosphorylation status of mammalian target of rapamycin and enhanced the bortezomib cytotoxicity. Similarly, a combination regimen of bortezomib and the histone deacetylase inhibitor trichostatin A abolished HDAC6 activity and decreased autophagy induction while significantly enhancing bortezomib-induced apoptosis in HNSCC cells. These data uncover a novel molecular mechanism indicating that HDAC6 may serve as a critical causal link between autophagy, apoptosis, and the cell survival response in HNSCC. A combination regimen resulting in regression of autophagy improves chemotherapeutic efficacy, thereby providing a new strategy to overcome chemoresistance and to improve the treatment and survival of HNSCC patients.

Topics: Apoptosis; Autophagy; Bortezomib; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Head and Neck Neoplasms; Histone Deacetylase 6; Histone Deacetylases; Humans; Hydroxamic Acids; Neoplasm Proteins

Inhibition of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACis) results in cancer cell growth inhibition, and HDACis have been revealed as potential anti-skin cancer agents. p21 is a cyclin-dependent kinase inhibitor and an essential regulator of growth inhibition. Recently, we reported that activating transcription factor 3 (ATF3) could significantly promote skin cancer cell growth. This study explored the relationship between ATF3 and HDACi-induced growth inhibition of epidermoid carcinoma cells. We found that trichostatin A (TSA) treatment inhibited cell growth in A431 epidermoid carcinoma cells in a dose-dependent manner. Simultaneously, p21 and ATF3 expression levels were upregulated and downregulated upon TSA stimulation, respectively. ATF3 overexpression promoted cell growth and downregulated p21 expression. In contrast, ATF3 depletion resulted in cell growth reduction and p21 transcriptional upregulation. More importantly, ATF3 overexpression partially antagonized TSA-induced growth inhibition and p21 activation. Collectively, these data demonstrate that ATF3 acts as an essential negative regulator of TSA-induced cell growth inhibition through interfering with TSA-induced p21 activation.

Topics: Activating Transcription Factor 3; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Transcriptional Activation; Up-Regulation

Lung cancer is clearly the primary cause of cancer-related deaths worldwide. Recent molecular-targeted strategy has contributed to improvement of the curative effect of adenocarcinoma of the lung. However, such current treatment has not been developed for squamous cell carcinoma (SCC) of the disease. The new genome-wide RNA analysis of lung-SCC may provide new avenues for research and the development of the disease. Our recent microRNA (miRNA) expression signatures of lung-SCC revealed that clustered miRNAs miR-1/133a were significantly reduced in cancer tissues. Here, we found that restoration of both mature miR-1 and miR-133a significantly inhibited cancer cell proliferation, migration and invasion. Coronin-1C (CORO1C) was a common target gene of the miR-1/133a cluster, as shown by the genome-wide gene expression analysis and the luciferase reporter assay. Silencing of CORO1C gene expression inhibited cancer cell proliferation, migration and invasion. Furthermore, CORO1C-regulated molecular pathways were categorized by using si-CORO1C transfectants. Further analysis of novel cancer signaling pathways modulated by the tumor-suppressive cluster miR-1/133a will provide insights into the molecular mechanisms of lung-SCC oncogenesis and metastasis.

Topics: Aged; Aged, 80 and over; Azacitidine; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Lung Neoplasms; Male; Microfilament Proteins; MicroRNAs; Middle Aged; Multigene Family; Neoplasm Invasiveness; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference

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

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

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

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

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

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

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

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

Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs). Moreover, evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alterations in the patterns of acetylation of histones and are a promising new group of anticancer agents. In this study, we investigated the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on two CD44+ cancer stem-like cell lines from squamous cell carcinoma of the head and neck (SCCHN) cultured in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. Histone deacetylase inhibitors inhibited the growth of SCCHN cell lines in a dose-dependent manner as measured by MTS assays. Moreover, HDACi induced cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2, on SCCHN cell lines was decreased by HDACi treatment. In addition, HDACi decreased mRNA expression levels of stemness-related genes and suppressed the epithelial-mesencymal transition phenotype of CSCs. As expected, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel, had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter the cancer stem cell phenotype in SCCHN, raising the possibility that HDACi may have therapeutic potential for cancer stem cells of SCCHN.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Epithelial-Mesenchymal Transition; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Hyaluronan Receptors; Hydroxamic Acids; Neoplasm Proteins; Neoplastic Stem Cells; Phenotype; Squamous Cell Carcinoma of Head and Neck

High activity of histone deacetylases (HDACs) has been documented in several types of cancer and may be associated with survival advantage. In a head and neck squamous cell carcinoma cell line, cisplatin-induced apoptosis was augmented by pretreatment with the HDAC inhibitor trichostatin A. Apoptosis was accompanied by lysosomal membrane permeabilization (LMP), as shown by immunoblotting of the lysosomal marker protease cathepsin B in extracted cytosol and by immunofluorescence. Moreover, LAMP-2 (lysosomal associated membrane protein-2) was translocated from lysosomal membranes and found in a digitonin extractable fraction together with cytosolic proteins and pretreatment with trichostatin A potentiated the release. Overall, protein level of LAMP-2 was decreased during cell death and, interestingly, inhibition of cysteine cathepsins, by the pan-cysteine cathepsin inhibitor zFA-FMK, prevented loss of LAMP-2. The importance of LAMP-2 for lysosomal membrane stability, was confirmed by showing that LAMP-2 knockout MEFs (mouse embryonic fibroblasts) were more sensitive to cisplatin as compared to the corresponding wildtype cells. Trichostatin A reduced lysosomal pH from 4.46 to 4.25 and cell death was prevented when lysosomal pH was increased by NH(4)Cl, or when inhibiting the activity of lysosomal proteases. We conclude that trichostatin A enhances cisplatin induced cell death by decreasing lysosomal pH, which augments cathepsin activity resulting in reduced LAMP-2 level, and might promote LMP.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydrogen-Ion Concentration; Hydroxamic Acids; Lysosomes; Up-Regulation

Histone acetylation is one of the key chromatin modifications that control gene transcription during development and tumorigenesis. Recently, it was reported that the histone deacetylase inhibitor, Trichostatin A (TSA), induces growth arrest and apoptosis in tumors. However, the molecular mechanisms responsible for its antitumor effects are not clear. The purpose of this study was to investigate the effect of TSA on human oral squamous carcinoma cells and to determine the mechanisms underlying the antitumor activity of TSA. MTT assays showed that TSA inhibited cell proliferation in YD-10B cells. TSA also effectively arrested cell cycle progression at the G2/M phase through the up-regulation of p21waf expression, down-regulation of Cyclin B1 and reduction of the inhibitory phophorylation of Cdc2. In addition, mitochondrial membrane destruction was induced by a 48 h TSA treatment. TSA also induced cytochrome c release and proteolytic activation of caspase 3 and caspase 7 in YD-10B cells. Taken together, these observations in YD-10B oral cancer cells reveal the potential value of TSA in inhibiting oral tumor growth.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mouth Neoplasms

Hypoxia is an essential feature of the microenvironment of solid tumors, which regulates a variety of transcription factors including hypoxia-inducible factor-1α (HIF-1α). HIF-1α overexpression enhances tumor angiogenesis via upregulation of vascular endothelial growth factor (VEGF) and some other hypoxia-inducible angiogenic factors, which lead to a more aggressive tumor phenotype, tumor metastasis and resistance to radiation and chemotherapy. In this study, we found that a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), inhibited cell proliferation and invasion, blocked the cell cycle, and induced cell apoptosis in a dose- and time-dependent manner in the human tongue squamous cell carcinoma (TSCC) SCC-6 cell line in vitro. Furthermore, TSA reduced both basal levels and hypoxia-induced HIF-1α protein accumulation but not HIF-1α mRNA levels, and both protein and mRNA levels of VEGF expression. These results showed that TSA had a potent anticancer activity on TSCC cells, suggesting that TSA could be a promising drug targeting tumor angiogenesis via inhibition of HIF-1α and VEGF expression in the development of an effective chemopreventive and anticancer agent on human TSCCs.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Tongue Neoplasms; Vascular Endothelial Growth Factor A

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

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

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

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

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

A group of histone deacetylase (HDAC) inhibitors has been shown to suppress the growth of a variety of human tumor lines in vitro and in vivo and they are among the most promising candidates for anti-cancer therapeutic agents. We investigated the ability of scriptaid, a novel HDAC inhibitor and trichostatin A (TSA) to enhance cell killing by radiation in radioresistant SQ-20B cells derived from human head and neck squamous carcinoma. SQ-20B cells were treated with scriptaid or TSA in combination with radiation. Cell survival was determined by a colony formation assay and protein levels were examined by Western blotting. DNA double strand breaks were measured by a gamma-H2AX focus assay. Radiosensitization was observed for SQ-20B cells incubated with scriptaid at 5 microM or TSA at 0.1 microM for 24 h. Radiosensitization by scriptaid was accompanied by a prolonged retention of gamma-H2AX foci, suggesting that the enhancement of radiation cell killing by scriptaid involved inhibition of DNA double strand break repair. In addition, treatment with scriptaid suppressed expression of Ku80, but not Ku70. Scriptaid may be a useful radiosensitizer in the treatment of radioresistant human carcinomas.

Topics: Antigens, Nuclear; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; DNA Breaks, Double-Stranded; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Hydroxylamines; Ku Autoantigen; Quinolines; Radiation; Radiation-Sensitizing Agents

Proteasome inhibitor PS-341 (also known as bortezomib) and histone deacetylase (HDAC) inhibitors have emerged as novel therapeutic agents for a variety of malignancies. In this study, we examined whether PS-341 and the HDAC inhibitor trichostatin A (TSA) induced apoptosis in head and neck squamous cell carcinoma (HNSCC), a common and lethal malignancy. We found that, although TSA treatment alone did not induce apoptosis in HNSCC cells, it significantly enhanced PS-341-induced apoptosis in HNSCC cells in vitro. Consistently, TSA significantly improved PS-341-mediated inhibition of HNSCC tumor growth in nude mice. Mechanistically, we found that TSA increased PS-341-induced Noxa expression and caspase activation in HNSCC cells. The knockdown of Noxa significantly reduced apoptosis induced by cotreatment of PS-341 and TSA. Taken together, our results provide new insight into the mechanisms of synergistic antitumor activity of the PS-341 and HDAC inhibitor regimen, offering a new therapeutic strategy for HNSCC patients.

Topics: Acetylation; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Northern; Blotting, Western; Boronic Acids; Bortezomib; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Mice; Mice, Nude; Proto-Oncogene Proteins c-bcl-2; Pyrazines; RNA Interference; Xenograft Model Antitumor Assays

Combining the use of a chemotherapeutic agent with oncolytic virotherapy is a useful way to increase the efficiency of the treatment of cancer. The effect of the histone diacetylase (HDAC) inhibitor trichostatin A (TSA) on the antitumor activity of a herpes simplex virus type-1 (HSV-1) mutant was examined in oral squamous cell carcinoma (SCC) cells. Immunoblotting analysis and immunoflourescence staining revealed that a cytoplasmic nuclear factor-kappaB (NF-kappaB) component, p65, translocated into the nucleus after infection with gamma(1)34.5 gene-deficient HSV-1 R849, indicating that R849 activated NF-kappaB. TSA induced acetylation of p65 and increased the amount of p65 in the nucleus of oral SCC cells. Treatment of R849-infected cells with TSA also increased the amount of nuclear p65 and binding of NF-kappaB to its DNA-binding site and an NF-kappaB inhibitor SN50 diminished the increase in nuclear p65. In the presence of TSA, the production of virus and the expression of LacZ integrated into R849 and glycoprotein D, but not ICP0, ICP6 and thymidine kinase, were increased. The viability of cells treated with a combination of R849 and TSA was lower than that of those treated with R849 only. After treatment with TSA, expression of the cell cycle kinase inhibitor p21 was upregulated and the cell cycle was arrested at G1. These results indicate that TSA enhanced the replication of the HSV-1 mutant through the activation of NF-kappaB and induced cell cycle arrest at G1 to inhibit cell growth. TSA can be used as an enhancing agent for oncolytic virotherapy for oral SCC with gamma(1)34.5 gene-deficient HSV-1.

Topics: Acetylation; Carcinoma, Squamous Cell; Cell Line, Tumor; Defective Viruses; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Gene Expression Regulation, Viral; Herpesvirus 1, Human; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Mouth Neoplasms; Neoplasm Proteins; NF-kappa B; Oncolytic Virotherapy; Peptides; Protein Processing, Post-Translational; Protein Transport; Transcription Factor RelA; Viral Proteins; Virus Activation; Virus Replication

Histone deacetylases (HDACs) inhibitors induce cell growth arrest and apoptosis in a wide variety of tumor cells. The purpose of this study was to evaluate the effects of trichostatin A (TSA), one of the HDACs inhibitors, on proliferation and apoptosis of oral squamous cell carcinoma cells. Exposure of Tca83 cells (established from human tongue squamous cell carcinoma) to TSA resulted in cell growth inhibition and apoptosis in a dose-dependent manner as measured with MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay and DAPI (4'6'diamidino-2-phenylindole dihydrochloride) staining. Western blot showed that both total PTEN and membrane-bound PTEN were induced by TSA treatment, whereas phosphorylation level (Ser 473) of AKT was correspondingly down-regulated by TSA treatment. Knock-down of PTEN expression with PTEN siRNA could sufficiently block 0.25mug/ml TSA induced inhibition of cell growth, but failed to block 0.5mug/ml TSA induced inhibition of cell growth and apoptosis. Moreover, induction of apoptosis by TSA treatment was also demonstrated by cytochrome C releasing and induction of caspase-3. Conclusively, the results suggested that PTEN/AKT pathway was involved in TSA induced cell growth inhibition and apoptosis of oral squamous cell carcinoma cells. HDACs inhibitors could be potential anticancer drugs for chemotherapy of oral squamous cell carcinoma.

Topics: Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Neoplasm Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Thiazoles; Tongue Neoplasms

Activator protein 2 alpha (AP-2alpha) is involved in a variety of physiological processes. Increased AP-2alpha expression correlates with progression in various squamous cell carcinomas, and a recent publication found AP-2alpha to be overexpressed in approximately 70% of Head and Neck Squamous Cell Carcinoma (HNSCC) patient samples. It was found to repress transcription of the tumor suppressor gene C/CAAT Enhancer Binding Protein alpha (C/EBPalpha), and its binding site correlated with upstream methylation of the C/EBPalpha promoter. Therefore, we investigated the potential for AP-2alpha to target methylation to additional genes that would be relevant to HNSCC pathogenesis.. Stable downregulation of AP-2alpha stable by shRNA in HNSCC cell lines correlated with decreased methylation of its target genes' regulatory regions. Furthermore, methylation of MLH1 in HNSCC with and without AP-2alpha downregulation revealed a correlation with microsatellite instability (MSI). ChIP analysis was used to confirm binding of AP-2alpha and HDAC1/2 to the targets. The effects of HDAC inhibition was assessed using Trichostatin A in a HNSCC cell line, which revealed that AP-2alpha targets methylation through HDAC recruitment.. These findings are significant because they suggest AP-2alpha plays a role not only in epigenetic silencing, but also in genomic instability. This intensifies the potential level of regulation AP-2alpha has through transcriptional regulation. Furthermore, these findings have the potential to revolutionize the field of HNSCC therapy, and more generally the field of epigenetic therapy, by targeting a single gene that is involved in the malignant transformation via disrupting DNA repair and cell cycle control.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gene Silencing; Head and Neck Neoplasms; Humans; Hydroxamic Acids; Microsatellite Instability; Microsatellite Repeats; Models, Biological; Protein Synthesis Inhibitors; PTEN Phosphohydrolase; Transcription Factor AP-2

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

The molecular mechanism for cisplatin (CDDP)-resistance of cancer cells has not yet been clarified, despite extensive studies. Here, we investigated whether death-associated protein (DAP) kinase, an apoptosis modulator, was involved in CDDP-resistance by examining the ME180 human cervical squamous cancer cell line and 6 monoclonal ME180-derived CDDP-resistant subclones. Co-treatment with CDDP and 5-aza-2'-deoxycytidine (5-aza-CdR), a demethylating agent, significantly enhanced the CDDP-sensitivities of the parent cells and CDDP-resistant subclones. Subsequent removal of 5-aza-CdR rapidly reversed the CDDP-sensitivity of the CDDP-resistant subclones to their original levels, whereas the parent cells retained the enhanced CDDP-sensitivity for at least 24 h. Quantitative RT-PCR revealed that the CDDP-resistant subclones expressed higher DNA methyltransferase (DNMT) mRNA levels than the parent cells, suggesting that increased DNMT expressions easily restored the CDDP-resistance of the CDDP-resistant subclones following 5-aza-CdR removal. Although the parent cells showed hypermethylation in the DAP kinase promoter region, corresponding methylated bands were not detected in 2 of the 6 CDDP-resistant subclones by methylation-specific PCR. All 6 CDDP-resistant subclones expressed higher DAP kinase mRNA levels than the parent cells, as evaluated by quantitative RT-PCR. Although DAP kinase protein expression was strongly suppressed in the parent cells and CDDP-resistant subclones, 5-aza-CdR treatment of the parent cells dose-dependently stimulated the DAP kinase protein expression, and this was synergistically enhanced by inhibiting histone deacetylation via trichostatin treatment in addition to 5-aza-CdR. However, DAP kinase protein expression in the CDDP-resistant subclones was not stimulated by treatment with 5-aza-CdR and/or trichostatin. These results indicate that post-transcriptional translation of DAP kinase mRNA is strongly suppressed and insensitive to treatment with 5-aza-CdR and trichostatin in the CDDP-resistant subclones established from ME180 human cervical squamous cancer cells. This CDDP-resistance is accompanied by molecular changes that disturb the post-transcriptional translation of the DAP kinase mRNA, and these molecular changes are transiently restored by demethylation.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Azacitidine; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Death-Associated Protein Kinases; Decitabine; DNA Methylation; DNA Modification Methylases; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Protein Biosynthesis; Protein Synthesis Inhibitors; RNA, Messenger; Uterine Cervical Neoplasms

Although RAD21 is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth, its role in cancer has been unclear. In this study, the relevance of RAD21 gene expression to the invasion and metastasis of oral squamous cell carcinoma was clarified using laser microdissection and real-time polymerase chain reaction (PCR). Using two different metastatic potential oral squamous cells [high-metastatic-potential squamous cell carcinoma cells (SAS-Ly) and low-metastatic-potential squamous cell carcinoma cells (SAS)], the relation of RAD21 gene expression to apoptosis, invasion, and metastasis was examined. The results showed that RAD21 gene expression was significantly decreased in oral squamous cell carcinoma when it expressed the INFbeta and INFgamma invasion patterns in comparison with the INFalpha invasion pattern (p<0.01). In addition, in comparison with SAS cells, SAS-Ly cells indicated tolerance to cell death induced by an apoptosis induction reagent, while the expression level of the RAD21 gene in SAS cells was increased by the apoptosis induction reagent. However, in SAS-Ly cells, the reagent induced no significant difference. Our findings indicate that the RAD21 gene was closely related to the invasion and metastasis of cancer cells.

Topics: Adult; Aged; Aged, 80 and over; Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Dactinomycin; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Male; Mice; Mice, Nude; Middle Aged; Mouth Neoplasms; Neoplasm Invasiveness; Nuclear Proteins; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The expression of survivin, a member of the inhibitor-of-apoptosis protein family, is elevated in many types of human cancer. High survivin expression has been associated with poor patient prognosis and tumor resistance to chemotherapy and radiotherapy. The purpose of this study was to compare the radiosensitizing effects of five agents that target survivin on their relative ability to downregulate survivin expression.. The human epidermoid carcinoma cell line A431 was treated with adenoviral-mediated wild-type p53, antisense to survivin, the mitogen-activated protein kinase inhibitor PD98059, the cyclin-dependent kinase inhibitor Purvalanol A, or the histone deacetylase inhibitor trichostatin A. The radiosensitizing effects of these treatments were determined by clonogenic survival curve analysis and their abilities to suppress survivin expression by Western blot analysis.. All the strategies were shown to radiosensitize A431 cells. This effect correlated with their abilities to downregulate survivin.. Expression of survivin appears to confer a radioresistant phenotype that can be overcome using several clinically achievable strategies that target survivin either specifically or nonspecifically.

Topics: Carcinoma, Squamous Cell; CDC2 Protein Kinase; Cell Line, Tumor; Down-Regulation; Flavonoids; Genes, p53; Humans; Hydroxamic Acids; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; Oligonucleotides, Antisense; Purines; Radiation Tolerance; Survivin; Tumor Suppressor Protein p53

The chemotherapeutic effects of all-trans-retinoic acid (atRA) are mediated by the retinoic acid receptor beta (RARbeta), but RARbeta expression is reduced in a number of head and neck carcinoma (HNSCC) cells which causes resistance to RA treatment in half the patients with HNSCC. The possible mechanism for the reduced RARbeta expression has been suggested as the methylation of the CpG islands adjacent to the RA response elements (RARE) in the RARbeta promoter and the loss of histone acetylation. The suppressed RARbeta expression can be reactivated by a demethylating agent (5-aza-2'-deoxycytidine, 5-AzaC) or a histone deacetylase inhibitor (trichostatin A, TSA). Therefore, we sought to determine if the restoration of RARbeta activity, or a combination of these drugs, could restore the sensitivity to RA in RARbeta-negative HNSCC cells with an epigenetically methylated RARbeta promoter region. SqCC/Y1 cells resistant to atRA showed methylated and unmethylated forms in the RARbeta promoter region. RARbeta expression of these cells was restored by 5-AzaC or TSA treatment. Also, treatment with TSA and atRA combined synergistically increased the growth-inhibitory effect and highly induced the transcriptional activation of the RARbeta promoter compared to atRA treatment in HNSCC cells. Additionally, TSA alone and the combination 5-AzaC and TSA increased lysine-9 (Lys-9) acetylation and Lys-4 methylation of the first exon at the RARbeta gene, while decreasing the methylation of Lys-9 in the HNSCC cells.

Topics: Acetylation; Antineoplastic Agents; Azacitidine; Carcinoma, Squamous Cell; Cell Line, Tumor; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lysine; Promoter Regions, Genetic; Receptors, Retinoic Acid; Tretinoin

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

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

The physiological and pathophysiological functions of PAI-1 are related to its expression by specific cell types in normal and diseased tissues. We analysed the contribution of DNA methylation to the variation in PAI-1 mRNA levels in five cell lines. We found varying frequencies of methylation of 25 CpGs in the -805/+152 region of the PAI-1 gene in Bowes, MCF-7 and U937 cells, while little or no methylation was detected in Hep2 and HT- 1080 cells. The methylation frequency was inversely correlated with PAI-1 mRNA level within its 20-fold range in Bowes, MCF-7, U937,and Hep2 cells, while the lack of methylation in both Hep2 and HT-1080 cells suggested another mechanism behind the 150-fold higher level in HT- 1080 cells than in Hep2 cells. However, all cell lines exhibited a high frequency of methylation of 10 CpGs in a CpG island at about--1800. Treatment with 5-aza-2'-deoxycytidine led up to circa a 40-fold increase in the PAI-1 mRNA level and a strong decrease in the frequency of methylation in the -805/+152 region in Bowes, MCF-7 and U937. The histone deacetylase inhibitor trichostatin A induced a several fold increase of the PAI-I mRNA level in cells with a high methylation frequency of the -805/+152 region. As compared with matched normal tissue, three samples of oral squamous cell carcinomas displayed decreased frequencies of methylation of the PAI-1 5' flanking region and increased levels of PAI-1 mRNA. These results for the first time implicate DNA methylation and histone acetylation in regulation of the PAI-1 gene, and indicate that without proper CpG islands in 5'-flanking region,trancription may be regulated by methylation of less dense CpGs in the 5'-flanking region rather than methylation of upstream CpG island.

Topics: 5' Flanking Region; Azacitidine; Carcinoma, Squamous Cell; CpG Islands; Decitabine; DNA; DNA Methylation; DNA Modification Methylases; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mouth Neoplasms; Plasminogen Activator Inhibitor 1; RNA, Messenger; Sequence Analysis, DNA; Time Factors; U937 Cells

Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents with potential for disruption of critical cellular processes in cancer cells. Transcriptional regulation, differentiation, cell cycle arrest, radiation sensitization, and apoptosis have been observed in response to exposure to HDAC inhibitors. In the present study, we observed that several potent HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, M344 (an analogue of hydroxamic acid), and the cyclic tetrapeptide, depsipeptide (FR90228), modulate cellular responses to ionizing radiation in cells of two human squamous carcinoma lines (SQ-20B and SCC-35), previously characterized as intrinsically resistant to radiation. Also exposure to IC(50) concentrations of these inhibitors, radiation sensitivities were enhanced in both cell lines. Depsipeptide exhibited the greatest effect on SQ-20B cells, decreasing D(0) values from 2.62 Gy to 1.64 Gy. M344 was the most active drug in sensitizing SCC-35 cells, decreasing D(0) values from 1.91 Gy to 1.21 Gy. The mechanisms underlying HDAC inhibitor-induced radiosensitization were further investigated by extending trichostatin A studies to assess cell cycle distributions and levels of apoptosis. Treatment of SQ-20B cells with radiosensitizing concentrations of trichostatin A resulted in cell cycle arrest in G(1) phase (>70%) and inhibition of DNA synthesis. Contrary to previous reports, induction of apoptosis was very low and caspase 3 and 9 were not activated. Taken together, these results implicate G(1) arrest and inhibition of DNA synthesis in the mechanisms underlying radiation sensitization by trichostatin A and support the use of HDAC inhibitors for targeting radioresistant cancers.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Survival; Depsipeptides; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Peptides, Cyclic; Radiation Tolerance; Radiation-Sensitizing Agents; Vorinostat

Head and neck squamous cell carcinoma (HNSCC) is the fifth most frequent cancer in the US. Several genetic and epigenetic alterations are associated with HNSCC tumorigenesis, including inactivation of CDKN2A, which encodes the p16 tumor suppressor, in cell lines and primary tumors by DNA methylation. Reactivation of tumor suppressor genes by DNA-demethylating agents and histone deacetylase (HDAC) inhibitors shows therapeutic promise for other cancers. Therefore, we investigated the ability of these agents to reactivate p16 in Tu159 HNSCC cells. Treatment of cells with 5-aza-2'deoxycytidine (5-aza-dC) increases CDKN2A expression and slightly increases histone H3 acetylation at this gene. No reactivation of CDKN2A is observed upon treatment with the HDAC inhibitor trichostatin A (TSA), but synergistic reactivation of CDKN2A is observed upon sequential treatment of Tu159 cells with both 5-aza-dC and TSA. Silencing of CDKN2A in Tu159 cells is correlated with increased methylation of histone H3 at lysine 9 and decreased methylation at lysine 4 relative to the upstream p15 gene promoter. Interestingly, global levels of H3-K9 methylation are decreased upon treatment with 5-aza-dC. Together these data indicate that DNA methylation is a dominant epigenetic mark for silencing of CDKN2A in Tu159 tumor cells. Moreover, changes in DNA methylation can reset the histone code by impacting multiple H3 modifications.

Topics: Animals; Antimetabolites, Antineoplastic; Azacitidine; Carcinoma, Squamous Cell; Decitabine; DNA Methylation; Gene Silencing; Genes, p16; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids

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

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

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