trichostatin-a and Glioma

The multiple physiological effects of the indoleamine melatonin, are mediated primarily by its two G protein-coupled MT

Topics: Animals; Azacitidine; Cell Line; Cell Line, Tumor; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Glioma; Histone Deacetylase Inhibitors; Hydroxamic Acids; Melatonin; Rats; Receptor, Melatonin, MT1; Receptors, Melatonin; RNA, Messenger; Transcriptional Activation; Valproic Acid

Herein we report novel hybrid compounds based on valproic acid and DNA-alkylating triazene moieties, 1, with therapeutic potential for glioblastoma multiforme chemotherapy. We identified hybrid compounds 1d and 1e to be remarkably more potent against glioma and more efficient in decreasing invasive cell properties than temozolomide and endowed with chemical and plasma stability. In contrast to temozolomide, which undergoes hydrolysis to release an alkylating metabolite, the valproate hybrids showed a low potential to alkylate DNA. Key physicochemical properties align for optimal CNS penetration, highlighting the potential of these effective triazene based-hybrids for enhanced anticancer chemotherapy.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Glioma; Humans; Molecular Structure; Structure-Activity Relationship; Triazenes; Tumor Cells, Cultured

Aberrant epigenetic histone modifications are implicated in cancer pathobiology, therefore histone modifying enzymes are emerging targets for anti-cancer therapy. There is a few evidence for deregulation of the histone modifying enzymes in glioblastomas. Glioma treatment is a clinical challenge due to its resistance to current therapies.. The effect of selected inhibitors on epigenetic modifications and viability of glioma C6 cells were studied using immunofluorescence and MTT metabolism test.. We found that VPA and TSA increase histone H4 acetylation in glioma cells, while chaetocin and BIX01294 at low concentrations reduce H3K9me3, and 3DZNep decreases H3K27me3. Long-term treatment with some epigenetic inhibitors affects viability of glioma cells.. We established the concentrations of selected inhibitors which in C6 glioma cells inhibit the enzyme activity, but do not decrease cell viability, hence allow to study the role of histone modifications in C6 glioma biology.

Topics: Acetylation; Adenosine; Animals; Azepines; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Epigenesis, Genetic; Glioma; Histones; Hydroxamic Acids; Piperazines; Quinazolines; Rats; Valproic Acid

The neurotoxic effects of excitatory amino acids (EAAs) are suggested to be connected with the chronic loss of neuronal cells, thereby being responsible for the age-related neurodegenerative diseases. Therefore, it seems conceivable that the excitatory amino acid transporters may contribute to the protection of neuronal cells against the excitotoxic damage by facilitating the removal of EAAs from the brain tissue. On the other hand, previous studies have suggested that glial cell differentiation may be involved in the protection and recovery of neural function probably through the elevation of BDNF gene expression in the brain. Based on these findings, histone deacetylase (HDAC) inhibitors are assumed to induce glutamate transporter-1 (GLT-1) gene expression probably through the promotion of glial cell differentiation. Then, we examined the effects of HDAC inhibitors on GLT-1 mRNA levels in rat C6 glioma cells and found that trichostatin A can induce GLT-1 gene transcription following steroid 5α-reductase and GFAP gene expression. Therefore, it seems conceivable that glial cell differentiation may play a potential role in the removal of EAAs probably through the expression of GLT-1, thereby being involved in the protection of neuronal cells against the chronic excitotoxic insults in the brain.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cholestenone 5 alpha-Reductase; Excitatory Amino Acid Transporter 2; Excitatory Amino Acids; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glioma; Histone Deacetylase Inhibitors; Hydroxamic Acids; Neuroglia; Neurotransmitter Agents; Rats; RNA, Messenger; Transcription, Genetic; Up-Regulation

NDRG2, a member of the N-Myc downstream-regulated gene family, was shown to be a putative tumor suppressor gene in glioblastoma and other cancers. Through a bioinformatic analysis, we found that NDRG2 protein contains an acyl carrier domain. In the current study, we therefore hypothesized that NDRG2 may play an important role in the regulation of histone acetylation. Treatment of U251 and U87 glioma cells with trichostatin A, an inhibitor of histone deacetylase, upregulated the expression of NDRG2 and acetylated forms of histones H3 and H4, reduced tumor cell viability and arrested the cell cycle at the G1/G0 phase. Overexpression of NDRG2 by transfecting glioma cells with adenovirus containing the NDRG2 gene upregulated the levels of acetylated forms of H3 and H4 whereas inhibition of NDRG2 expression by siRNA-mediated knockdown downregulated the level of histone acetylation. Furthermore, NDRG2 siRNA significantly reduced the level of histone acetylation induced by trichostatin A. Taken together, these data demonstrate that NDRG2 can regulate the level of histone acetylation to control glioma cell growth.

Topics: Acetylation; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Glioma; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; RNA, Messenger; RNA, Small Interfering; Tetrazolium Salts; Thiazoles; Transfection; Tumor Suppressor Proteins

It has been suggested that reduced glutamate receptor expression protects glioma cells from glutamate toxicity. GluR2 is the critical subunit of the GluR2 subtype of AMPA glutamate receptors as this subunit determines the Ca(2+) permeability of the receptor. The gene encoding the GluR2 subtype of AMPA receptors has been described as a target gene for the transcription repressor REST. However, we recently showed that the GluR2 gene is not regulated by REST in several neuronal and neuroendocrine cell lines, due to a repressive chromatin environment. Here, we show that the GluR2 gene has an open chromatin configuration in human glioma cells. Overexpression of REST reduced GluR2 mRNA levels while shRNA-mediated depletion of REST or expression of a REST mutant, that contained a transcriptional activation domain, enhanced GluR2 expression. Incubation with trichostatin A (TSA), a histone deacetylase inhibitor, induced acetylation of histone 4 of the GluR2 locus in glioma cells, leading to an upregulation of GluR2 expression. Together, these data suggest that REST is responsible for the reduced expression of GluR2 in glioma cells. The transcription factor Sp1 additionally binds under physiological conditions to the GluR2 gene in human glioma cells and expression of a dominant-negative mutant of Sp1 reduced expression of GluR2. Thus, the regulation via Sp1 represents a further control point for GluR2 expression in glioma cells. Together, we show that the GluR2 gene is embedded into an open chromatin configuration in glioma cells and expression of GluR2 is controlled by REST and Sp1.

Topics: Acetylation; Cell Line, Tumor; Chromatin; Chromatin Immunoprecipitation; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Glioma; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Molecular Conformation; Promoter Regions, Genetic; Protein Binding; Receptors, AMPA; Repressor Proteins; Sp1 Transcription Factor; Transcriptional Activation; Up-Regulation

To investigate the effect of monoamine oxidase inhibitor tranylcypromine (TCP) on the differentiation of human U251 glioma cells, we treated U251 cells with TCP and/or 100 nmol/L histone deacetylase inhibitor trychostatin A (TSA). The differentiation of U251 cells was observed with inverted microscopy. The cell proliferation and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Apoptosis was observed by Hoechst 33258 staining. The levels of differentiation-related genes were assessed by real-time PCR and Western blotting. TCP-induced differentiation was characterized by typical morphological changes, inhibition of cellular proliferation, accumulation of cells in the G1 phase of the cell cycle, decreased expression of the pluripotency transcription factors Oct4 and Sox2, and increased expression of glial fibrillary acid protein (GFAP). The combination of TCP and TSA treatment also triggered an over-expression of GFAP. These findings suggest that TCP may induce differentiation of U251 glioma cells, and the differentiation process may be promoted by histone deacetylase inhibitor TSA.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Glioma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Monoamine Oxidase Inhibitors; Tranylcypromine

Histone deacetylase (HDAC) inhibitors were demonstrated to induce cell cycle arrest, promote cell differentiation or apoptosis, and inhibit metastasis. HDAC inhibitors have thus emerged as a new class of anti-tumor agents for various types of tumors. However, the mechanisms by which HDAC inhibition-induced cell death remain to be fully defined.. In the present study, we explored the apoptotic actions of trichostatin A (TSA), a HDAC inhibitor, in C6 glioma cells.. TSA activated p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation and activation. P53, a proapoptotic transcription factor, in turn transactivated the expression of a proapoptotic protein, Bax. In addition, survivin, a member of inhibitor of apoptotic protein, was significantly decreased in TSA-treated C6 cells. P53 recruited to the endogenous survivin promoter region was increased and accompanied by decreasing recruitment of SP1 in response to TSA. TSA was also shown to induce IKK dephosphorylation and to suppress NF-κB reporter activity.. TSA may cause C6 cell apoptosis through activating p38MAPK-p53 cascade resulting in Bax expression and survivin suppression. Negative regulation of IKK-NF-κB signaling may also lead to p53 activation and contribute to TSA apoptotic actions.. TSA-induced p53 activation may occur through p53 modification by phosphorylation or by acetylation via IKK inactivation. The present study delineates, in part, the signaling pathways involved in TSA-induced glioma cell death.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cell Survival; Chromatin Immunoprecipitation; Dose-Response Relationship, Drug; Flow Cytometry; Glioma; Histone Deacetylase Inhibitors; Hydroxamic Acids; I-kappa B Kinase; Microtubule-Associated Proteins; Models, Biological; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Rats; Survivin; Time Factors; Tumor Suppressor Protein p53

Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines.. To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays.. We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation.. This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.

Topics: Azacitidine; Binding Sites; CCCTC-Binding Factor; Cell Line, Tumor; Decitabine; DNA Methylation; DNA, Neoplasm; Down-Regulation; Genes, Reporter; Genes, Retinoblastoma; Glioma; HeLa Cells; Humans; Hydroxamic Acids; K562 Cells; Mutation; Nucleic Acid Conformation; Promoter Regions, Genetic; Repressor Proteins; Sequence Analysis, DNA; Transgenes

The adrenergic and serotonergic stimulations of rat C6 glioma cells have previously been shown to induce the activation of steroid 5alpha-reductase (5alpha-R) gene expression, resulting in their differentiation through the production of neuroactive 5alpha-reduced steroid metabolites. In addition, progesterone and histone deacetylase (HDAC) inhibitors have also been reported to promote the glial cell differentiation with the enhancement of serotonin-stimulated brain-derived neurotrophic factor gene transcription through the production of 5alpha-reduced neurosteroids, thus suggesting that glial cell differentiation is probably implicated in the protection and survival of neuronal cells in the brain. Therefore, the expression of 5alpha-R gene in glial cells seems physiologically important in maintaining the neural function in the brain, but little is known about the mechanism underlying the regulation of 5alpha-R gene transcription. In the present study, the effect of a HDAC inhibitor trichostatin A (TSA) on 5alpha-R gene transcription in the glioma cells was examined, and TSA was shown to induce the elevation of 5alpha-R mRNA levels through the activation of the 5alpha-R promoter via a mechanism involving Sp1 and Sp3 transcription factors in a time- and concentration-dependent manner. Thus, both Sp1 and Sp3 are considered to play a physiological role in the regulation of 5alpha-R gene expression, and hence the production of 5alpha-reduced neurosteroids in glial cells.

Topics: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Animals; Base Sequence; Cell Line, Tumor; Gene Deletion; Gene Expression; Genes, Reporter; Glioma; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Molecular Sequence Data; Mutagenesis, Site-Directed; Rats; Sp1 Transcription Factor; Sp3 Transcription Factor

Tumor suppressor genes (TSGs) are sometimes inactivated by transcriptional silencing through promoter hypermethylation. To identify novel methylated TSGs in melanoma, we carried out global mRNA expression profiling on a panel of 12 melanoma cell lines treated with a combination of 5-Aza-2-deoxycytidine (5AzadC) and an inhibitor of histone deacetylase, Trichostatin A. Reactivation of gene expression after drug treatment was assessed using Illumina whole-genome microarrays. After qRT-PCR confirmation, we followed up 8 genes (AKAP12, ARHGEF16, ARHGAP27, ENC1, PPP1R3C, PPP1R14C, RARRES1, and TP53INP1) by quantitative DNA methylation analysis using mass spectrometry of base-specific cleaved amplification products in panels of melanoma cell lines and fresh tumors. PPP1R3C, ENC1, RARRES1, and TP53INP1, showed reduced mRNA expression in 35-59% of the melanoma cell lines compared to melanocytes and which was correlated with a high proportion of promoter methylation (>40-60%). The same genes also showed extensive promoter methylation in 6-25% of the tumor samples, thus confirming them as novel candidate TSGs in melanoma.

Topics: Azacitidine; Carrier Proteins; Cell Line, Tumor; Colonic Neoplasms; CpG Islands; Decitabine; DNA Methylation; Esophageal Neoplasms; Gene Expression Profiling; Gene Silencing; Genes, Tumor Suppressor; Glioma; Heat-Shock Proteins; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Melanoma; Membrane Proteins; Microfilament Proteins; Neuropeptides; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re-expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at -271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor-Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co-immunoprecipitation (co-IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation.

Topics: Acetylation; Animals; Binding Sites; Brain; Brain Neoplasms; Cell Adhesion Molecules; Cell Line, Tumor; Cell Proliferation; CpG Islands; DNA Methylation; Female; Glioma; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoglobulins; Membrane Proteins; Methylation; Mice; Mice, Nude; Neoplasm Transplantation; p300-CBP Transcription Factors; Promoter Regions, Genetic; RNA, Messenger; Sequence Analysis, DNA; Sp1 Transcription Factor

C6 glioma cells were treated with clinically relevant concentrations of valproic acid (0.5 or 1.0 mM) for 1-7 days and RT-PCR used to examine expression of the melatonin MT(1) receptor and selected epigenetic modulators. Valproic acid caused significant time-dependent changes in the mRNA expression of the melatonin MT(1) receptor, histone deacetylase (HDAC) 1, 2 and 3, and methyl CpG binding protein 2 (MeCP2). A structurally distinct HDAC inhibitor, trichostatin A, also caused a significant concentration-dependent induction of melatonin MT(1) receptor mRNA expression, suggesting involvement of an epigenetic mechanism. The ability of clinical concentrations of valproic acid to significantly alter melatonin MT(1) receptor expression, suggests a role for this receptor in the diverse neuropharmacological and oncostatic effects of this agent.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glioma; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Methyl-CpG-Binding Protein 2; Rats; Receptor, Melatonin, MT1; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Valproic Acid

FBXW7 has been reported to be a candidate tumor suppressor gene on 4q31. Three isoforms (alpha-form, beta-form, and gamma-form) of FBXW7 are produced from mRNAs with distinct 5' exons. Our previous study identified the specific suppression of the mRNA expression of the FBXW7 beta-form in human gliomas. Because this form is the major FBXW7 isoform in the human brain, we elucidated the silencing mechanisms for the FBXW7 beta-form in gliomas. No genetic alterations were found in the whole FBXW7 gene including putative promoter region of the beta-form. Treatments with 5-azacytidine and trichostatin A did not induce re-expression. A sodium bisulfite-modification assay indicated that CpG sequences in the promoter of FBXW7 beta-form were not methylated in glioma cells. Meanwhile we searched for the expression of FBXW7 and the sodium bisulfite sequences in normal human peripheral blood cells, and we surprisingly found that the mRNA expression of the FBXW7 beta-form was highly suppressed and the CpG sequences in the promoter region of the FBXW7 beta-form were heavily methylated. Our data suggest that the inactivation of the FBXW7 beta-form plays an important role in the pathogenesis of gliomas and that an unknown mechanism(s) other than mutation and methylation is the major cause of the suppression of the FBXW7 beta-form in gliomas.

Topics: Azacitidine; Base Sequence; Brain Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; DNA Methylation; DNA Mutational Analysis; Enzyme Inhibitors; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioma; Humans; Hydroxamic Acids; Isoenzymes; Molecular Sequence Data; Promoter Regions, Genetic; Ubiquitin-Protein Ligases

The effects of combining histone deacetylase (HDAC) inhibitors and proteasome inhibitors were evaluated in both established glioblastoma multiforme (GBM) cell lines and short-term cultures derived from the Mayo Clinic xenograft GBM panel. Coexposure of LBH589 and bortezomib at minimally toxic doses of either drug alone resulted in a striking induction of apoptosis in established U251, U87, and D37 GBM cell lines, as well as in GBM8, GBM10, GBM12, GBM14, and GBM56 short-term cultured cell lines. Synergism of apoptosis induction was also observed in U251 cells when coexposing cells to other HDAC inhibitors, including LAQ824 and trichostatin A, with the proteasome inhibitor MG132, thus demonstrating a class effect. In U251 cells, bortezomib alone or in combination with LBH589 decreased Raf-1 levels and suppressed Akt and Erk activation. LBH589 or bortezomib alone increased expression of the cell cycle regulators p21 and p27. Additionally, the combination, but not the individual agents, markedly enhanced JNK activation. Synergistic induction of apoptosis after exposure to LBH589 and bortezomib was partially mediated by Bax translocation from the cytosol to the mitochondria resulting from Bax conformational changes. Bax translocation precedes cytochrome c release and apoptosis, and selective down-regulation of Bax using siRNA significantly mitigates the cytotoxicity of LBH589 and bortezomib. This combination regimen warrants further preclinical and possible clinical study for glioma patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Boronic Acids; Bortezomib; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Glioma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Mitochondria; Panobinostat; Proteasome Inhibitors; Protein Transport; Pyrazines; RNA, Small Interfering

Recent advances in cancer cell biology have focused on histone deacetylase inhibitors (HDACi's) because they target pathways critical to the development and progression of disease. In particular, HDACi's can induce expression of epigenetically silenced genes that promote growth arrest, differentiation and cell death. In glioma cells, one such repressed gene is the tetraspanin CD81, which regulates cytostasis in various cell lines and in astrocytes, the major cellular component of gliomas. Our studies show that HDACi's, trichostatin and sodium butyrate, promote growth arrest and differentiation with negligible cell death in glioma cells and induce expression of CD81 and cyclin-dependent kinase inhibitor 1A (p21(CIP/WAF-1)), another regulator of cytostasis in astrocytes. Interference RNA knock-down of CD81 abrogates cytostasis promoted by HDAC inhibition indicating that HDACi-induced CD81 is responsible for growth arrest. Induction of CD81 expression through HDAC inhibition is a novel strategy to promote growth arrest in glioma cells.

Topics: Animals; Antigens, CD; Brain Neoplasms; Butyrates; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, cdc; Glioma; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Rats; Rats, Inbred F344; Rats, Wistar; RNA Interference; Tetraspanin 28

Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2'-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis.

Topics: Azacitidine; Brain Neoplasms; Decitabine; DNA Methylation; Gene Expression; Gene Expression Profiling; Gene Silencing; Genes, Tumor Suppressor; Genome, Human; Glioma; Histones; Humans; Hydroxamic Acids; Nerve Tissue Proteins; Promoter Regions, Genetic

Histone deacetylase (HDAC) inhibitors have both apoptotic and differentiating effects on various tumor cells. However, the mechanisms underlying the effect of HDAC inhibitors remain unclear. In this study, we investigated the function of anti-proliferative effects of HDAC inhibitors, N-butyric acid and trichostatin A, on human malignant glioma cell lines, U251-MG and D54. MTT assay showed a dose-dependent inhibition of cellular proliferation in both cell lines. Cell cycle analysis revealed increased sub-G1 population in both lines, and G1 arrest only in U251-MG cells. Induction of apoptosis was also supported by the occurrence of DNA fragmentation in tumor cells treated with HDAC inhibitors. Furthermore, caspase inhibition assay indicated that HDAC inhibitor-induced apoptosis was caspase-dependent. Neither mitochondrial membrane potential nor the expression of caspase-9 was changed by treatment with HDAC inhibitors, suggesting the possibility that HDAC inhibitor-induced apoptosis was not mediated by the mitochondrial cell death pathway. On the other hand, immunoblot assay confirmed increased expression of caspase-8 in both lines, and elevation of p21 but not p27 protein in U251-MG cells following HDAC inhibitor treatment. Taken together, the HDAC inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis with or without p21-mediated G1 arrest in human malignant glioma cells.

Topics: Apoptosis; Brain Neoplasms; Butyric Acid; Caspase 8; Caspase 9; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Induction; Enzyme Inhibitors; Glioma; Histamine Antagonists; Humans; Hydroxamic Acids

Trichostatin A (TSA) is a histone deacetylase inhibitor that causes growth inhibition of malignant cells. The authors' goal was to evaluate its effect on cell growth and cell cycle regulation in a large panel of glioma cell lines, as well as in human astrocytes, fibroblasts, and endothelial cells.. Cell growth in response to TSA was evaluated using a tetrazolium colorimetric assay and a clonogenic assay. Cell cycle effects were examined using flow cytometry. A DNA fragmentation assay was used to evaluate induction of apoptosis. Histone acetylation status and the expression of p21WAF1, phosphorylated retinoblastoma protein (Rb), poly(adenosine diphosphate-ribose) polymerase (PARP), and caspase-3 were studied using Western blot analysis. In the glioma cell lines, there was significant inhibition of cell growth and detection of increased levels of acetylated histones after TSA treatment. The mechanisms underlying the growth inhibition include cell cycle arrest at the G2/M phase and apoptosis induction. The expression of p21WAF1 was activated, with a temporally related decrease in levels of phosphorylated Rb. Apoptosis was preceded by detection of cleaved PARP and activated caspase-3. The effects of TSA were less pronounced or absent in human astrocytes, fibroblasts, and endothelial cells.. The TSA caused inhibition of glioma cell growth by both cell cycle arrest and apoptosis. Cell cycle arrest was associated with an increase in p21WAF1 expression and a decrease in phosphorylated Rb. Apoptosis was mediated at least partly through the activation of caspase-3. Because of the differential effects in glioma cells compared with nonneoplastic cells, TSA may provide a novel strategy for achieving tumor growth inhibition and cytotoxicity. Further investigation is warranted.

Topics: Acetylation; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Inhibitors; Glioma; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Topics: Azacitidine; Brain Neoplasms; Chromatin; Chromosomal Proteins, Non-Histone; CpG Islands; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA-Binding Proteins; Enzyme Inhibitors; Gene Silencing; Glioblastoma; Glioma; Glycoproteins; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Methyl-CpG-Binding Protein 2; Polymerase Chain Reaction; Promoter Regions, Genetic; Repressor Proteins

Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine, which is the first and rate-limiting step in catecholamine biosynthesis. In the present study, we report that treatment with the histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) or sodium butyrate, prominently induces the TH promoter activity in both non-neuronal and neuronal cell lines. By analyzing a series of deletional reporter constructs, we also determined that the proximal 151bp region of the TH promoter is largely responsible for TSA-mediated activation. Finally, we found that mutation of the Sp1 or CRE site, residing in the proximal area, abolishes TSA-mediated activation, strongly suggesting that the Sp1 and CRE sites may mediate TH promoter activation by inhibition of HDAC. In summary, our results provide a novel regulatory frame in which modulation of chromatin structure by histone deacetylase may contribute to transcriptional regulation of the TH via the Sp1 and/or CRE site.

Topics: Animals; Butyrates; Carcinoma, Hepatocellular; Gene Expression Regulation, Enzymologic; Glioma; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mice; Multienzyme Complexes; Neuroblastoma; NIH 3T3 Cells; Promoter Regions, Genetic; Rats; Transcriptional Activation; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

The histone deacetylase inhibitor, trichostatin A(TSA), was shown to induce apoptosis in transformed cells at submicromolar concentrations. However, the effect of TSA on brain tumor cells is still unknown. This study was designed to investigate whether TSA posses antitumor activity and if any, its mechanism.. A p53 mutant human glioma cell line T98G and a p53 wild type human neuroblastoma cell line SKNSH were exposed to TSA. Cell proliferation was assessed by sulforhodamine B (SRB) cytotoxicity assay. Apoptosis was quantified by flow cytometry and confirmed by apoptotic ladder formation. Expression patterns of accumulation of highly acetylated histone H3, H4; p53 and cell cycle-associated p21waf, p27 which were induced by TSA were determined by using Western blot analysis.. TSA inhibited the proliferation of brain tumor cell lines at nanomolar concentrations and induced accumulation of highly acetylased histone moleculars. Treatment with TSA at 0.33 microM for 24 h significantly induced cell apoptosis. In addition to the suppression of cell growth, the up regulation of p21waf and p27 expression was observed within 48 h after the treatment. p21 protein levels were increased at early time points and reached maximal levels at 8 h, while p27 protein levels were increased after 8 h. However, there was no significant changes of acetylased p53 and endogenous p53 protein levels were observed.. TSA may inhibit brain tumor cell growth in vitro, which is otherwise particularly resistant to chemotherapy. TSA acts as an anti-tumor agent could be through co-operation between p21 and p27 in growth inhibition, irrespective of endogenous p53 status.

Topics: Apoptosis; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; DNA Fragmentation; Enzyme Inhibitors; Glioma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

While cyclooxygenase (COX)-2 is a highly inducible gene, COX-1 is widely known as a noninducible gene and is constitutively expressed in a variety of cell lines and human tissues. Recently, several reports have indicated that COX-1 is also regulated at the transcriptional level by various stimuli. We present evidence that histone deacetylase (HDAC) inhibitors induce COX-1 transcription and translation in normal human astrocyte (NHA) cells and glioma cell lines. HDAC inhibitors increased acetylated histone H4 protein expression in NHA cells. The levels of COX-1 mRNA and protein were maximal at 24 and 48 h, respectively, after treatment with the specific HDAC inhibitor, trichostatin A (TSA). In addition, TSA-treated NHA cells produced prostaglandin E(2) as determined by enzyme-linked immunosorbent assay after incubation with 10 microm exogenous arachidonic acid, indicating that the induced COX-1 is functionally active. In addition to NHA cells, this up-regulation of COX-1 after treatment with HDAC inhibitors was observed in 5 different glioma cell lines. The nucleotide sequence of the inducible COX-1 cDNA was confirmed identical to human COX-1 that was previously reported. HDAC inhibitors stimulated COX-1 promoter activity as measured by luciferase reporter assays, suggesting that the induction of COX-1 is regulated at the transcriptional level. Furthermore, mutation analysis of the COX-1 promoter suggests that TSA-responsive element exists in the proximal Sp1-binding site at +25 to +31. In conclusion, COX-1 is an inducible gene in glial-derived cells including immortalized cells, and appears to be transcriptionally regulated by a unique mechanism associated with histone acetylation.

Topics: Acetylation; Arachidonic Acid; Astrocytes; Binding Sites; Blotting, Northern; Cell Nucleus; Cyclooxygenase 1; Dinoprostone; DNA Mutational Analysis; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Glioma; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Isoenzymes; Membrane Proteins; Mutation; Neuroglia; Oligonucleotides; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sp1 Transcription Factor; Time Factors; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation

Histone deacetylase inhibitors that increase histone acetylation on transformed cells are being investigated as unique anticancer drugs. The aim of this investigation was to evaluate an antiproliferative activity of the histone deacetylase inhibitors sodium butyrate (NaBT) and trichostatin A on 5 glioma cell lines, T98G, A172, U-87 MG, U-118 MG, and U-373 MG, with the examination of the altered expressions in p21 and gelsolin genes. Treatment with 5-mM NaBT and 40 ng/ml trichostatin A for 48 h caused more than a 50% growth inhibition in 5 cell lines as measured by cell proliferation assays. An increase in histone acetylation was confirmed in each cell line. After treatment with 5 mM NaBT, T98G, A172, and U118 cells undergo apoptosis as indicated by DNA ladder formation. Treatment with NaBT and trichostatin A also decreased DNA synthesis as examined by the fluorescence-activated cell sorting analysis in T98G and U87 cells. In addition to the suppression of cell growth, the up regulation of p21 and gelsolin expression was observed after treatment with NaBT, especially in T98G cells. Maximum expression of p21 and gelsolin was observed within 24 h after treatment. Results from our in vitro studies indicate that the treatment of human glioma cells with one of the histone deacetylase inhibitors suppresses cell growth with decreasing DNA synthesis and stimulates apoptosis, and that associated molecular mechanisms responsible for these effects include increased histone acetylation as well as enhanced expression of p21 and gelsolin.

Topics: Acetylation; Apoptosis; Butyrates; Cell Cycle; Cell Division; Central Nervous System Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Flow Cytometry; Gelsolin; Gene Expression Regulation, Neoplastic; Glioma; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Tumor Cells, Cultured; Up-Regulation