trichostatin-a and thiazolyl-blue

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

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

Topics: Acetylation; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dogs; Female; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Mast-Cell Sarcoma; Mice; Mice, Nude; Tetrazolium Salts; Thiazoles; Trypan Blue

To investigate the effects of mitomycin C and the histone deacetylase inhibitors sodium butyrate and trichostatin on the viability and growth of conjunctival melanoma cell lines and Tenon capsule fibroblasts.. Cells were treated with a range of concentrations of sodium butyrate, trichostatin, and mitomycin C. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assays were performed 48 hours after treatment. Treated cells were stained with acridine orange/ethidium bromide to assess for cell death. Cell-cycle changes in histone deacetylase inhibitor-treated melanoma cells were quantified using flow cytometry.. All agents induced dose-dependent cell death in the melanoma cell lines; however, sodium butyrate and trichostatin were relatively nontoxic to Tenon capsule fibroblasts. Acridine orange/ethidium bromide staining indicated that sodium butyrate and trichostatin induced apoptotic cell death. At low doses, sodium butyrate and trichostatin induced a G1 cell-cycle block in the melanoma cells.. Sodium butyrate and trichostatin induced cell death in melanoma cells, comparable with mitomycin C, with minimal effect on Tenon capsule fibroblasts. In addition, they induced a G1 cell-cycle block. These findings support the need for further investigation into the in vivo efficacy of these agents.

Topics: Apoptosis; Butyrates; Cell Cycle; Cell Proliferation; Cell Survival; Conjunctival Neoplasms; Connective Tissue Cells; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Melanoma; Mitomycin; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

5-FU is the drug most frequently used to treat biliary tract cancer, while dihydropyrimidine dehydrogenase (DPD) is known to be a principal factor in 5-FU drug resistance. However, whether DPD activity and mRNA levels correlate with response to 5-FU is unknown for biliary tract cancers. The precise mechanism of DPD regulation also remains to be elucidated. In the present study, we quantitatively analyzed DPD mRNA in 8 biliary tract cancer cell lines using real-time RT-PCR, and assessed whether DPD mRNA levels correlate with DPD activity or the sensitivity to 5-FU. Finally, we examined the epigenetic gene silencing of DPD using one of the 8 lines, a gallbladder cancer cell line with deficient DPD expression, KMG-C. Strong correlation was found between DPD activity and DPD mRNA expression in the 8 cancer cell lines (R=0.797, P=0.0148). DPD mRNA expression and DPD activity exhibited positive correlation with the IC50 for 5-FU (R=0.658, R=0.644, respectively), although these relationships were not statistically significant. In the KMGC cells with deficient DPD mRNA levels, restoration of DPD expression was observed by 5-Aza-2' deoxycytidine (5-aza-C) treatment in a dose-dependent manner, suggesting gene suppression by promoter hypermethylation. Combined bisulfite restriction analysis was performed to analyze the methylation on CpG islands around the 5'-flanking region and intron 1 of the DPD gene, however, no methylated CpG sites were identified in these regions. In addition, the restored DPD expression level was more strongly induced by the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), than 5-aza-C treatment. These findings suggest that other mechanisms, including histone modification, may be important for DPD suppression. In conclusion, these results may aid the selection of 5-FU chemotherapy following determination of DPD expression in biliary tract cancers. Furthermore, epigenetic gene silencing appears to be an important mechanism of DPD suppression in biliary tract cancer.

Topics: Antimetabolites, Antineoplastic; Base Sequence; Biliary Tract Neoplasms; Cell Line, Tumor; Enzyme Inhibitors; Epigenesis, Genetic; Fluorouracil; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Molecular Sequence Data; Protein Synthesis Inhibitors; Tetrazolium Salts; Thiazoles

We demonstrate that the histone deacetylase (HDAC) inhibitor drug trichostatin A (TSA) reduces spinal cord inflammation, demyelination, neuronal and axonal loss and ameliorates disability in the relapsing phase of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). TSA up-regulates antioxidant, anti-excitotoxicity and pro-neuronal growth and differentiation mRNAs. TSA also inhibits caspase activation and down-regulates gene targets of the pro-apoptotic E2F transcription factor pathway. In splenocytes, TSA reduces chemotactic, pro-Th1 and pro-proliferative mRNAs. A transcriptional imbalance in MS may contribute to immune dysregulation and neurodegeneration, and we identify HDAC inhibition as a transcriptional intervention to ameliorate this imbalance.

Topics: Animals; Cell Death; Cells, Cultured; Cerebral Cortex; Cytokines; Disease Models, Animal; Drug Administration Schedule; Drug Interactions; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Profiling; Gene Expression Regulation; Glycoproteins; Hydroxamic Acids; Immunohistochemistry; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurons; Oligonucleotide Array Sequence Analysis; Peptide Fragments; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Spleen; Tetrazolium Salts; Thiazoles; Time Factors

Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA.. To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody.. The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, alpha2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, alpha7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes.. This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromatin; Coloring Agents; DNA, Complementary; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; In Situ Nick-End Labeling; Liver Neoplasms; Oligonucleotide Array Sequence Analysis; Precipitin Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tetrazolium Salts; Thiazoles; Transcription, Genetic; Up-Regulation

By preventing deacetylation of histones, histone deacetylase inhibitors (HDIs) transcriptionally induce p21. Here we show that the HDIs sodium butyrate (Bu), trichostatin A (TSA) and depsipeptide (FR901228) all induced p21, but only TSA and FR901228 caused mitotic arrest (in addition to arrest in G1 and G2). The ability to cause mitotic arrest correlated with the higher cytotoxicity of these compounds. Although causing mitotic arrest, TSA and FR901228 (unlike paclitaxel) did not affect tubulin polymerization. Unlike FR9012208, TSA caused acetylation of tubulin at lysine 40; both soluble tubulin and microtubules were acetylated. Whereas the induction of p21 reached a maximum by 8 h, tubulin was maximally acetylated after only 1 h of TSA treatment. Tubulin acetylation was detectable after treatment with 12-25 ng/ml TSA although acetylation plateaued at 50 ng/ml TSA, coinciding with G2-M arrest, appearance of cells with a sub-2N DNA content, poly(ADP-ribose) polymerase cleavage, and rapid cell death. We conclude that HDIs have differential effects on non-histone deacetylases and that rapid acetylation of tubulin caused by TSA is a marker of nontranscriptional effects of TSA.

Topics: Anesthetics, Intravenous; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Apoptosis; Butyrates; Cell Cycle; Cell Line; Coloring Agents; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Depsipeptides; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; G2 Phase; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Immunoblotting; Jurkat Cells; Mitosis; Peptides; Peptides, Cyclic; Protein Synthesis Inhibitors; Sodium Oxybate; Tetrazolium Salts; Thiazoles; Time Factors; Transcription, Genetic; Tubulin; Tumor Cells, Cultured