trichostatin-a and pyrimidin-2-one-beta-ribofuranoside

Topics: Animals; Cytidine; DNA Methylation; DNA Modification Methylases; Gene Knockout Techniques; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasms; RNA Interference; Valproic Acid

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

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

A pattern of epigenetic modifications and changes, DNA methylation and histone modification, is central to many human cancers. A variety of tumor suppressor genes (TSGs) have been demonstrated to be silenced because of histone deacetylation and DNA hypermethylation in several cancers. Recent in vitro studies have shown that two known mechanisms of epigenetic alteration consisting of methylation and histone deacetylation seem to be the best candidate mechanisms for inactivation of CIP/KIP family (p21Cip1/Waf1/Sdi1, and p27Kip1) in numerous cancers. Numerous investigations have indicated that DNA demethylating and histone deacetylase inhibitors (HDACIs) can restore the CIP/KIP family gene expression. Previously, we evaluated the effect of trichostatin A (TSA) and 5-aza-2'-deoxycytidine (5-AZA-CdR) on hepatocellular carcinoma (HCC). The present study was designed to investigate the effect of zebularine in comparison to and in combination with trichostatin A on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNMT1, DNMT3a and DNMT3b, Class I HDACs (HDACs 1, 2, 3) and Class II HDACs (HDACs 4, 5, 6) gene expression, cell growth inhibition and apoptosis induction in colon cancer LS 174T cell line.. The colon cancer LS 174T cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively.. Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively.. The zebularine and trichostatin A can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.

Topics: Apoptosis; Biomarkers, Tumor; Calcium-Binding Proteins; Cell Proliferation; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytidine; DNA (Cytosine-5-)-Methyltransferases; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Tumor Cells, Cultured

Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression.. In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression.. These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

Topics: Adenoma; Animals; Antineoplastic Agents; Cell Line; Chromatin Assembly and Disassembly; CpG Islands; Cytidine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; HMGA Proteins; HMGA2 Protein; Humans; Hydroxamic Acids; MicroRNAs; Pituitary Gland; Pituitary Neoplasms; Rats; Rats, Sprague-Dawley; Up-Regulation

Retinoic acid (RA)-induced expression of bone morphogenetic protein-4 (BMP-4) inhibits in vitro and in vivo cell proliferation and ACTH synthesis in corticotroph-derived tumor cells. Reduced expression of BMP-4 in this adenoma subtype is associated with epigenomic silencing, and similar silencing mechanisms are also associated with the RA-responsive dopamine D2 receptor (D2R) in somatolactotroph cells. We now show that preincubation with the epidrugs zebularine and trichostatin A is obligate and permissive for RA-induced expression of the BMP-4 and the D2R genes in pituitary tumor cells. Combined epidrug challenges are associated with marginal reduction in CpG island methylation. However, significant change to histone tail modifications toward those associated with expression-competent genes is apparent, whereas RA challenge alone or in combined incubations does not have an impact on these modifications. Epidrug-mediated and RA-augmented expression of endogenous BMP-4 increased or decreased cell proliferation and colony-forming efficiency in GH3 and AtT-20 pituitary tumor cells, respectively, recapitulating recent reports of challenges of these cells with exogenous ligand. The specificity of the BMP-4-mediated effects was further supported by knock-down experiments of the BMP-4 antagonist noggin (small interfering RNA [siRNA]). Knock-down of noggin, in the absence and the presence of epidrugs, induced and augmented BMP-4 expression, respectively. In cell proliferation assays, challenge with either epidrugs or siRNA led to significant increase in cell numbers at the 72-hour time point; however, in siRNA-treated cells coincubated with epidrugs, a significant increase was apparent at the 48-hour time point. These studies show the potential of combined drug challenges as a treatment option, where epidrug renders silenced genes responsive to conventional therapeutic options.

Topics: Adenoma; Animals; Bone Morphogenetic Protein 4; Cell Culture Techniques; Cytidine; Drug Synergism; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Pituitary Neoplasms; Prodrugs; Receptors, Dopamine D2; Time Factors; Tretinoin; Tumor Cells, Cultured

The orexin system plays a central role in the integration of sleep/wake and feeding behaviors in a broad spectrum of neural-metabolic physiology. Orexin-A and orexin-B are produced by the cleavage of prepro-orexin, which is encoded on the Hcrt gene. To date, methods for generating other peptide neurons could not induce orexin neurons from pluripotent stem cells. Considering that the metabolic status affects orexin expression, we supplemented the culture medium with a nutrient factor, ManNAc, and succeeded in generating functional orexin neurons from mouse ES cells. Because DNA methylation inhibitors and histone deacetylase inhibitors could induce Hcrt expression in mouse ES cells, the epigenetic mechanism may be involved in this orexin neurogenesis. DNA methylation analysis showed the presence of a tissue-dependent differentially methylated region (T-DMR) around the transcription start site of the Hcrt gene. In the orexin neurons induced by supplementation of ManNAc, the T-DMR of the Hcrt gene was hypomethylated in association with higher H3/H4 acetylation. Concomitantly, the histone acetyltransferases p300, CREB-binding protein (CBP), and Mgea5 (also called O-GlcNAcase) were localized to the T-DMR in the orexin neurons. In non-orexin-expressing cells, H3/H4 hypoacetylation and hyper-O-GlcNAc modification were observed at the T-DMRs occupied by O-GlcNAc transferase and Sirt1. Therefore, the results of the present study suggest that the glucose metabolite, ManNAc, induces switching from the inactive state by Ogt-Sirt1 to the active state by Mgea5, p300, and CBP at the Hcrt gene locus.

Topics: Acetylation; Animals; Cell Differentiation; Cytidine; Deoxycytidine; DNA Methylation; DNA-Cytosine Methylases; Embryonic Stem Cells; Epigenesis, Genetic; Female; Glycosylation; Hexosamines; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Neurons; Neuropeptides; Orexins; Protein Processing, Post-Translational; Sequence Analysis, DNA; Sirtuin 1; Spheroids, Cellular; Transcription Initiation Site

Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.

Topics: Butyrates; Cell Membrane; Cytidine; Epigenesis, Genetic; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Polysaccharides

Epigenetic changes play a role in all major events during tumorigenesis and changes in glycan structures are hallmarks of virtually every cancer. Also, proper N-glycosylation of membrane receptors is important in cell to cell and cell-environment communication. To study how modulation of epigenetic information can affect N-glycan expression we analyzed effects of epigenetic inhibitors on HeLa cell membrane N-glycome.. HeLa cells were treated with DNA methylation (zebularin and 5-aza-2-deoxycytidine) and histone deacetylation (trichostatin A and Na-butyrate) inhibitors. The effects on HeLa cell membrane N-glycome were analyzed by hydrophilic interaction high performance liquid chromatography (HILIC).. Each of the four epigenetic inhibitors induced changes in the expression of HeLa cell membrane N-glycans that were seen either as an increase or a decrease of individual glycans in the total N-glycome. Compared to DNA methylation inhibitors, histone deacetylation inhibitors showed more moderate changes, probably due to their higher gene target selectivity.. The results clearly show that composition of HeLa cell membrane N-glycome can be specifically altered by epigenetic inhibitors.. Glycans on the cell membrane are essential elements of tumor cell's metastatic potential and are also an entry point for nearly all pathogenic microorganisms. Since epigenetic inhibitors used in this work are registered drugs, our results provide a new line of research in the application of these drugs as anticancer and antimicrobial agents. This article is part of a Special Issue entitled Glycoproteomics.

Topics: Antineoplastic Agents; Azacitidine; Butyrates; Carbohydrate Sequence; Cytidine; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Glycomics; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Molecular Sequence Data; Polysaccharides

The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine, can reverse p15INK4B promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies.. We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples.. Zebularine treatment of AML193, which has a densely methylated p15INK4B promoter, results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation, arrests cells at G(2)/M, and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis.. Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies.

Topics: Acetylation; Acute Disease; Apoptosis; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p15; Cytidine; DNA Methylation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; G2 Phase; Gene Expression Profiling; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia, Myeloid; Phosphorylation; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

Inhibitors of DNA methyltransferases (DNMT) and histone deacetylases can reactivate epigenetically silenced tumor suppressor genes and thereby decrease tumor cell growth. Little, however, is known on the effects of these compounds in endothelial cell biology and tumor angiogenesis. Here, we show that the DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine markedly decrease vessel formation in different tumor models. We show that DNMT inhibitors are antiproliferative for tumor-conditioned endothelial cells, without affecting endothelial cell apoptosis and migration. Furthermore, these compounds inhibit angiogenesis in vitro and in vivo as shown by inhibition of endothelial cells sprouting in a three-dimensional gel and inhibition of microvessel formation in the chorioallantoic membrane, respectively. 5-Aza-2'-deoxycytidine, as well as the histone deacetylase inhibitor trichostatin A, reactivates the growth-inhibiting genes TSP1, JUNB, and IGFBP3, which are suppressed in tumor-conditioned endothelial cells. Despite enhanced DNMT activity and increased overall genomic methylation levels in tumor-conditioned endothelial cells, silencing of these genes seemed not to be regulated by direct promoter hypermethylation. For IGFBP3, gene expression in endothelial cells correlated with histone H3 acetylation patterns. In conclusion, our data show that DNMT inhibitors have angiostatic activity in addition to their inhibitory effects on tumor cells. This dual action of these compounds makes them promising anticancer therapeutics.

Topics: Acetylation; Angiogenesis Inhibitors; Animals; Azacitidine; Cell Movement; Cytidine; DNA Modification Methylases; Endothelial Cells; Enzyme Inhibitors; Gene Expression; Histones; Humans; Hydroxamic Acids; Insulin-Like Growth Factor Binding Protein 3; Melanoma, Experimental; Mice; Mice, Mutant Strains; Neovascularization, Pathologic; Neovascularization, Physiologic; Thrombospondin 1