trichostatin-a and Kidney-Neoplasms

Metabolome analysis is an approach to investigate cell characteristics from the metabolites that are constantly produced and changed by those cells. We conducted a metabolome analysis of the response of 786-O renal cell carcinoma (RCC) cells to histone deacetylase (HDAC) inhibitors, which are expected to increase anticancer drug sensitivity, and compared the response with that of drug-resistant cells. Trichostatin A (TSA), an HDAC inhibitor, increased the sensitivity of 786-O cells to sunitinib. Moreover, TCA cycle and nucleotide metabolism of the cells were promoted. The findings that acetylated p53 (active form) and early apoptotic cells were increased suggests that the mechanism involved enhancement of mitochondrial metabolism and function. In addition, established sunitinib-resistant RCC cells were exposed to a combination of sunitinib and TSA, resulting in significant growth inhibition. Principal component analysis revealed that the parent and resistant cells were obviously different, but approximately half their fluctuations were illustrated by the same pathways. In summary, it was suggested that TSA reduced sunitinib resistance by triggering intracellular metabolome shifts in energy metabolism. This was the first recognized mechanism of action of TSA as an HDAC inhibitor.

Topics: Antineoplastic Agents; Carcinoma, Renal Cell; Drug Resistance, Neoplasm; Energy Metabolism; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kidney Neoplasms; Metabolome; Metabolomics; Mitochondria; Sunitinib; Tumor Cells, Cultured; Tumor Suppressor Protein p53

In this study, our aim was to exploring the influences of DNA methylation of PON1 on cell proliferation, migration and apoptosis of renal cancer cells. The genome-wide methylation array of renal cell carcinoma samples and adjacent tissues were obtained from the cancer genome atlas (TCGA) database. By analysing the DNA methylation and conducting the CpG islands array, methylation status expressed in renal tumour samples and normal renal tissue samples were detected. Methylation-specific PCR (MS-PCR) and qRT-PCR were employed to detect the methylation level and mRNA expression of PON1. Wound-healing assay, transwell assay and MTT assay were utilized to detecting the migration, invasion and proliferation abilities, respectively. The cell apoptosis was testified by Tunnel assay. In addition, the effect of PON1 on renal cancer cells was verified by experiments in vivo. The methylation status of different genes in renal cell carcinoma samples was obtained by CpG islands arrays and hypermethylated PON1 was selected for further study. PON1 was down-regulated in renal cell carcinoma tissues detected by qRT-PCR and Western blot. Both in vitro and vivo experiments indicated that the sunitinib-resistant in renal cancer cells could be suppressed by treat with 5-Aza-dC or TSA, and the effect came out more obvious after 5-Aza-dC and TSA co-treatment. In detail, the demethylation of PON1 inhibited the migration, invasion and proliferation of renal cancer cells and also arrested more cells in G0/G1 phase. The vivo experiment indicated that demethylated PON1 suppressed the growth of tumour. Hypermethylated PON1 promoted migration, invasion and proliferation of sunitinib-resistance renal cancer cells and arrested more cells in G0/G1 phase.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aryldialkylphosphatase; Azacitidine; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; CpG Islands; Databases, Genetic; Disease Progression; DNA Methylation; Drug Resistance, Neoplasm; Epigenesis, Genetic; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Kidney Neoplasms; Mice; Mice, Nude; Sunitinib; Transplantation, Heterologous

The majority of clear cell renal cell carcinomas (ccRCCs) are caused by an accumulation of hypoxia‑inducible factor (HIF) and the overexpression of downstream genes in response to the von Hippel‑Lindau (VHL) gene becoming inactivated. In the present study, our hypothesis was that BNIP3, a gene positioned downstream of HIF, would be expressed at a higher level in ccRCC; however, instead, lower levels of BNIP3 expression were identified in RCC tumor tissues compared with adjacent non‑tumor tissues. These changes were associated with lower levels of VHL, and higher levels of HIF and vascular endothelial growth factor. BNIP3 was also undetectable in three investigated RCC cell lines (786‑O, ACHN, A498) and GRC‑1‑1 cells. Methylation of the BNIP3 promoter was not detected, and neither did treatment with a methylation inhibitor cause cell proliferation. However, treatment with a histone deacetylation inhibitor, trichostatin A (TSA), inhibited cultured RCC cell proliferation, promoted apoptosis and restored BNIP3 expression. Furthermore, histone deacetylation of the BNIP3 promoter was identified in ACHN and 786‑O cells, and the acetylation status was restored following TSA treatment. Taken together, the results of the present study suggest that histone deacetylation, but not methylation, is most likely to cause BNIP3 inactivation in RCC. The data also indicated that restoration of BNIP3 expression by a histone deacetylation inhibitor led to growth inhibition and apoptotic promotion in RCC.

Topics: Acetylation; Aged; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Neoplasms; Male; Membrane Proteins; Middle Aged; Promoter Regions, Genetic; Proto-Oncogene Proteins; Vascular Endothelial Growth Factor A; Von Hippel-Lindau Tumor Suppressor Protein

Although sunitinib is the first-line drug for progressive renal cell carcinoma (RCC), most patients experience its tolerance. One possible way of overcoming drug resistance is combination therapy. Epigenetic modifier is one of the candidate drug group. A recent evidence suggests that cell metabolism is regulated by epigenetic mechanisms. Epigenetic abnormalities lead to changes in metabolism and may contribute to drug resistance and progression of RCC. Consequently, we investigated whether trichostatin A (TSA), a potent histone-deacetylase (HDAC) inhibitor, alters sunitinib-induced cytotoxicity and metabolism in RCC cells at epigenetic regulatory concentrations. Combined metabolome and transcriptome analysis suggested that TSA impacts on energy productive metabolic pathways, such as those involving TCA cycle and nucleotide metabolism especially for increase of hyperphosphorylated form. Combination of sunitinib and TSA increased cell death with PARP cleavage, an early marker of mitochondrial apoptosis, whereas receptor tyrosine kinase signaling, which is the target of sunitinib, was not altered by TSA. Finally, the established sunitinib resistant-RCC cell (786-O Res) was also exposed to sunitinib and TSA combination, resulting in significant growth inhibition. In summary, it was suggested that TSA reduces sunitinib resistance by triggering intracellular metabolome shifts regarding energy metabolism, that is the first recognized mechanism as an HDAC inhibitor.

Topics: Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Drug Resistance, Neoplasm; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kidney Neoplasms; Metabolic Networks and Pathways; Receptor Protein-Tyrosine Kinases; Signal Transduction; Sunitinib

Interferon regulatory factor 8 (IRF8), as a central element of IFN-γ-signaling, plays a critical role in tumor suppression. However, its expression and underlying molecular mechanism remain elusive in renal cell carcinoma (RCC). Here, we examined IRF8 expression and methylation in RCC cell lines and primary tumors, and further assessed its tumor suppressive functions. We found that IRF8 was widely expressed in human normal tissues including kidney, but frequently downregulated by promoter methylation in RCC cell lines. IRF8 methylation was detected in 25% of primary tumors, but not in adjacent non-malignant renal tissues, and associated with higher tumor nuclear grade of RCC. Ectopic expression of IRF8 inhibited colony formation and migration abilities of RCC cells, through inducing cell cycle G2/M arrest and apoptosis. IFN-γ could induce IRF8 expression in RCC cells, together with increased cleaved-PARP. We further found that IRF8 inhibited expression of oncogenes YAP1 and Survivin, as well as upregulated expression of tumor suppressor genes CASP1, p21 and PTEN. Collectively, our data demonstrate that IRF8 as a functional tumor suppressor is frequently methylated in RCC, and IRF8-mediated interferon signaling is involved in RCC pathogenesis.

Topics: Azacitidine; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Decitabine; DNA Methylation; DNA Modification Methylases; Down-Regulation; Female; G2 Phase Cell Cycle Checkpoints; Genes, Tumor Suppressor; HEK293 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Interferon Regulatory Factors; Kidney Neoplasms; M Phase Cell Cycle Checkpoints; Male; Prognosis; Promoter Regions, Genetic

To study the effect of histone deacetylase inhibitors trichostatin A (TSA) and LBH589 on the growth of human renal cell carcinoma OS-RC-2 cells in vitro and explore the underlying molecular mechanism.. OS-RC-2 cells were treated with LBH589 or TSA with or without SP600125 pretreatment, and the cell viability was measured by MTT assay. The changes of cell cycle distribution and apoptosis of OS-RC-2 cells were examined by flow cytometry, and the expressions of c-Jun, p-c-Jun, Bcl-2, and Bax were quantified by Western blotting.. TSA and LBH589 both inhibited the growth of OS-RC-2 cells in a dose- and time-dependent manner. TSA at 1 µnmol/L and LBH589 at 50 nmol/L caused obvious cell cycle arrest in G2/M phase and cell apoptosis, and significantly increased the protein levels of phosphorylated c-Jun. TSA treatment obviously increased Bax expression but decreased Bcl2 expression in the cells. The growth inhibitory effect of TSA was attenuated by the JNK inhibitor SP600125 in OS-RC-2 cells. TSA-induced phosphorylation of c-Jun and Bax upregulation was partially counteracted by SP600125.. TSA and LBH589 can cause cell cycle arrest and induce apoptosis in OS-RC-2 cells, in which process P-JNK pathway plays an important role.

Topics: Anthracenes; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Renal Cell; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; JNK Mitogen-Activated Protein Kinases; Kidney Neoplasms; MAP Kinase Signaling System; Panobinostat; Phosphorylation; Proto-Oncogene Proteins c-bcl-2

The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5-Aza-2'-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5-Aza-2'-deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T-cell factor/lymphoid enhancer factor activity and nuclear beta-catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c-Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta-catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Azacitidine; Blotting, Western; Carcinoma, Renal Cell; Caspase 3; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin-Dependent Kinase Inhibitor p21; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Male; Promoter Regions, Genetic; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Wnt Proteins

Wnt/beta-catenin signaling is involved in renal cancer. DKK2, a Wnt antagonist, is silenced in some cancers, although its function has not been investigated. We hypothesized that DKK2 may be epigenetically silenced and inhibits progression of renal cell carcinoma (RCC).. RCC cell lines and a normal kidney cell line were used for methylation and chromatin immunoprecipitation assays. To assess various functions of DKK2, we established stable DKK2-transfected cells and examined them with regard to cell viability, colony formation, apoptosis, cell cycle, and invasive capability. A total of 52 patients with confirmed conventional RCC were enrolled in this study.. RCC cell lines had decreased levels of DKK2, which were significantly increased after treatment with 5-Aza-2'-deoxycytidine alone or 5-Aza-2'-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the levels of acetyl H3, acetyl H4, and dimethylated H3K4 were decreased, whereas the level of dimethylated H3K9 was increased in RCC cell lines compared with HK2 cells. Increased methylation in RCC tissues was associated with higher grades, pathologic stages, and pathologic tumor in RCC. Functional analysis showed that the numbers of viable A498 cells were significantly decreased in DKK2-transfected cells compared with mock cells. The number of apoptotic cells and S/G(2)-M phase cells was significantly increased and decreased after DKK2 transfection, respectively. Corresponding to these results, Bcl2 and cyclin D1 expression were also decreased in DKK2-overexpressing cells.. DKK2 is epigenetically silenced by methylation in higher grades and stages of RCC. These results suggest that DKK2 inhibits renal cancer progression through apoptotic and cell cycle pathways.

Topics: Adult; Aged; Apoptosis; Azacitidine; Carcinoma, Renal Cell; Cell Cycle; Chromatin Immunoprecipitation; Decitabine; Disease Progression; Female; Gene Silencing; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Male; Middle Aged; Wnt Proteins

Secreted frizzled-related protein 2 (sFRP2) is a negative modulator of the Wingless-type (Wnt) signaling pathway, and shown to be inactivated in renal cell carcinoma (RCC). However, the molecular mechanism of silencing of sFRP2 is not fully understood. Our study was designed to elucidate the silencing mechanism of sFRP2 in RCC. Expression of sFRP2 was examined in 20 pairs of primary cancers by immunohistochemistry. Kidney cell lines (HK-2, Caki-1, Caki-2, A-498 and ACHN) were analyzed for sFRP2 expression using real-time RT-PCR and Western blotting. The methylation status at 46 CpG sites of the 2 CpG islands in the sFRP2 promoter was characterized by bisulfite DNA sequencing. Histone modifications were assessed by chromatin immunoprecipitation (ChIP) assay using antibodies against AcH3, AcH4, H3K4 and H3K9. sFRP2 was frequently repressed in primary cancers and in RCC cells. The majority of sFRP2 negative cells had a methylated promoter. Meanwhile, sFRP2 expression was repressed by a hypomethylated promoter in Caki-1 cells, and these cells had a repressive histone modification at the promoter. In Caki-1 cells, sFRP2 was reactivated by trichostatin A (TSA). Repressive histone modifications were also observed in RCC cells with hypermethylated promoters, but sFRP2 was reactivated only by 5-aza-2'-deoxycytidine (DAC) and not by TSA. However, the activation of the silenced sFRP2 gene could be achieved in all cells using a combination of DAC and TSA. This is the first report indicating that aberrant DNA methylation and histone modifications work together to silence the sFRP2 gene in RCC cells.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Blotting, Western; Carcinoma, Renal Cell; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Immunoprecipitation; Kidney Neoplasms; Membrane Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Wnt Proteins

Aberrant promoter hypermethylation is a common mechanism for inactivation of tumor suppressor genes in cancer cells. To generate a global profile of genes silenced by hypermethylation in renal cell cancer (RCC), we did an expression microarray-based analysis of genes reactivated in the 786-0, ACHN, HRC51, and HRC59 RCC lines after treatment with the demethylating drug 5-aza-2 deoxycytidine and histone deacetylation inhibiting drug trichostatin A. Between 111 to 170 genes were found to have at least 3-fold up-regulation of expression after treatment in each cell line. To establish the specificity of the screen for identification of genes, epigenetically silenced in cancer cells, we validated a subset of 12 up-regulated genes. Three genes (IGFBP1, IGFBP3, and COL1A1) showed promoter methylation in tumor DNA but were unmethylated in normal cell DNA. One gene (GDF15) was methylated in normal cells but more densely methylated in tumor cells. One gene (PLAU) showed cancer cell-specific methylation that did not correlate well with expression status. The remaining seven genes had unmethylated promoters, although at least one of these genes (TGM2) may be regulated by RASSF1A, which was methylated in the RCC lines. Thus, we were able to show that up-regulation of at least 6 of the 12 genes examined was due to epigenetic reactivation. The IGFBP1, IGFBP3, and COL1A1 gene promoter regions were found to be frequently methylated in primary renal cell tumors, and further study will provide insight into the biology of the disease and facilitate translational studies in renal cancer.

Topics: Adult; Aged; Azacitidine; Base Sequence; Carcinoma, Renal Cell; Cell Line, Tumor; Collagen Type I; Collagen Type I, alpha 1 Chain; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hydroxamic Acids; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 3; Kidney Neoplasms; Male; Middle Aged; Molecular Sequence Data; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Up-Regulation

Therapy for advanced renal cell carcinoma (RCC) is ineffective in the majority of patients. We have previously reported that retinoid-induced up-regulation of retinoic acid receptor beta (RARbeta) correlated with antitumor effects in RCCs. Recent studies show that there is a reduction in the level of RARbeta2 expression in cancer cells due in part to histone hypoacetylation. Therefore, we tested whether combining histone deacetylase inhibitors with retinoic acid (RA) would restore RARbeta2 receptor expression, leading to increased growth inhibition in RCC cells.. Cell proliferation, Western blot, and reverse transcription-PCR analyses of two RA-resistant RCC cell lines, SK-RC-39 and SK-RC-45, were assessed in the presence of all-trans retinoic acid (ATRA), trichostatin A (TSA), or the combination of ATRA and TSA. Analysis of apoptosis was also done on SK-RC-39 cells treated with these combinations. Additionally, a xenograft tumor model (SK-RC-39) was used in this study to investigate the efficacy of a liposome-encapsulated, i.v. form of ATRA (ATRA-IV) plus TSA combination therapy.. Enhanced inhibition of the proliferation of RCC cell lines and of tumor growth in a xenograft model was observed with the combination of ATRA plus TSA. Reactivation of RARbeta2 mRNA expression was observed in SK-RC-39 and SK-RC-45 cells treated with TSA alone or TSA in combination with ATRA. A partial G0-G1 arrest and increased apoptosis were observed with SK-RC-39 cells on treatment with ATRA and TSA.. The combination of ATRA and the histone deacetylase inhibitor TSA elicits an additive inhibition of cell proliferation in RCC cell lines. These results indicate that ATRA and histone deacetylase inhibitor therapies should be explored for the treatment of advanced RCC.

Topics: Acetylation; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Kidney Neoplasms; Mice; Mice, Nude; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Xenograft Model Antitumor Assays

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.

Topics: Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Kidney Neoplasms; Liver Neoplasms; Nasopharyngeal Neoplasms; Neoplasms; Oligonucleotide Array Sequence Analysis; Tumor Cells, Cultured; Vorinostat

We investigated the in vitro effect of trichostatin (histone deacetylase inhibitor) on cell proliferation, cell cycle regulation and apoptosis in renal cell carcinoma cell lines. Trichostatin significantly inhibited the proliferation of all six cell lines examined in dose-dependent manner with IC50 of about 125-250 nM. Trichostatin (72-h incubation) induced a G1 phase arrest in ACHN, Caki-1, Caki-2 and Renca cell lines and a G2-M phase arrest in A498 cells. When we examined the effects of this drug on ACHN cells, trichostatin decreased the levels of CDK4, CDK6, cyclin D1 and cyclin A proteins. p27 protein was increased by trichostatin. In addition, trichostatin markedly enhanced the binding of p27 with CDK2 and CDK4. Furthermore, the activities of CDK2, CDK4- and CDK6-associated kinase were reduced and the lack of the CDK activity was paralleled by increased hypophosphorylation of Rb protein. Trichostatin also induced apoptosis in all the renal cell carcinoma cell lines. Apoptotic process of ACHN cells was associated with the changes of Bcl-2, caspase-9, caspase-3, caspase-7 proteins as well as mitochondria transmembrane potential (deltapsim) loss. Taken together, these results demonstrate that trichostatin inhibits the growth of renal cell carcinoma cells via cell cycle arrest or apoptosis.

Topics: Apoptosis; Carcinoma, Renal Cell; Cell Cycle; Cell Division; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kidney Neoplasms; Microfilament Proteins; Muscle Proteins; Tumor Cells, Cultured