trichostatin-a and Prostatic-Hyperplasia

Prostate cancer is the second most common malignancy in men worldwide. Abnormal epigenetic alterations such as DNA methylation and histone modification play an important role in tumor initiation, progression and regulation of cancer-related genes such as integrin α4 and E-cadherin. Expression of these genes was determined by semi-quantitative reverse transcriptase-PCR in prostate cancer cell lines, DU145 and PC3, before and after treatment with 5-aza-2-deoxycytidine and trichostatin A. Laser capture microdissection microscopy was used to obtain exclusively affected epithelial cells from prostate gland biopsies of 30 patients with prostate cancer and 40 with benign prostate hyperplasia. DNA bisulfite modifications followed by methylation-specific PCR were used to evaluate the promoter methylation status of E-cadherin and α4 integrin genes in extracted DNA from patients and aforementioned cell lines. The integrin α4 promoter in DU145 was fully methylated, whereas in PC3 cells, partial methylation was detected. E-cadherin was expressed in both cell lines; trichostatin A and 5-aza-2-deoxycytidine treatment had no effect on E-cadherin expression, however the combined treatment of both drugs or 5-aza-2-deoxycytidine alone increased integrin α4 expression. Integrin α4 and E-cadherin were hypermethylated in 66.6 % and 6.6 % of prostate cancer cases, respectively; no hypermethylation was observed in patients with benign prostate hyperplasia. These results together suggest that aberrant DNA methylation is one of the mechanisms involved in integrin α4 expression and may play an important role in human prostate carcinogenesis. In addition, the higher rate of integrin α4 gene methylation in prostate cancer patients elects it as a potential molecular tumor marker.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Biomarkers, Tumor; Cadherins; CpG Islands; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Integrin alpha4; Male; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction.. Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2β1integrin(hi)/CD133(+)), transit-amplifying (TA, α2β1integrin(hi)/CD133(-)) and committed basal (CB, α2β1integrin(lo)) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser.. Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation.. Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.

Topics: Aged; Aged, 80 and over; Cell Differentiation; Comet Assay; DNA Damage; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Middle Aged; Neoplastic Stem Cells; Prostatic Hyperplasia; Prostatic Neoplasms; Radiation-Sensitizing Agents; Tumor Cells, Cultured

Transcriptional silencing associated with aberrant promoter methylation has been established as an alternate pathway for the development of cancer by inactivating tumor suppressor genes. TMS1 (Target of Methylation induced Silencing), also known as ASC (Apoptosis Speck like protein containing a CARD) is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to promote apoptosis directly and by activation of downstream caspases. This study describes the methylation induced silencing of TMS1/ASC gene in prostate cancer cell lines. We also examined the prevalence of TMS1/ASC gene methylation in prostate cancer tissue samples in an effort to correlate race and clinico-pathological features with TMS1/ASC gene methylation.. Loss of TMS1/ASC gene expression associated with complete methylation of the promoter region was observed in LNCaP cells. Gene expression was restored by a demethylating agent, 5-aza-2'deoxycytidine, but not by a histone deacetylase inhibitor, Trichostatin A. Chromatin Immunoprecipitation (ChIP) assay showed enrichment of MBD3 (methyl binding domain protein 3) to a higher degree than commonly associated MBDs and MeCP2. We evaluated the methylation pattern in 66 prostate cancer and 34 benign prostatic hyperplasia tissue samples. TMS1/ASC gene methylation was more prevalent in prostate cancer cases than controls in White patients (OR 7.6, p 0.002) while no difference between the cases and controls was seen in Black patients (OR 1.1, p 0.91).. Our study demonstrates that methylation-mediated silencing of TMS1/ASC is a frequent event in prostate cancer, thus identifying a new potential diagnostic and prognostic marker for the treatment of the disease. Racial differences in TMS1/ASC methylation patterns implicate the probable role of molecular markers in determining in susceptibility to prostate cancer in different ethnic groups.

Topics: Aged; Aged, 80 and over; Azacitidine; CARD Signaling Adaptor Proteins; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; Cytoskeletal Proteins; Decitabine; DNA Methylation; DNA Modification Methylases; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Middle Aged; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction

Androgen and progesterone receptors (AR and PR) are two determining factors in gonadal differentiation that are highly expressed in developing and mature gonads. Loss of AR results in XY sex reversal and mutations causing reduced AR activity lead to varying degrees of defects in masculinization. Female PR knockout mice are infertile due to ovarian defects. While much has been discovered about positive regulation of these receptors by coactivators little is known about repression of the transcriptional activity of AR and PR in the presence of agonists. In this study we assessed the effect of SMRT and DAX-1 on AR and PR activity in the presence of both agonists and partial antagonists. We show that SMRT and DAX-1 repress agonist-dependent activity of both receptors, and the mechanism of repression includes disruption of the receptor dimer interactions rather than recruitment of histone deacetylases. We demonstrate that endogenous agonist-bound PR and DAX-1 in T47D breast cancer cells and endogenous AR and DAX-1 in LNCaP prostate cancer cells can be coimmunoprecipitated suggesting that the interaction is physiological. Surprisingly, although DAX-1 represses partial antagonist activity of AR, it was ineffective in repressing partial antagonist induced activity of PR. In contrast to most reported repressors, the expression of DAX-1 is restricted. We found that although DAX-1 is expressed in normal human prostate, its expression is strongly reduced in benign prostatic hyperplasia suggesting that DAX-1 plays a role in limiting AR activity in prostate.

Topics: Animals; Binding Sites; Breast Neoplasms; COS Cells; DAX-1 Orphan Nuclear Receptor; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Hormone Antagonists; Humans; Hydroxamic Acids; Male; Metribolone; Mifepristone; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Co-Repressor 2; Promoter Regions, Genetic; Prostate; Prostatic Hyperplasia; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Receptors, Androgen; Receptors, Calcitriol; Receptors, Interferon; Receptors, Progesterone; Receptors, Retinoic Acid; Repressor Proteins; Testosterone Congeners; Tumor Suppressor Protein p53

Transcriptional silencing of cancer-related genes by DNA methylation is observed in various cancers. To identify genes controlled by methylation in prostate cancer, we used cDNA microarray analysis to investigate gene expression in prostate cancer cell lines LNCaP and DU145 treated with a methyltransferase inhibitor alone or together with a histone deacetylase inhibitor. We detected significant changes (3.4-5.7%) in gene expression in prostate cancer cell lines with the drug treatments. Among the affected genes, that for the vascular endothelial growth factor receptor 1 (VEGFR-1) was re-expressed in LNCaP and DU145 after the drug treatments. Bisulfite sequencing revealed the promoter and exon 1 of the VEGFR-1 to be hypermethylated in the cell lines. These results support the idea that methylation is associated with loss of VEGFR-1 mRNA expression in prostate cancer cell lines. Combined bisulfite restriction analysis (COBRA) showed the gene to be methylated in 24 (38.1%) of 63 primary local prostate cancer samples, while in all 13 benign prostate samples it was not. These findings indicate that methylation of VEGFR-1 is related with prostatic carcinogenesis.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Sulfites; Tumor Cells, Cultured; Vascular Endothelial Growth Factor Receptor-1

Expression of the KAI1 gene, a metastasis-suppressor for prostate cancer, is reduced in all foci of prostatic metastasis. The altered regulatory mechanism is not strongly related to mutations or allelic losses of the KAI1 gene in prostate tumors. Since transcriptional silencing of genes has been found to be caused by epigenetic mechanisms, we have investigated the involvement of this epigenetic regulation of KAI1 expression in prostate cancers. The methylation status of the KAI1 promoter region was examined by restriction-enzyme digestion and sequencing, after amplifying a 331-bp fragment in the GC-rich promoter region from 4 human prostate cancer cell lines treated with bisulfite. The same 4 cell lines were also exposed to various concentrations of the demethylating agent, 5-aza-2'-deoxycytidine (5-AzaC) and / or the histone deacetylase inhibitor, trichostatin A (TSA). To clarify the influence of epigenetic modification on reduced KAI1 mRNA expression in the tumor cells, RT-PCR and northern-blot analyses were performed. Bisulfite-sequencing data showed a few methylated CpG islands in the promoter. RT-PCR analysis of 5-AzaC and / or TSA-treated cells indicated reversal of suppression of KAI1 transcription in two cell lines (PC-3 and DU-145), although the expression could not be detected by northern blots. From these results, it is suggested that epigenetic change is not the main mechanism of KAI1 down-regulation, though there remains a possibility that methylation in a more upstream region might be associated with this regulation.

Topics: Adenocarcinoma; Antigens, CD; Azacitidine; Base Sequence; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kangai-1 Protein; Male; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Proto-Oncogene Proteins; Sulfites; Tumor Cells, Cultured