RESUMO
BACKGROUND: Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Tumor suppressor genes retinoic acid receptor beta (RARbeta) and PDLIM4 are hypermethylated and silenced in prostate cancer (PCa) tissues and PCa cell lines compared to normal prostate cells. METHODS: In this study, a benign prostate epithelial cell line RWPE1 was used as a model to study the epigenetic regulation of Myc on the RARbeta and PDLIM4 promoters. Forced Myc overexpression inhibited the RARbeta and PDLIM4 expression. RESULTS: Pyrosequencing study showed that Myc overexpression increased methylation in several CpG sites of both promoters. A DNA methylation inhibitor 5-aza-2'-deoxycytidine reversed the epigenetic alteration effect of Myc on both RARbeta and PDLIM4. CONCLUSION: The epigenetic regulation of Myc may be related to its up-regulation of the DNA methyltransferase DNMT3a and DNMT3b.
Assuntos
Proteínas de Ligação a DNA/genética , Genes myc , Próstata/fisiologia , Receptores do Ácido Retinoico/genética , Western Blotting , Processos de Crescimento Celular/genética , Linhagem Celular , DNA/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Epigênese Genética , Perfilação da Expressão Gênica , Humanos , Proteínas com Domínio LIM , Masculino , Reação em Cadeia da Polimerase , Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Análise de Sequência de DNA , Transfecção , DNA Metiltransferase 3BRESUMO
We investigated the molecular function of PDLIM4 in prostate cancer cells. PDLIM4 mRNA and protein-expression levels were reduced in LNCaP, LAPC4, DU145, CWR22, and PC3 prostate cancer cells. The re-expression of PDLIM4 in prostate cancer cells has significantly reduced the cell growth and clonogenicity with G1 phase of cell-cycle arrest. We have shown the direct interaction of PDLIM4 with F-actin. Restoration of PDLIM4 expression resulted in reduction of tumor growth in xenografts. These results suggest that PDLIM4 may function as a tumor suppressor, involved in the control of cell proliferation by associating with actin in prostate cancer cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Neoplasias da Próstata/prevenção & controle , Proteínas Supressoras de Tumor/fisiologia , Actinas/análise , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Humanos , Proteínas com Domínio LIM , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos , Neoplasias da Próstata/patologiaRESUMO
To identify the relevant CpG sites as molecular markers, for the diagnosis and to distinguish the indolent and aggressive prostate tumors, we have determined the methylation status of 8 genes, including FLNC, EFS, ECRG4, RARB2, PITX2, GSTP1, PDLIM4, and KCNMA1 in 32 nonrecurrent, 32 recurrent primary prostate tumors, and 32 benign prostate tissues using EpiTYPER technology. Specific CpG site hypermethylation of RARB2 and GSTP1 CpG sites were useful for diagnosis of prostate cancer. Furthermore, CpG site hypermethylation of genes FLNC, EFS, ECRG4, PITX2, PDLIM4, and KCNMA1 were associated with prediction of biochemical, local, and systemic recurrence of prostate cancer.
Assuntos
Biomarcadores Tumorais/genética , Ilhas de CpG , Metilação de DNA , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/genética , Reação em Cadeia da Polimerase , Hiperplasia Prostática/diagnóstico , Hiperplasia Prostática/genética , Fatores de Risco , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
This study was to investigate the effect of phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, on the expression of alpha- and beta-tubulins, which are the main components of microtubules in prostate cancer cells. Flow cytometry, light microscopy and western blot were used to study the cell cycle distribution, morphology changes and the expression of alpha- and beta-tubulins in prostate cancer cells treated with PEITC. The results showed that PEITC-induced G2-M cell phase arrest and inhibited the expression of alpha- and beta-tubulin proteins in a number of human prostatic carcinoma cell lines. Further, it is showed that this inhibitory effect could be reversed by antioxidant N-acetyl cysteine and proteasome inhibitor MG132. Finally, it is concluded that PEITC inhibited the expression of alpha- and beta-tubulins in prostate cancer cells, which is at least related to the oxygen reaction species and protein degradation.
Assuntos
Anticarcinógenos/farmacologia , Ciclo Celular/efeitos dos fármacos , Isotiocianatos/farmacologia , Neoplasias da Próstata/metabolismo , Tubulina (Proteína)/metabolismo , Antioxidantes/farmacologia , Western Blotting , Linhagem Celular Tumoral , Cisteína/farmacologia , Citometria de Fluxo , Fase G2/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Masculino , Isoformas de Proteínas/metabolismoRESUMO
PURPOSE: We analyzed the expression of genes to identify reliable molecular markers in the diagnosis and progression of prostate cancer. EXPERIMENTAL DESIGN: Gene expression profiling was done using HG-U133 set microarrays in 32 prostate cancer and 8 benign tissues of patients with cancer. Expression levels of 11 genes were selected for quantitative real-time PCR evaluation in 52 prostate cancer and 20 benign tissues. Further, to assess transcriptional inactivation, we analyzed the promoter methylation of genes by quantitative methylation-specific PCR in 62 tumor and 36 benign tissues. RESULTS: Our results showed a significant down-regulation in the mRNA expression levels of PRIMA1, TU3A, PDLIM4, FLJ14084, SVIL, SORBS1, C21orf63, and KIAA1210 and up-regulation of FABP5, SOX4, and MLP in prostate cancer tissues by TaqMan real-time PCR. Quantitative methylation-specific PCR of PDLIM4, SVIL, PRIMA1, GSTP1, and PTGS2 detected prostate carcinoma with a sensitivity of 94.7%, 75.4%, 47.4%, 89.5%, and 87.7%, and a specificity of 90.5%, 75%, 54.2%, 95.8%, and 90.2%, respectively. Using this panel of methylation markers in combination, we were able to distinguish between prostate cancer and adjacent benign tissues with sensitivities and specificities of about 90% to 100%. Our data provide evidence of transcriptional repression of the putative tumor suppressor gene PDLIM4 by hypermethylation. CONCLUSIONS: Our analysis revealed differential expression of eight down-regulated and three up-regulated genes, implicating their role in prostate cancer development and progression. We further showed that the hypermethylation of PDLIM4 gene could be used as a sensitive molecular tool in detection of prostate tumorigenesis.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Neoplasias da Próstata/patologia , Idoso , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa S-Transferase pi/genética , Humanos , Proteínas com Domínio LIM , Masculino , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase/métodos , Neoplasias da Próstata/genéticaRESUMO
We profiled the expression of genes in benign and untreated human prostate cancer tissues using oligonucleotide microarrays. We report here 50 genes with distinct expression patterns in metastatic and confined tumors (Gleason score 6 and 9; lymph node invasive and noninvasive). Validation of expression profiles of 6 genes by quantitative PCR revealed a strong inverse correlation in the expression of zinc finger protein 185 (ZNF185), bullous pemphigoid antigen gene (BPAG1), and prostate secretory protein (PSP94) with progression of prostate cancer. Treatment of prostate cancer cell lines with 5-aza-2'-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, restored ZNF185 expression levels. Moreover, methylation-specific PCR confirmed methylation of the 5'CpG islands of the ZNF185 gene in all of the metastatic tissues and 44% of the localized tumor tissues, as well as in the prostate cancer cell lines tested. Thus, transcriptional silencing of ZNF185 by methylation in prostate tumor tissues implicates the ZNF185 gene in prostate tumorigenesis.
Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso , Colágenos não Fibrilares , Neoplasias da Próstata/genética , Idoso , Autoantígenos/biossíntese , Autoantígenos/genética , Colágeno/biossíntese , Colágeno/genética , Metilação de DNA , Proteínas de Ligação a DNA/biossíntese , Progressão da Doença , Regulação para Baixo , Distonina , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Proteínas com Domínio LIM , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Secretadas pela Próstata/biossíntese , Proteínas Secretadas pela Próstata/genética , Transcrição Gênica , Colágeno Tipo XVIIRESUMO
In the ligand-binding inactive state, the steroid receptor heterocomplex contains Hsp90, Hsp70, high-molecular weight immunophilins, and other proteins. Hsp90 acts in association with co-chaperones to maintain the native state of the receptor within the cells. It was reported earlier that Hsp90 might not be as important for the androgen receptor (AR) activity as for the glucocorticoid receptor (GR) and the progesterone receptor (PR) activities. We used the Hsp90 inhibitor geldanamycin (GA) to explore the role of Hsp90 in the function of the AR heterocomplex. GA selectively binds to Hsp90 and inhibits its activity, leading to the loss of steroid receptor activity, and frequently, its degradation. In our study, LNCaP prostate cancer cells were treated with GA for 30 minutes or 24 hours, in the presence of mibolerone, a synthetic androgen. GA reduced the androgen-induced AR protein levels to 15% after 24 hours of treatment. Several androgen up-regulated genes, including immunophilin FKBP51 and prostate specific antigen (PSA), were reduced by GA treatment. In cells treated with GA after transfection with a PSA promoter or an androgen response element-driven reporter gene, AR-mediated transactivation of reporter gene expression was reversibly inhibited by GA. Loss of androgen-binding ability and AR levels was attributed to reduced transcription of AR-regulated gene expression. Degradation rate of 35S-labeled AR was significantly increased by GA in the presence or absence of mibolerone. GA induced the degradation of AR through the proteasomal pathway. AR in cells treated with proteasomal inhibitor lactacystin, was insoluble in Nonidet P-40 (NP40)-based buffer and could not restore the androgen-binding ability. We report here that GA treatment disrupted both hormone-binding activity and receptor protein stability, resulting in a dramatic loss of androgen-induced gene activation. These results show that Hsp90 activity is important for both the chaperone-mediated folding of the AR into a high-affinity ligand-binding conformation and the functional activity of the AR.