| Objective: In recent years, many studies have found that DNA methy-lation isone of the important mechanisms of cancerdevelopment.DNA methylation is oftenoccurred in a tumor-suppressor gene promoter region (CpG island). By inhibiting thesilencing tumor suppressor gene expression promoter function, it can lead totumorigenesis. To explore the methylation status of the secreted frizzled-relatedprotein-1(SFRP-1) gene promoter and the potential demethylation of this promoter witharsenic trioxide treatment in androgen-independent prostate cancer PC-3cells.Methods: Cholecystokinin-8(CCK8) was utilized to examine the inhibitory roleof arsenic trioxide on the proliferation of PC-3cells, and flow cytometry was employedto investigate the effect of arsenic trioxide on apoptosis in PC-3cells. Bisulfitesequencing was used to assess the methylation status of CpG islands in the SFRP-1gene promoter in PC-3cells before and after arsenic trioxide treatments. Quantitativereal-time PCR (RT-PCR) was performed to measure SFRP-1mRNA expression levelsin PC-3cells before and after arsenic trioxide treatments. Immunofluorescence andWestern blot analyses were utilized to quantify the protein expression levels of SFRP-1.Results: CCK8detection results showed that arsenic trioxide can signify-cantlyinhibit prostate cancer PC-3cell proliferation, in a time-and dose-dependent manner (P<0.05); Flow cytometry results showed that the function on PC-3cell withconcentration of arsenic trioxide for (0,1,2.5,5,10,20) μmol／Ls after24h and48h,apoptosis rate is respectively:(5.5±0.87,8.8±1.43,15.5±2.72,26.9±1.63,36.7±2.12and55.8±3.87)%;(7.9±1.32,11.2±1.56,7.9±2.84,38.7±3.56,57.2±2.53,70.9±5.04)%, with significant difference (P <0.05), suggesting that arsenic trioxide can inducesPC-3cells apoptosis, with the increase of arsenic trioxide concentration and action time,apoptosis rate increased significantly; BSP analyses revealed that both CpG islands inthe SFRP-1promoter were methylated. In particular, the methylation levels of the first and second CpG islands of this promoter were85%and71%, respectively, prior toarsenic trioxide treatment and were reduced to54.2%and69.8%,; Immunofluorescenceanalyses demonstrated that the SFRP-1protein expression levels were low in the normalcontrol group of PC-3cells; by contrast, these expression levels were significantlyincreased in PC-3cells following treatment with10μmol/L arsenic trioxide for24h (P<0.05); Quantitative RT-PCR results revealed that at24h after arsenic trioxideintervention, increased SFRP-1mRNA expression levels were observed in cells in theintervention group relative to cells in the control group. Increasing SFRP-1mRNAexpression levels were observed as the arsenic trioxide concentration increased; athigher arsenic trioxide concentrations (of2.5,5, or10μmol/L), SFRP-1mRNAexpression levels were significantly higher in the intervention groups than in theuntreated groups (P <0.05); Western blot results indicated that SFRP-1proteinexpression levels were low in normal control cells. Arsenic trioxide treatment increasedSFRP-1protein expression levels in a dosage-dependent manner. These expressionlevels was observed between the control group and each of the remaining interventiongroups (P <0.05).Conclusion: In PC-3prostate cancer cells, CpG islands in the SFRP-1genepromoter are methylated, and arsenic trioxide reduced methylation and increasedSFRP1gene expression. Arsenic trioxide induces apoptosis of prostate cancer may beinstead of SFRP-1gene methylation. |
PDF Full Text Request