5-Aza-2′-Deoxycytidine Enhances Maspin Expression and Inhibits Proliferation, Migration, and Invasion of the Bladder Cancer T24 Cell Line

Background: Downregulation of maspin expression has been linked to bladder cancer development, and that DNA methylation may be important for regulating maspin gene activation in bladder cancer cells. Thus, we attempted to explore the effects of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR), on the maspin expression and the biological behaviors in bladder cancer T24 cells. Method: The methylation status of maspin in T24 cells was investigated by methylation-specific polymerase chain reaction (PCR). After treated with different concentrations of 5-Aza-CdR (0, 0.25, 0.5, 1, and 2 lM), the maspin gene mRNA expression and protein expression were examined by real-time PCR and western blotting analysis. Cell proliferations were evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was used to identify the apoptosis rates. Migration and invasive ability were determined by the transwell assay. Using the western blotting analysis, the changes of Cyclin D1, VEGF-C, VEGFR-3, MMP-2, MMP-9, caspase-3 p17, Bax, and Bcl-2 expression were measured. Results: Promoter DNA methylation of maspin was observed in T24 cells. The expression levels of maspin mRNA and protein in T24 cells were increased in a dose manner after treatment with increasing 5-Aza-CdR (p < 0.05). The proliferation, migration, and invasion of cells were significantly inhibited with increasing 5-Aza-CdR, whereas the apoptosis was greatly increased (p < 0.05). These were associated with the decreased ratio of Bcl-2/Bax, activation of caspase-3, and decreased expression of Cyclin D1, VEGF-C, VEGFR-3, MMP-2 and MMP-9. Conclusions: The present study demonstrates that maspin is silenced by DNA methylation in bladder T24 cells, and its expression can be reactivated by treatment with 5-Aza-CdR. 5-Aza-CdR could result in obvious inhibitions of the proliferation, migration, and invasion of T24 cells, which may serve as a potential strategy for the treatment of bladder cancer.