Effects of DNMT and MEK inhibitors on the expression of RECK, MMP-9, -2, uPA and VEGF in response to arsenite stimulation in human uroepithelial cells

Abstract

It has been shown that the ingestion of arsenic-contaminated drinking water is closely correlated with risk of several cancers. The mechanism of arsenic-induced carcinogenesis is still unclear. The RECK, MMP-9, -2, uPA and VEGF are the most common dysregulation in human tumors and cancer cell lines. However, the effect of arsenite on these markers expression and the molecular mechanism are still unclear. The purpose of the study was to investigate the relationship between the expression of RECK, MMP-9, -2, uPA and VEGF in arsenite-treated human and rat uroepithelial cells. In addition, we also observed and compared the expression of these markers in urothelial carcinoma (UC) from Blackfoot disease (BFD) areas and non-Blackfoot disease (non-BFD) areas. We analyzed the arsenite causing cell proliferation, RECK, MMP-9, -2, uPA and VEGF expression by Western blotting, immunocytochemistry (ICC), RT-PCR, and gelatin zymography. We demonstrated the effect of arsenite on methylation status of RECK promoter as determined by using methylation-specific PCR (MSP). Our results show that arsenite downregulation of RECK is caused by epigenetic inactivation via promoter hypermethylation, and that levels of MMP-9, -2, uPA and VEGF were increased in human uroepithelial cells (SV-HUC-1). However, when the cells were pretreated with inhibitors (5-aza-CdR or U0126) for 24 h, the effects of arsenite on RECK, MMP-9, -2, uPA and VEGF expression were suppressed. Indeed, we also found significant differences between the expression of RECK, MMP-9, -2, uPA and VEGF in UC from the BFD areas and non-BFD areas ( p = 0.006, 0.007, 0.003, <0.001 and 0.001 respectively), as detected by immunohistochemistry (IHC). In in vivo study, our results showed the RECK protein expression was reduced and the expression of MMP-9, -2, uPA and VEGF increased in arsenite treatment groups. In conclusion, our results support the notion that arsenite might cause the histologic changes, RECK, MMP-9, -2, uPA and VEGF dysregulation through epigenetic inactivation and ERK1/2 activation in SV-HUC-1 cells. These findings may provide a better understanding of the urothelial carcinogenesis of arsenite.