Jian-hua Zhang, Jun Liu, Yun-jian Liao, Nan-lin Xiang, You-wei Huang, Zhi-quan Bai, Yun Luo, Jie-ying Wu, Zeng-Wu, Xi Lin and Jin-ming Di
NPPB3-(4-5-nitro-2-(3-phenylpropylamino) benzoic acid, as one of the most commonly used chloride channel blockers, is very essential for chloride channel studies. However, the role of NPPB in mediating apoptosis is uncertain. The purpose of this study was to investigate the apoptosis mechanism of NPPB in human cancer DU145 cell lines. Cell viability was examined by MTT assay. JC-1 fluorescent probe was used for detecting l membrane potential. Cell apoptosis was determined by flow method (FCM). The expression of apoptosis-related protein Caspase-3, Cleaved caspase-3, PARP, Cleaved PARP, Bcl-2, Mcl-1, Bcl-xl, Bcl-w and Bax was detected by western blotting. Cell proliferation rate was remarkably inhibited 2 h after treating with NPPB(50,100 μmol/L) (p<0.01); Flow cytometry method analysis showed that the apoptosis rate in each treated group were significantly higher, especially for the promotion of early apoptosis rate effect was significantly (P<0.05); Images of JC-1 showed NPPB led to the decrease of intracellular mitochondrial membrane potential; Western blot results showed that caspase-3/PARP signaling pathway was activated, while the expression of cleaved caspase-3, cleaved PARP was significantly increased (P<0.01).PARP, as substrate of caspase-3, was significantly decreased (P<0.05).Bcl-2, Bcl-xl compared with the control group had no significant difference, and anti-apoptotic protein Mcl-1 (P<0.05) and Bcl-w (P<0.01) decreased significantly, pro-apoptotic protein Bax (P<0.01) were obviously upregulated. Our findings indicated that NPPB could increase apoptosis in DU145 cell lines via mitochondria-related apoptotic pathway, and following results presented that ClC-3 Chloride Channels involved in the process of NPPB induced DU145 cells apoptosis.
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