Abstract
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.
Highlights
Prostate cancer (PCa) is one of the most commonly diagnosed malignant cancer in men, with 220,800 (26%) incident cases in US alone. 27,540 PCa cases was reported in the United States in 2015, and it was predicted as the second leading cause of cancer deaths among men [1]
NPPB inhibited cell survival and proliferation in DU145 cells The cytotoxic effect of NPPB on human prostate cancer DU145 cells was examined with varying concentrations of NPPB and times by MTT assay
We evaluated the proliferation inhibition and apoptosis promotion effects of chloride channel blocker NPPB in human prostate cancer DU145 cells, along its mechanism of action
Summary
Prostate cancer (PCa) is one of the most commonly diagnosed malignant cancer in men, with 220,800 (26%) incident cases in US alone. 27,540 PCa cases was reported in the United States in 2015, and it was predicted as the second leading cause of cancer deaths among men [1]. Prostate cancer (PCa) is one of the most commonly diagnosed malignant cancer in men, with 220,800 (26%) incident cases in US alone. 27,540 PCa cases was reported in the United States in 2015, and it was predicted as the second leading cause of cancer deaths among men [1]. Overexpression of anti-apoptotic oncoprotein Bcl-2 in androgendependent prostate cancer epithelial cells resulted in the increase of swelling-activated Clcurrent and the enhancement of endogenous expression of ClC-3 protein [4]. It prompted a potential mechanism between ClC channel and apoptotic mediation of prostate cancer
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