JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells

Abstract

Reactive oxygen species (ROS) are chemical species that alter redox status, and are responsible for inducing carcinogenesis. The purpose of the present study was to assess the effects of the glutathione S transferase-activated nitric oxide donor prodrug, JS-K, on ROS accumulation and on proliferation and apoptosis in human prostate cancer cells. Cell proliferation and apoptosis, ROS accumulation and the activation of the mitochondrial signaling pathway were measured. The results demonstrated that JS-K may inhibit prostate cancer cell growth in a dose- and time-dependent manner, and induce ROS accumulation and apoptosis in a dose-dependent manner. With increasing concentrations of JS-K, expression of pro-apoptotic proteins increased, but Bcl-2 expression decreased. Additionally, the antioxidant N-acetylcysteine reversed JS-K-induced cell apoptosis; conversely, the pro-oxidant glutathione disulfide exacerbated JS-K-induced apoptosis. In conclusion, the data suggest that JS-K induces prostate cancer cell apoptosis by increasing ROS levels.

Keywords: JS-K; apoptosis; nitric oxide; prostate cancer cells; reactive oxygen species.

Figure 1.

Inhibition of cell proliferation of prostate cancer cells following JS-K treatment. Cells were exposed to various concentrations of JS-K (1, 2, 5, 10 or 20 µM) for 12, 24 or 48 h, and the inhibition rate of cells without JS-K treatment was defined as 0%. Each sample was duplicated, and the figures present three independent assays (n=6). Results are presented as mean ± standard deviation.

Figure 2.

JS-K-induced apoptosis in prostate cancer cells, analyzed by flow cytometry with the Annexin V staining method. Untreated cells were analyzed as the control group. PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 3.

Effect of JS-K on ROS, RNS, GSH/GSSG ratio, mitochondrial membrane potential, ATP production and apoptotic-associated proteins in prostate cancer cells. (A) Prostate cancer cells were treated with JS-K (0, 1, 2 or 5 µM) for 6 h prior to assessment of the intracellular levels of (a) total ROS, (b) RNS and (c) superoxide, and (d) the GSH/GSSG ratio. (B) Following 6 h JS-K treatment, (a) the mitochondrial membrane potential and (b) ATP production were measured. (C) The levels of BAK, Bax, Bcl-2, caspase-9 and PARP proteins were detected by western blotting following JS-K (0, 1, 2 or 5 µM) treatment for 24 h. Results are presented as mean ± standard deviation for at least three independent assays. *P<0.05 and **P<0.01 vs. the corresponding untreated group (0 µM). ROS, reactive oxygen species; RNS, reactive nitrogen species; GSH, glutathione; GSSG, glutathione disulfide; ATP, adenosine triphosphate; PARP, poly ADP ribose polymerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4.

Effects of NAC and GSSG on JS-K-induced cell growth suppression and apoptosis in prostate cancer cells. (A) Cells were cultured with 100 µM NAC or 5 µM GSSG for 24 h, and then treated with or without 5 µM JS-K for 24 h. Cell survival was measured by cell counting kit-8 assay. (B) Cells were pretreated with 100 µM NAC or 5 µM GSSG for 24 h, treated with or without 5 µM JS-K for 6 h and ROS production was measured. (C) Cell apoptosis was analyzed in the indicated groups. The mean ± standard deviation is reported for at least three independent experiments. *P<0.05; **P<0.01 vs. specified group. NAC, N-acetylcysteine; GSSG, glutathione disulfide; ROS, reactive oxygen species.