The Specific Inhibition of SOD1 Selectively Promotes Apoptosis of Cancer Cells via Regulation of the ROS Signaling Network

Figure 5

The specific SOD1 inhibition activates mitochondria-dependent apoptosis of cancer cells. (a, b) RT-qPCR (a) and western blotting (b) examination of p53 mRNA levels and protein content in 50 (RT-qPCR) or 0-100 μM (western blotting) LD100-treated HeLa, DU145, and RWPE-1 cells. (c) The protein levels of p21 and p19 in HeLa and DU145 cells are evaluated by western blotting analysis, after the cells are incubated with varied concentrations of LD100 for 24 h. β-Actin is used as an internal control (b, c). (d) The protein levels of proto- and cleaved caspase-9 and -3 in HeLa cells are evaluated by western blotting analysis, after HeLa cells are incubated with varied concentrations of LD100 for 24 h. (e) The time dependence of the protein levels of proto- and cleaved caspase-9 is evaluated by western blotting analysis in 50 μM LD100-treated HeLa cells. (f, h) HeLa cells are incubated with varied concentrations of LD100 for 24 h prior to western blotting evaluation of Bcl-2 (f) and cleaved PARP1 (h) protein levels. (g) Rh123 fluorescence that is positively correlated with mitochondrial membrane potential is measured by flow cytometry in HeLa, DU145, and RWPE-1 cells after incubated with varied concentrations of LD100 for 24 h. (i) DNA fragmentation in HeLa cells is assayed using agarose gel electrophoresis after incubated with varied concentrations of LD100 for 24 h. (j) The peroxynitrite content in HeLa cells is presented by DHR123 fluorescence measured with flow cytometry after incubation for 24 h, respectively, with LD100 (0, 50, and 100 μM) and L-NMMA (0, 1 mM), a cell membrane-permeable competitive NOS inhibitor. (k) After incubation for 24 h, respectively, with LD100 (0, 100 μM) and L-NMMA (1 mM L-NMMA), flow cytometric analysis of HeLa cell apoptosis is carried out with Annexin V and propidium iodide staining. To obtain more significant results, 100 μM LD100 was used here. The relative intensity of protein bands () in the western blotting is quantified by using the ImageJ software and normalized through the negative control, respectively (b–g). Representative results from three independent experiments are shown (b–g and k). Data are mean of triplicate (ns: not statistically significant; ; unpaired Student’s -test), and all error bars are SD.

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