Figure 3.

ROS are involved in apoptosis by WA in MCF-7 cells. (A) Effect of overexpression of Mn-SOD on WA-mediated activation of MAPK. The MCF-7 cells stably transfected with pcDNA3.1 empty vector or pcDNA3.1 vector encoding for Mn-SOD were treated with the desired concentrations of WA for 2 hours. The total lysates from these cells were subjected to western blotting for phosphorylated MAPK. Quantitation relative to DMSO-treated empty vector transfected cells is shown. (B) Detection of apoptosis (histone-associated DNA fragment release into the cytosol) in MCF-7 cells stably transfected with pcDNA3.1 empty vector or pcDNA3.1 vector encoding for Mn-SOD. The cells were treated with the indicated doses of WA for 24 h and then processed for apoptosis assay. Statistical significance (P<0.05) was analyzed one-way ANOVA with Bonferroni’s multiple comparison test. ^aSignificantly different (P<0.05) compared with each control. ^bSignificantly different (P<0.05) between groups with the same dose. The results shown are mean ± S.D (n= 3). (C) Effect of NAC on WA-mediated activation of ERK. MCF-7 cells (5×10⁵ cells per dish) were plated in 6-cm dish and allowed to attach. After overnight incubation, the cells were pretreated with 5 mM NAC for 2 h and then treated with indicated doses of WA in the absence or presence of NAC for 2 h. After treatment, the cells were collected and processed for immunoblotting. (D) Effect of NAC on WA-mediated apoptosis induction in MCF-7 cells. The cells were pretreated with 5 mM NAC for 2 h and then treated with 2.5 μM WA in the absence or presence of NAC for 24 h. Statistical significance (P<0.05) was analyzed by one-way ANOVA with Bonferroni’s multiple comparison test. ^aSignificantly different (P<0.05) compared with each control. ^bSignificantly different (P<0.05) between groups with the same dose. The results shown are mean ± S.D. (n= 2–3).