Figure 3.

CVA21-Induced Cell Death of Bladder Cancer Cell Lines

(A) Determination of levels of apoptosis in three bladder cancer cell lines left untreated or treated with Mitomycin-C, CVA21, or the combination at 24, 48, and 72 hr post-treatment by means of annexin-V- and 7AAD-based flow cytometry. Graphs represent pooled data from two independent experiments, and the statistical significance of the treatment group comparisons was analyzed using two-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (B) Representative dot plots recorded for TCCSUP cells following the different treatments at 24, 48, and 72 hr post-treatment are shown. (C) Apoptosis (quantified via caspase 3/7 activity) in three bladder cancer cell lines left untreated or treated with Mitomycin-C, CVA21, or the combination at 48 and 72 hr post-treatment is shown. Graphs represent pooled data from two independent experiments, and the statistical significance of the treatment group comparisons was analyzed using two-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Increase in percentage cell survival was seen in CVA21 and combination-treated cells with Z-VAD-FMK, apoptosis inhibitor, whereas the necroptosis inhibitor, necrostatin-1, failed to increase cell survival in bladder cancer cell lines treated with CVA21 alone or in combination with Mitomycin-C. Graphs represent pooled data from two independent experiments, and the statistical significance of the treatment group comparisons was analyzed using two-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (E) Western blot analysis demonstrating absence of RIP1 and RIP3 necroptosis markers but upregulation of cleaved PARP in TCCSUP cells 72 hr post-CVA21 infection is shown.