Figure 6

NRC-03- and NRC-07-mediated cytotoxicity is associated with mitochondrial membrane damage and ROS generation. MDA-MB-231 breast cancer cells were cultured in the absence or presence of NRC-03 or NRC-07 for 30 minutes. (a) DiOC6 or (b) DHE was added to cells to detect a loss of mitochondrial transmembrane potential and ROS generation, respectively. Solid peak, medium alone. Open peak, 50 μM NRC-03 or NRC-07 treatment. (c) Mitochondria were isolated from MDA-MB-231 cells and treated with NRC-03 or NRC-07, as described earlier, for 10 minutes. Cytochrome c was detected in the supernatant and mitochondria fractions with Western blot analysis. Mitochondrial Hsp70 was detected to confirm equal protein loading. Data shown are from a representative experiment (n = 3). MDA-MB-231 cells were pretreated with (d) 40 μM Boc-D-FMK or (e) 5 mM GSH before exposure to 50 μM NRC-03 or NRC-07. Percentage cytotoxicity was assessed with MTT and acid phosphatase viability assays, respectively. Data shown are the mean ± SEM of three independent experiments and are not statistically significant (p > 0.05) in comparison with controls with the Bonferroni multiple comparisons test.