Figure 5

D6 and D1 triggered-apoptosis is caspase-dependent and involves the apoptotic intrinsic pathway. (A) and (B): Caspase 3/7 activation was assessed on GI-LI-N cells (A) and LB24 cells (B), untreated (control), or treated with D1 (10 μM) and D6 (5 μM) for 24 hours. Cells received an 1 hour pre-treatment with a pan-caspases inhibitor (z-VAD-FMK). RFU: Relative Fluorescence Unit. ***, P < 0.001 vs control. (C) To assess the effects of drugs on mitochondrial membrane permeability, neuroblastoma IMR-32 and melanoma LB24 cells were untreated (control) or treated with D1 and D6, as above, for 18 hours, harvested, and analyzed for changes in mitochondrial membrane potential. Results are expressed as the mean percentage of cells with polarized mitochondria from four independent experiments; error bars, 95% CIs. *, P < 0.05; ***, P < 0.001 vs control. (D) IMR-32 and LB24 cells were treated for 24 hours as above and fractionated cellular lysates were analyzed by western blot with a specific primary antibody for cytochrome c. GAPDH was used as loading control. (E) SH-SY5Y, HTLA-230, GI-LI-N neuroblastoma cell lines and LB24, M14, WM melanoma cell lines were treated as in panel D for 48 hours and total cellular lysates were analyzed by western blot with specific primary antibodies for procaspase 9, procaspase 3 and poly(ADP)ribose polymerase (PARP). As for (D), blots are representative of three independent experiments and data were normalized against GAPDH.