Fig. 4. Naftopidil sensitizes ovarian cancer cells to ABT-737 through BH3-only protein-induced expression.

Effects of Naftopidil (50 µM) and increasing concentrations of ABT-737 (0, 1, 5 µM) combination treatments were investigated in SKOV3 (left column) and IGROV1-R10 (right column) cell lines after 24 and 48 h a on proliferation by the Trypan blue exclusion test (curves represent the relative viable cells number in the treated cells normalized to that in T0 condition); and only after 48 h b on apoptosis by analyzing the rate of sub-G1 events using flow cytometry (upper panel) and PARP and caspase 3 cleavages by western blot (lower panel), and c on the expression of BH3-only proteins by western blot (the same western blot was used for actin protein expression in b, c). Twenty-four hours before Naftopidil/ABT-737 (5 µM) treatment, SKOV3 cells were transfected with control (Si-Ct) or Noxa (Si-Noxa) siRNAs (20 nM) while IGROV1-R10 cells were transfected with control (Si-Ct) or Noxa (Si-Noxa) and Bim (Si-Bim) siRNAs (20 nM), d after a 48-h combination treatment, target protein expression and PARP and Caspase 3 cleavages as markers of apoptosis were observed by western blot and e the number of viable cells was assessed by the Trypan blue exclusion test (histograms represent the relative viable cells number in treated cells transfected with Si-Noxa for SKOV3 cells or Si-Noxa and Si-Bim for IGROV1-R10 cells normalized to that in transfected with Si-Ct) (left panel), percentage of sub-G1 events was obtained by flow cytometry (histograms represent the percentage of sub-G1 events in treated cells transfected with Si-Noxa for SKOV3 cells normalized to that in transfected with Si-Ct) (middle panel) and the cell morphology of treated cells transfected with Si-Noxa for SKOV3 cells or Si-Noxa and Si-Bim for IGROV1-R10 cells was observed by optical microscopy (scale bar = 100 μm) (right panel) (N = 3). Results were considered statistically different if *p < 0.05, **p < 0.01, ***p < 0.001.