Figure 4

Induction of apoptosis by RKA182, FBEG100 and ARS in HL-60 cells. (A) Cells were exposed to the indicated concentrations of RKA182 (black circles) or FBEG100 (white circles) for 24 h. The number of cells with reduced mitochondrial membrane potential, indicative of depolarization, was quantified by TMRE staining and flow cytometry, and expressed as percentage of total cells. Data represent mean + SD, n = 3. (B) Cells were pretreated (triangles) or not (circles) with the caspase inhibitor Z.VAD.FMK (100 µmol/L, 1 h) and then exposed to the indicated concentrations of ARS (gray symbols), RKA182 (black symbols) or FBEG100 (white symbols) for a further 24 h. As an index of caspase activity, cleavage of a luminogenic caspase 3/7 substrate was quantified, and expressed as a percentage of the activity in cells exposed only to vehicle (methanol, 0.5 %). Data represent mean + SD, n = 3. (C) Cells were exposed to methanol (MeOH, 0.5 %), RKA182 (3, 5, 7.5 or 10 µmol/L), FBEG100 (20, 30, 40, 50 µmol/L) or ARS (0.1, 0.3, 1, 3 µmol/L) for 24 h. The processing of caspase 3 from its inactive 32 kDa form to its proteolytically active 17 kDa form was determined in whole cell lysates by Western blotting. Staurosporine (STS; 1 µmol/L) was used as a positive control for caspase 3 activation. β-Actin was probed as a loading control. (D) Cells were exposed to RKA182 (55 µmol/L), FBEG100 (75 µmol/L) or ARS (10 µmol/L) for the indicated times. Caspase 3 processing was detected by Western blotting. (E) Cells were exposed to the indicated concentrations of RKA182 (black circles) or FBEG100 (white circles) for 24 h. The number of cells in sub-G₀/G₁ phase of the cell cycle was quantified by PI staining and flow cytometry, and expressed as percentage of total cells. Data represent mean + SD, n = 3. (F) Cells were pretreated or not with the caspase inhibitor Z.VAD.FMK (100 µmol/L, 1 h) and then exposed to STS (1 µmol/L), methanol (0.5 %), ARS (1 µmol/L), RKA182 (10 µmol/L) or FBEG100 (40 µmol/L) for a further 24 h. Extracted genomic DNA was separated by electrophoresis on an agarose gel stained with ethidium bromide. (G) Cells were pretreated (gray bars) or not (black bars) with the caspase inhibitor Z.VAD.FMK (100 µmol/L 1 h) and exposed to RKA182, FBEG100 or ARS for a further 24 h. Cytotoxicity was determined by quantification of cellular ATP content. IC₅₀ values were calculated from concentration-response curves. Unpaired t test, N.S. no statistically significant increase in IC₅₀ value with Z.VAD.FMK pretreatment. Data represent mean + SD, n = 3.