Synergistic activity between vorinostat and PEITC in various myeloid leukemia cell lines. (A) Dose-dependent induction of apoptosis in U937 by vorinostat in the absence and presence of PEITC (2.5μM, 48 hours) was analyzed with annexin V/PI assay. Data represent mean ± SD from 3 experiments. Addition of 2.5μM PEITC significantly enhanced the cytotoxicity of vorinostat alone. *P < .01 (Student t test). (B) Combination index (CI) of vorinostat and PEITC in U937 cells were analyzed by median dose-effect method using Calcusyn Version 2.0 software (Biosoft). U937 cells were incubated with various concentrations of vorinostat (0.3-4μM) and PEITC (0.5-3μM) for 72 hours. Drug effect on cell viability was determined by MTT assay as described in “Cytotoxicity assays.” CI = 1 indicates additive effect; CI < 1 indicates synergistic effect; and CI > 1 indicates antagonist effect. (C) HL60 cells were treated with 1μM vorinostat alone, 1μM PEITC alone, or vorinostat plus PEITC (V + P) for 48 hours, and killing effect was analyzed with annexin V/PI assay. Bars represent mean ± SD from 3 experiments. (D) Growth inhibition by vorinostat and PEITC in U937 and ML1 cells. Cells in exponential growth were treated with vorinostat or/and PEITC at the indicated concentration. Cell culture was continued for up to 72 hours, and total cell numbers were counted every 24 hours using a Coulter Z2 Particle Counter & Size Analyzer. (E) Sequence-dependent effect of vorinostat combined with PEITC in U937 and ML1 cells. U937 and ML1 cells were both treated with 2μM vorinostat and 2.5μM PEITC concomitantly for 48 hours (V + P) or treated with vorinostat for 24 hours followed by PEITC for another 24 hours (V/P), or treated with PEITC for 24 hours followed by vorinostat for another 24 hours (P/V). Cytotoxic effect of each compound alone or their combination was analyzed with annexin V/PI assay.
