Figure 7
Figure 7. Combining NVP-BEZ235 with Obatoclax or UO126 eradicates proliferation of human HMC-1.2 leukemia cell line. (A) Whole-cell lysates from HMC-1.2 cells treated or not (−; DMSO) for 4 hours with IM (10μM), PP1 (10μM), or PP2 (10μM) were analyzed by immunoblotting with the indicated Abs. Data are from 1 representative experiment of 3. (B) HMC-1.2 cells were plated at 4 × 105 cells/mL and cultured in the absence (−; DMSO) or in the presence of indicated concentrations of NVP-BEZ235, Obatoclax (OBX), or UO126. Number of living cells, percentage of dead cells (trypan blue exclusion), and percentage of apoptotic cells (flow cytometry using anti-active caspase 3 Abs) were determined at 48 hours. Bar is mean ± SEM (n = 3). Statistical differences from the value of living cells control (−) are indicated as follows: ***P < .001; **P < .01; *P < .05 (by Student t test). Mortality after NVP-BEZ235 treatment was not significantly different (P > .05 by Student t test). (C) Representative cell-cycle distribution of HMC-1.2 cells treated or not (−; DMSO) with NVP-BEZ235 (50nM) for 48 hours. The percentages of living cells in the G0/G1, S, and G2/M phases of the cell cycle are indicated below. Data are from 1 of 3 independent experiments. (D) HMC-1.2 cells were plated at 4 × 105 cells/mL and cultured in the absence (V: DMSO) or in the presence of NVP-BEZ235 (25nM), Obatoclax (150nM), or UO126 (15μM) and the combination of NVP-BEZ235 (25nM) with Obatoclax (150nM) or NVP-BEZ235 (25nM) with UO126 (15μM). Viable cells were scored at 24 and 48 hours. Data are means ± SEM (n = 3). P < .001 comparing control cells with cells grown in the presence of NVP-BEZ235, Obatoclax, and/or UO126 (by Student t test). (E) The effects of BEZ and OBX or UO126 on cell proliferation were analyzed according to the Chou-Talalay method using CalcuSyn software.30 HMC-1.2 cells were treated with suboptimal concentrations of BEZ combined with suboptimal concentrations of OBX or UO126. The combination index values were determined from these dose-response curves for each combination. Combination index values of 0.9-1.1 indicate additive, 0.7-0.9 moderately synergistic, 0.3-0.7 synergistic, and less than 0.3 strongly synergistic effects.

Combining NVP-BEZ235 with Obatoclax or UO126 eradicates proliferation of human HMC-1.2 leukemia cell line. (A) Whole-cell lysates from HMC-1.2 cells treated or not (−; DMSO) for 4 hours with IM (10μM), PP1 (10μM), or PP2 (10μM) were analyzed by immunoblotting with the indicated Abs. Data are from 1 representative experiment of 3. (B) HMC-1.2 cells were plated at 4 × 105 cells/mL and cultured in the absence (−; DMSO) or in the presence of indicated concentrations of NVP-BEZ235, Obatoclax (OBX), or UO126. Number of living cells, percentage of dead cells (trypan blue exclusion), and percentage of apoptotic cells (flow cytometry using anti-active caspase 3 Abs) were determined at 48 hours. Bar is mean ± SEM (n = 3). Statistical differences from the value of living cells control (−) are indicated as follows: ***P < .001; **P < .01; *P < .05 (by Student t test). Mortality after NVP-BEZ235 treatment was not significantly different (P > .05 by Student t test). (C) Representative cell-cycle distribution of HMC-1.2 cells treated or not (−; DMSO) with NVP-BEZ235 (50nM) for 48 hours. The percentages of living cells in the G0/G1, S, and G2/M phases of the cell cycle are indicated below. Data are from 1 of 3 independent experiments. (D) HMC-1.2 cells were plated at 4 × 105 cells/mL and cultured in the absence (V: DMSO) or in the presence of NVP-BEZ235 (25nM), Obatoclax (150nM), or UO126 (15μM) and the combination of NVP-BEZ235 (25nM) with Obatoclax (150nM) or NVP-BEZ235 (25nM) with UO126 (15μM). Viable cells were scored at 24 and 48 hours. Data are means ± SEM (n = 3). P < .001 comparing control cells with cells grown in the presence of NVP-BEZ235, Obatoclax, and/or UO126 (by Student t test). (E) The effects of BEZ and OBX or UO126 on cell proliferation were analyzed according to the Chou-Talalay method using CalcuSyn software.30  HMC-1.2 cells were treated with suboptimal concentrations of BEZ combined with suboptimal concentrations of OBX or UO126. The combination index values were determined from these dose-response curves for each combination. Combination index values of 0.9-1.1 indicate additive, 0.7-0.9 moderately synergistic, 0.3-0.7 synergistic, and less than 0.3 strongly synergistic effects.

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