American Society of Hematology

Figure 1

$Combined treatment with sorafenib and obatoclax results in a marked induction of cell death in association with profound mitochondrial injury and caspase activation, and diminishes the colony-formation capacity of human leukemia cells. (A) U937 cells were exposed to the designated concentrations of sorafenib alone (○) or in combination with 1.5μM obatoclax (Ob, ●) for 48 hours after which the percentage of apoptotic cells was determined by the 7-AAD staining assay. *Significantly greater than sorafenib alone; P < .05; **P < .01. (B) U937 cells were exposed to the designated concentrations of obatoclax alone (○) or in combination with 7.5μM sorafenib (●), for 48 hours after which cell death was determined as in panel A. *Significantly greater than obatoclax alone; P < .02; **P < .01. (C) Cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) alone or in combination for the indicated intervals, after which the percentage of dead cells was determined as above. *Significantly greater than either agent alone; P < .05; **P < .002. (D) Median dose effect analysis of cell death induction by sorafenib and obatoclax. U937 cells were exposed to varying concentrations of obatoclax and sorafenib at a fixed ratio (1:5), for 48 hours after which extent of cell death was monitored with the 7-AAD staining assay. Combination Index (CI) values were determined in relation to the fractional effect using the Calcusyn software program. CI values < 1.0 correspond to a synergistic interaction. (E) MV4-11 and HL-60 cells were exposed to sorafenib (75nM and 7.5μM, respectively) and obatoclax (0.5μM and 2μM, respectively), either individually or in combination for 48 hours, after which the percentage of dead cells was determined by the 7-AAD assay. *Significantly greater than values for either agent alone; P < .02. (F) U937 cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) alone or in combination for 24 hours after which mitochondria-free cytosolic fractions were obtained and subjected to Western blot analysis to monitor the release of cytochrome c and AIF into the cytosol. For this and all subsequent Western blot analysis, blots were subsequently reprobed with antitubulin (Tub) Abs to document equivalent loading and transfer, and the blots shown are representative of at least 3 separate experiments. (G) U937, MV4-11 and HL-60 cells were exposed to sorafenib and obatoclax individually or in combination as in panels E and F for 48 hours after which protein lysates were prepared and subjected to Western blot analysis using the designated Abs. (H) U937 cells were plated in methylcellulose in the presence of sorafenib (7.5μM) and obatoclax (75nM) alone or in combination for 10 days after which CFUs were enumerated and expressed as a percentage of untreated cells. *Significantly less than values for either agent alone; P < .02. For panels A, B, C, E, and H, values represent the means ± SD for 3 separate experiments in which each sample was analyzed in triplicate.$

Combined treatment with sorafenib and obatoclax results in a marked induction of cell death in association with profound mitochondrial injury and caspase activation, and diminishes the colony-formation capacity of human leukemia cells. (A) U937 cells were exposed to the designated concentrations of sorafenib alone (○) or in combination with 1.5μM obatoclax (Ob, ●) for 48 hours after which the percentage of apoptotic cells was determined by the 7-AAD staining assay. *Significantly greater than sorafenib alone; P < .05; **P < .01. (B) U937 cells were exposed to the designated concentrations of obatoclax alone (○) or in combination with 7.5μM sorafenib (●), for 48 hours after which cell death was determined as in panel A. *Significantly greater than obatoclax alone; P < .02; **P < .01. (C) Cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) alone or in combination for the indicated intervals, after which the percentage of dead cells was determined as above. *Significantly greater than either agent alone; P < .05; **P < .002. (D) Median dose effect analysis of cell death induction by sorafenib and obatoclax. U937 cells were exposed to varying concentrations of obatoclax and sorafenib at a fixed ratio (1:5), for 48 hours after which extent of cell death was monitored with the 7-AAD staining assay. Combination Index (CI) values were determined in relation to the fractional effect using the Calcusyn software program. CI values < 1.0 correspond to a synergistic interaction. (E) MV4-11 and HL-60 cells were exposed to sorafenib (75nM and 7.5μM, respectively) and obatoclax (0.5μM and 2μM, respectively), either individually or in combination for 48 hours, after which the percentage of dead cells was determined by the 7-AAD assay. *Significantly greater than values for either agent alone; P < .02. (F) U937 cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) alone or in combination for 24 hours after which mitochondria-free cytosolic fractions were obtained and subjected to Western blot analysis to monitor the release of cytochrome c and AIF into the cytosol. For this and all subsequent Western blot analysis, blots were subsequently reprobed with antitubulin (Tub) Abs to document equivalent loading and transfer, and the blots shown are representative of at least 3 separate experiments. (G) U937, MV4-11 and HL-60 cells were exposed to sorafenib and obatoclax individually or in combination as in panels E and F for 48 hours after which protein lysates were prepared and subjected to Western blot analysis using the designated Abs. (H) U937 cells were plated in methylcellulose in the presence of sorafenib (7.5μM) and obatoclax (75nM) alone or in combination for 10 days after which CFUs were enumerated and expressed as a percentage of untreated cells. *Significantly less than values for either agent alone; P < .02. For panels A, B, C, E, and H, values represent the means ± SD for 3 separate experiments in which each sample was analyzed in triplicate.

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