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. Author manuscript; available in PMC: 2009 Oct 1.

Published in final edited form as: Cancer Res. 2008 Oct 1;68(19):7985–7994. doi: 10.1158/0008-5472.CAN-08-1418

ABT-737 restores sensitivity to paclitaxel in paclitaxel-resistant MDA-MB-468 cells. A, MDA-MB-468 cells were treated with 100 nM paclitaxel for 48 h. MDA-MB-468 TaxR cells were treated with paclitaxel (100 nM), ABT-737 (200 nM) or combination of paclitaxel (100 nM) and ABT-737 (200 nM) for 48 h. The percent apoptotic response was evaluated by Annexin V staining. Columns, mean of three independent experiments; bars, SE. **, P < 0.01, paclitaxel plus ABT-737 treated with respect to paclitaxel-only treated. B, MDA-MB-468 cells were treated with paclitaxel (100 nM) for 0-48 h, MDA-MB-468 TaxR cells were treated as in A. Cytosolic and mitochondrial fractions were blotted for cytochrome c. CoxIV was probed as a loading control for mitochondrial fractions. C, MDA-MB-468 TaxR cells were treated with paclitaxel (100 nM), ABT-737 (200 nM) or combination of paclitaxel (100 nM) and ABT-737 (200 nM) for 12 h. Activation of BAX and BAK was analyzed by immunoprecipitation with active conformation-specific anti BAX (6A7) and anti-BAK (Ab-2) antibodies followed by immunoblot analysis of BAX and BAK. 5% of the input for immunoprecipitation was also subjected to immunoblot analysis. β-Actin was probed as a loading control. D, (left) MDA-MB-468 TaxR cells were treated as in A, and activation of caspase-3, caspase-9 and caspase-8 was evaluated by fluorometric caspase activation assays. Data were expressed as relative fluorescence units (RFU). Columns, mean of three independent experiments; bars, SE. (middle) Total cell extracts were analyzed for the activation of caspase-3, caspase-9 and caspase-8 by immunoblotting. β-Actin was probed as a loading control. (right) MDA-MB-468 TaxR cells were pretreated with 20 μM pancaspase inhibitor (Z-VAD-FMK), 20 μM caspase-9 inhibitor (Z-LEHD-FMK) and 20 μM caspase-8 inhibitor (Z-IETD-FMK) before treatment with paclitaxel plus ABT-737 for 48 h. The percent apoptotic response was evaluated by Annexin V staining. Columns, mean of three independent experiments; bars, SE.

ABT-737 restores sensitivity to paclitaxel in paclitaxel-resistant MDA-MB-468 cells. A, MDA-MB-468 cells were treated with 100 nM paclitaxel for 48 h. MDA-MB-468 TaxR cells were treated with paclitaxel (100 nM), ABT-737 (200 nM) or combination of paclitaxel (100 nM) and ABT-737 (200 nM) for 48 h. The percent apoptotic response was evaluated by Annexin V staining. Columns, mean of three independent experiments; bars, SE. **, P < 0.01, paclitaxel plus ABT-737 treated with respect to paclitaxel-only treated. B, MDA-MB-468 cells were treated with paclitaxel (100 nM) for 0-48 h, MDA-MB-468 TaxR cells were treated as in A. Cytosolic and mitochondrial fractions were blotted for cytochrome c. CoxIV was probed as a loading control for mitochondrial fractions. C, MDA-MB-468 TaxR cells were treated with paclitaxel (100 nM), ABT-737 (200 nM) or combination of paclitaxel (100 nM) and ABT-737 (200 nM) for 12 h. Activation of BAX and BAK was analyzed by immunoprecipitation with active conformation-specific anti BAX (6A7) and anti-BAK (Ab-2) antibodies followed by immunoblot analysis of BAX and BAK. 5% of the input for immunoprecipitation was also subjected to immunoblot analysis. β-Actin was probed as a loading control. D, (left) MDA-MB-468 TaxR cells were treated as in A, and activation of caspase-3, caspase-9 and caspase-8 was evaluated by fluorometric caspase activation assays. Data were expressed as relative fluorescence units (RFU). Columns, mean of three independent experiments; bars, SE. (middle) Total cell extracts were analyzed for the activation of caspase-3, caspase-9 and caspase-8 by immunoblotting. β-Actin was probed as a loading control. (right) MDA-MB-468 TaxR cells were pretreated with 20 μM pancaspase inhibitor (Z-VAD-FMK), 20 μM caspase-9 inhibitor (Z-LEHD-FMK) and 20 μM caspase-8 inhibitor (Z-IETD-FMK) before treatment with paclitaxel plus ABT-737 for 48 h. The percent apoptotic response was evaluated by Annexin V staining. Columns, mean of three independent experiments; bars, SE.