Alteration of the mitochondrial apoptotic pathway is key to acquired paclitaxel resistance and can be reversed by ABT-737

Abstract

Paclitaxel is a microtubule-targeting antineoplastic drug widely used in human cancers. Even when tumors are initially responsive, progression of disease despite continued taxane therapy is all too common in the treatment of many of the most common epithelial cancers, including breast cancer. However, the mechanisms underlying paclitaxel resistance in cancer cells are not completely understood. Our hypothesis is that changes in the intrinsic (or mitochondrial) cell death pathway controlled by the BCL-2 family are key to the development of acquired paclitaxel resistance. Here we show that paclitaxel activates the mitochondrial apoptosis pathway, which can be blocked by BCL-2 overexpression. Treatment with ABT-737, a small-molecule BCL-2 antagonist, restores sensitivity to paclitaxel in BCL-2-overexpressing cells. To investigate the importance of changes in the intrinsic apoptotic pathway in the absence of enforced BCL-2 expression, we generated two independent breast cancer cell lines with acquired resistance to apoptosis induced by paclitaxel. In these lines, acquired resistance to paclitaxel is mediated either by increased antiapoptotic BCL-2 proteins or decreased proapoptotic BCL-2 proteins. In both cases, ABT-737 can engage the mitochondrial apoptosis pathway to restore sensitivity to paclitaxel to cell lines with acquired paclitaxel resistance. In summary, these findings suggest that alterations in the intrinsic apoptotic pathway controlled by BCL-2 protein family members may be crucial to causing paclitaxel resistance. Furthermore, our results suggest that combining small-molecule BCL-2 antagonists with paclitaxel may offer benefit to patients with paclitaxel-resistant tumors, an oncologic problem of great prevalence.

Figure 1

BCL-2 protects against paclitaxel-induced apoptosis and ABT-737 might overcome BCL-2-mediated paclitaxel resistance. A, MCF-7 cells were stably transfected with control vector (MCF-7 Mock) or pCI.Neo.FlagBCL-2 (MCF-7 BCL-2). The expression of FLAG-BCL-2 in transfected cells were verified by immunoblotting with anti-BCL-2 or anti-FLAG antibody. β-Actin was probed as a loading control. B, cells were treated with treated with paclitaxel (100 nM), ABT-737 (100 nM), combination of paclitaxel (100 nM) and ABT-737 (100 nM), enantiomer of ABT-737 (100 nM) or combination of enantiomer of ABT-737 (100 nM) and paclitaxel (100 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. C, T47-D cells were stably transfected with control vector (T47D Mock) or pCI.Neo.FlagBCL-2 (T47D BCL-2). The expression of FLAG-BCL-2 in transfected cells were verified by immunoblotting with anti-BCL-2 or anti-FLAG antibody. β-Actin was probed as a loading control. D, cells were treated with treated with paclitaxel (100 nM), ABT-737 (100 nM), combination of paclitaxel (100 nM) and ABT-737 (100 nM), enantiomer of ABT-737 (100 nM) or combination of enantiomer of ABT-737 (100 nM) and paclitaxel (100 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.

Figure 1

BCL-2 protects against paclitaxel-induced apoptosis and ABT-737 might overcome BCL-2-mediated paclitaxel resistance. A, MCF-7 cells were stably transfected with control vector (MCF-7 Mock) or pCI.Neo.FlagBCL-2 (MCF-7 BCL-2). The expression of FLAG-BCL-2 in transfected cells were verified by immunoblotting with anti-BCL-2 or anti-FLAG antibody. β-Actin was probed as a loading control. B, cells were treated with treated with paclitaxel (100 nM), ABT-737 (100 nM), combination of paclitaxel (100 nM) and ABT-737 (100 nM), enantiomer of ABT-737 (100 nM) or combination of enantiomer of ABT-737 (100 nM) and paclitaxel (100 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. C, T47-D cells were stably transfected with control vector (T47D Mock) or pCI.Neo.FlagBCL-2 (T47D BCL-2). The expression of FLAG-BCL-2 in transfected cells were verified by immunoblotting with anti-BCL-2 or anti-FLAG antibody. β-Actin was probed as a loading control. D, cells were treated with treated with paclitaxel (100 nM), ABT-737 (100 nM), combination of paclitaxel (100 nM) and ABT-737 (100 nM), enantiomer of ABT-737 (100 nM) or combination of enantiomer of ABT-737 (100 nM) and paclitaxel (100 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.

Figure 2

ABT-737-mediated sensitization of MCF-7 BCL-2 cells to paclitaxel-induced apoptosis via mitochondrial apoptosis pathway. A, MCF-7 BCL-2 cells were treated with paclitaxel (100 nM), ABT-737 (100 nM), or combination of paclitaxel (100 nM) and ABT-737 (100 nM) for 48 h. Cytosolic and mitochondrial fractions from MCF-7 BCL-2 cells were immunoblotted for cytochrome c. CoxIV was probed as a loading control for mitochondrial fractions. B, MCF-7 BCL-2 cells were treated with paclitaxel (100 nM), ABT-737 (100 nM), or the combination of paclitaxel (100 nM) and ABT-737 (100 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. C, MCF-7 BCL-2 cells were treated with paclitaxel (100 nM), ABT-737 (100 nM), or combination of paclitaxel (100 nM) and ABT-737 (100 nM) for 48 h. Activation of caspase-9 and caspase-8 by the combination of paclitaxel and ABT-737 treatment was evaluated by fluorometric caspase activation assays as described in Materials and methods. Data were expressed as relative fluorescence units (RFU). Columns, mean of three independent experiments; bars, SE. D, MCF-7 BCL-2 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.

Figure 2

ABT-737-mediated sensitization of MCF-7 BCL-2 cells to paclitaxel-induced apoptosis via mitochondrial apoptosis pathway. A, MCF-7 BCL-2 cells were treated with paclitaxel (100 nM), ABT-737 (100 nM), or combination of paclitaxel (100 nM) and ABT-737 (100 nM) for 48 h. Cytosolic and mitochondrial fractions from MCF-7 BCL-2 cells were immunoblotted for cytochrome c. CoxIV was probed as a loading control for mitochondrial fractions. B, MCF-7 BCL-2 cells were treated with paclitaxel (100 nM), ABT-737 (100 nM), or the combination of paclitaxel (100 nM) and ABT-737 (100 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. C, MCF-7 BCL-2 cells were treated with paclitaxel (100 nM), ABT-737 (100 nM), or combination of paclitaxel (100 nM) and ABT-737 (100 nM) for 48 h. Activation of caspase-9 and caspase-8 by the combination of paclitaxel and ABT-737 treatment was evaluated by fluorometric caspase activation assays as described in Materials and methods. Data were expressed as relative fluorescence units (RFU). Columns, mean of three independent experiments; bars, SE. D, MCF-7 BCL-2 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.

Figure 3

Acquired resistance to paclitaxel is mediated by BCL-2 protein family members. A, EC₅₀ values of parental (MCF-7 and MDA-MB-468) and paclitaxel-resistant cell lines (MCF-7 TaxR30, MCF-7 TaxR50 and MDA-MB-468 TaxR, respectively) were evaluated with MTT assay after treatment with various doses of paclitaxel for 48 h. EC₅₀ values were determined by nonlinear regression analysis using GraphPad Prism software. Bars, SE. B and C, Western blot analysis of BCL-2 protein family members in parental and paclitaxel-resistant cell lines. Paclitaxel-resistant cell lines marked with “+” were grown in continuous exposure to paclitaxel (30 nM for MCF-7 TaxR30, 50 nM for MCF-7 TaxR50, 15 nM for MDA-MB-468 TaxR) or paclitaxel was withdrawn from growth media for 24 hours to reduce the possibility of acute paclitaxel-mediated variations in protein levels (marked with “-”). Total cellular proteins were isolated either in the presence or in the absence of paclitaxel in growth media and analyzed in parallel. β-Actin was probed as a loading control.

Figure 3

Acquired resistance to paclitaxel is mediated by BCL-2 protein family members. A, EC₅₀ values of parental (MCF-7 and MDA-MB-468) and paclitaxel-resistant cell lines (MCF-7 TaxR30, MCF-7 TaxR50 and MDA-MB-468 TaxR, respectively) were evaluated with MTT assay after treatment with various doses of paclitaxel for 48 h. EC₅₀ values were determined by nonlinear regression analysis using GraphPad Prism software. Bars, SE. B and C, Western blot analysis of BCL-2 protein family members in parental and paclitaxel-resistant cell lines. Paclitaxel-resistant cell lines marked with “+” were grown in continuous exposure to paclitaxel (30 nM for MCF-7 TaxR30, 50 nM for MCF-7 TaxR50, 15 nM for MDA-MB-468 TaxR) or paclitaxel was withdrawn from growth media for 24 hours to reduce the possibility of acute paclitaxel-mediated variations in protein levels (marked with “-”). Total cellular proteins were isolated either in the presence or in the absence of paclitaxel in growth media and analyzed in parallel. β-Actin was probed as a loading control.

Figure 4

ABT-737 restores sensitivity to paclitaxel in paclitaxel-resistant MCF-7 cells. A, MCF-7 TaxR30 and MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM), or combination of paclitaxel (300 nM) and ABT-737 (300 nM) for 48 h. The percent apoptotic response was evaluated by Annexin V staining. Columns, mean of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01, paclitaxel plus ABT-737 treated with respect to paclitaxel-only treated. B, MCF-7 cells were treated with paclitaxel (100 nM) for 0-48 h, MCF-7 TaxR50 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, MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM) or combination of paclitaxel (300 nM) and ABT-737 (300 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) MCF-7 TaxR50 cells were treated as in A, and activation of 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-9 and caspase-8 by immunoblotting. β-Actin was probed as a loading control. (right) MCF-7 TaxR50 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.

Figure 4

ABT-737 restores sensitivity to paclitaxel in paclitaxel-resistant MCF-7 cells. A, MCF-7 TaxR30 and MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM), or combination of paclitaxel (300 nM) and ABT-737 (300 nM) for 48 h. The percent apoptotic response was evaluated by Annexin V staining. Columns, mean of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01, paclitaxel plus ABT-737 treated with respect to paclitaxel-only treated. B, MCF-7 cells were treated with paclitaxel (100 nM) for 0-48 h, MCF-7 TaxR50 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, MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM) or combination of paclitaxel (300 nM) and ABT-737 (300 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) MCF-7 TaxR50 cells were treated as in A, and activation of 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-9 and caspase-8 by immunoblotting. β-Actin was probed as a loading control. (right) MCF-7 TaxR50 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.

Figure 4

ABT-737 restores sensitivity to paclitaxel in paclitaxel-resistant MCF-7 cells. A, MCF-7 TaxR30 and MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM), or combination of paclitaxel (300 nM) and ABT-737 (300 nM) for 48 h. The percent apoptotic response was evaluated by Annexin V staining. Columns, mean of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01, paclitaxel plus ABT-737 treated with respect to paclitaxel-only treated. B, MCF-7 cells were treated with paclitaxel (100 nM) for 0-48 h, MCF-7 TaxR50 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, MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM) or combination of paclitaxel (300 nM) and ABT-737 (300 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) MCF-7 TaxR50 cells were treated as in A, and activation of 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-9 and caspase-8 by immunoblotting. β-Actin was probed as a loading control. (right) MCF-7 TaxR50 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.

Figure 5

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.

Figure 5

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.

Figure 5

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.

Figure 6. The effect of ABT-737 and paclitaxel on colony formation in paclitaxel-resistant MCF-7 and MDA-MB-468 cells

A, MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM) or combination of paclitaxel (300 nM) and ABT-737 (300 nM) for 16 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 16 h. Clonogenic survival was assessed by colony-forming assay. Data presented are percentage of colony formation normalized to untreated control cells. B, Model of alterations in BCL-2 protein family members governing paclitaxel resistance. I, paclitaxel sensitive cells. II, Paclitaxel-resistant cells, similar to MCF-7 TaxR50. III, Paclitaxel resistant cells, similar to MDA-MB-468 TaxR.

Figure 6. The effect of ABT-737 and paclitaxel on colony formation in paclitaxel-resistant MCF-7 and MDA-MB-468 cells

A, MCF-7 TaxR50 cells were treated with paclitaxel (300 nM), ABT-737 (300 nM) or combination of paclitaxel (300 nM) and ABT-737 (300 nM) for 16 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 16 h. Clonogenic survival was assessed by colony-forming assay. Data presented are percentage of colony formation normalized to untreated control cells. B, Model of alterations in BCL-2 protein family members governing paclitaxel resistance. I, paclitaxel sensitive cells. II, Paclitaxel-resistant cells, similar to MCF-7 TaxR50. III, Paclitaxel resistant cells, similar to MDA-MB-468 TaxR.