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Displacement of Bim by Bmf and Puma rather than increase in Bim level mediates paclitaxel-induced apoptosis in breast cancer cells.

Kutuk O, Letai A - Cell Death Differ. (2010)

Bottom Line: However, the signaling pathways that connect paclitaxel-induced microtubule perturbation to mitochondrial outer membrane permeabilization and cytochrome c release are not well characterized.Bim and either Puma or Bmf are required for paclitaxel toxicity.This novel mechanism suggests the potential usage of novel therapies targeted at altering BH3-only protein heterodimerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

ABSTRACT
Taxanes exert their antitumor effect through stabilizing microtubule dynamics and initiating G2/M arrest in cancer cells followed by apoptotic cell death. However, the signaling pathways that connect paclitaxel-induced microtubule perturbation to mitochondrial outer membrane permeabilization and cytochrome c release are not well characterized. Here, we show that in breast cancer cells, paclitaxel induces a novel displacement mechanism: prodeath BH3-only proteins Bmf and Puma competitively displace prodeath BH3-only protein Bim from antiapoptotic proteins to activate Bax and Bak and commit the cell to apoptotic death. Bim and either Puma or Bmf are required for paclitaxel toxicity. Although prior mechanisms of apoptosis induced by taxol have focused on changes in Bim levels, we find that an increase is not required for paclitaxel killing of breast cancer cells. Rather, competitive displacement of Bim from antiapoptotic proteins is the important step committing the cell to death. This novel mechanism suggests the potential usage of novel therapies targeted at altering BH3-only protein heterodimerization.

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Bmf is required for paclitaxel-induced apoptosis in breast cancer cells. (a) MDA-MB-468 and (b) BT20 cells were treated with paclitaxel (100 nM) for 0, 4, 8, and 12 h and the interaction of Bmf with Bcl-2 and Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (c) T47D cells were treated with paclitaxel (100 nM) 12 h and the interaction of Puma with Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (d) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. T47D cells were transiently transfected with Puma siRNA or Scrambled siRNA for 48 h. The efficiency of knockdowns was monitored by immunoblots. Untransfected and siRNA-transfected cells were treated with paclitaxel (100 nM) for 48 h and apoptosis was evaluated by using Annexin V staining (mean ± SEM, n = 4, *p< 0.05, **p< 0.01 by two-tailed t test). (e) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. Cells were treated with paclitaxel (100 nM) for 12 h and 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. (e) Fluorescence polarization assays were performed using recombinant GST-Bcl-2 (100 μM) and FITC-labeled Bim and Bmf BH3 peptides (10 nM). Unlabeled Bim and Bmf BH3 peptides were used to compete with binding of FITC-labeled Bim and Bmf BH3 peptides to test the ability of Bim and Bmf to displace each other from Bcl-2. Data shown are mean ± SEM of four independent experiments in duplicate.
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Figure 6: Bmf is required for paclitaxel-induced apoptosis in breast cancer cells. (a) MDA-MB-468 and (b) BT20 cells were treated with paclitaxel (100 nM) for 0, 4, 8, and 12 h and the interaction of Bmf with Bcl-2 and Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (c) T47D cells were treated with paclitaxel (100 nM) 12 h and the interaction of Puma with Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (d) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. T47D cells were transiently transfected with Puma siRNA or Scrambled siRNA for 48 h. The efficiency of knockdowns was monitored by immunoblots. Untransfected and siRNA-transfected cells were treated with paclitaxel (100 nM) for 48 h and apoptosis was evaluated by using Annexin V staining (mean ± SEM, n = 4, *p< 0.05, **p< 0.01 by two-tailed t test). (e) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. Cells were treated with paclitaxel (100 nM) for 12 h and 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. (e) Fluorescence polarization assays were performed using recombinant GST-Bcl-2 (100 μM) and FITC-labeled Bim and Bmf BH3 peptides (10 nM). Unlabeled Bim and Bmf BH3 peptides were used to compete with binding of FITC-labeled Bim and Bmf BH3 peptides to test the ability of Bim and Bmf to displace each other from Bcl-2. Data shown are mean ± SEM of four independent experiments in duplicate.

Mentions: To evaluate the involvement of Bmf in paclitaxel-induced apoptosis in MDA-MB-468 and BT20 cells, we initially monitored whether Bmf interacts with antiapoptotic Bcl-2 family proteins in response to paclitaxel treatment for 0-12 hours using coimmunoprecipitation experiments. As demonstrated in Figure 6a, both Bcl-2 and Bcl-xL interact weakly with Bmf in untreated MDA-MB-468 cells, which was more clearly shown with reciprocal Bmf immunoprecipitation experiments. Dramatically increased Bmf/Bcl-2 and Bmf/Bcl-xL interactions in MDA-MB-468 were detected after 8 h of paclitaxel treatment. Similarly, paclitaxel treatment resulted in induction of Bmf/Bcl-2 and Bmf/Bcl-xL protein complexes in BT20 cells (Figure 6b). In neither case was an interaction between Bmf and Mcl-1 detectable (not shown). Note that this pattern of Bmf binding to Bcl-2 and Bcl-xL matches the displacement of Bim from the same proteins with the same kinetics. Because increased Puma levels and decreased Bim/Bcl-xL interaction were observed in T47D cells, we tested whether Puma can contribute to paclitaxel-induced apoptosis in this cell line. Thereby, we investigated the interaction of Puma with Bcl-xL in response to paclitaxel treatment for 12 h. In Figure 6c, we show that paclitaxel induces increased Bcl-xL-bound Puma levels, which was also evident in reciprocal immunoprecipitation experiments.


Displacement of Bim by Bmf and Puma rather than increase in Bim level mediates paclitaxel-induced apoptosis in breast cancer cells.

Kutuk O, Letai A - Cell Death Differ. (2010)

Bmf is required for paclitaxel-induced apoptosis in breast cancer cells. (a) MDA-MB-468 and (b) BT20 cells were treated with paclitaxel (100 nM) for 0, 4, 8, and 12 h and the interaction of Bmf with Bcl-2 and Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (c) T47D cells were treated with paclitaxel (100 nM) 12 h and the interaction of Puma with Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (d) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. T47D cells were transiently transfected with Puma siRNA or Scrambled siRNA for 48 h. The efficiency of knockdowns was monitored by immunoblots. Untransfected and siRNA-transfected cells were treated with paclitaxel (100 nM) for 48 h and apoptosis was evaluated by using Annexin V staining (mean ± SEM, n = 4, *p< 0.05, **p< 0.01 by two-tailed t test). (e) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. Cells were treated with paclitaxel (100 nM) for 12 h and 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. (e) Fluorescence polarization assays were performed using recombinant GST-Bcl-2 (100 μM) and FITC-labeled Bim and Bmf BH3 peptides (10 nM). Unlabeled Bim and Bmf BH3 peptides were used to compete with binding of FITC-labeled Bim and Bmf BH3 peptides to test the ability of Bim and Bmf to displace each other from Bcl-2. Data shown are mean ± SEM of four independent experiments in duplicate.
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Figure 6: Bmf is required for paclitaxel-induced apoptosis in breast cancer cells. (a) MDA-MB-468 and (b) BT20 cells were treated with paclitaxel (100 nM) for 0, 4, 8, and 12 h and the interaction of Bmf with Bcl-2 and Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (c) T47D cells were treated with paclitaxel (100 nM) 12 h and the interaction of Puma with Bcl-xL was evaluated by coimmunoprecipitation assays. Inputs for coimmunoprecipitations were also subjected to immunoblot analysis and actin was probed as a loading control. (d) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. T47D cells were transiently transfected with Puma siRNA or Scrambled siRNA for 48 h. The efficiency of knockdowns was monitored by immunoblots. Untransfected and siRNA-transfected cells were treated with paclitaxel (100 nM) for 48 h and apoptosis was evaluated by using Annexin V staining (mean ± SEM, n = 4, *p< 0.05, **p< 0.01 by two-tailed t test). (e) MDA-MB-468 and BT20 cells were transiently transfected with Bmf siRNA or Scrambled siRNA for 48 h. Cells were treated with paclitaxel (100 nM) for 12 h and 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. (e) Fluorescence polarization assays were performed using recombinant GST-Bcl-2 (100 μM) and FITC-labeled Bim and Bmf BH3 peptides (10 nM). Unlabeled Bim and Bmf BH3 peptides were used to compete with binding of FITC-labeled Bim and Bmf BH3 peptides to test the ability of Bim and Bmf to displace each other from Bcl-2. Data shown are mean ± SEM of four independent experiments in duplicate.
Mentions: To evaluate the involvement of Bmf in paclitaxel-induced apoptosis in MDA-MB-468 and BT20 cells, we initially monitored whether Bmf interacts with antiapoptotic Bcl-2 family proteins in response to paclitaxel treatment for 0-12 hours using coimmunoprecipitation experiments. As demonstrated in Figure 6a, both Bcl-2 and Bcl-xL interact weakly with Bmf in untreated MDA-MB-468 cells, which was more clearly shown with reciprocal Bmf immunoprecipitation experiments. Dramatically increased Bmf/Bcl-2 and Bmf/Bcl-xL interactions in MDA-MB-468 were detected after 8 h of paclitaxel treatment. Similarly, paclitaxel treatment resulted in induction of Bmf/Bcl-2 and Bmf/Bcl-xL protein complexes in BT20 cells (Figure 6b). In neither case was an interaction between Bmf and Mcl-1 detectable (not shown). Note that this pattern of Bmf binding to Bcl-2 and Bcl-xL matches the displacement of Bim from the same proteins with the same kinetics. Because increased Puma levels and decreased Bim/Bcl-xL interaction were observed in T47D cells, we tested whether Puma can contribute to paclitaxel-induced apoptosis in this cell line. Thereby, we investigated the interaction of Puma with Bcl-xL in response to paclitaxel treatment for 12 h. In Figure 6c, we show that paclitaxel induces increased Bcl-xL-bound Puma levels, which was also evident in reciprocal immunoprecipitation experiments.

Bottom Line: However, the signaling pathways that connect paclitaxel-induced microtubule perturbation to mitochondrial outer membrane permeabilization and cytochrome c release are not well characterized.Bim and either Puma or Bmf are required for paclitaxel toxicity.This novel mechanism suggests the potential usage of novel therapies targeted at altering BH3-only protein heterodimerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

ABSTRACT
Taxanes exert their antitumor effect through stabilizing microtubule dynamics and initiating G2/M arrest in cancer cells followed by apoptotic cell death. However, the signaling pathways that connect paclitaxel-induced microtubule perturbation to mitochondrial outer membrane permeabilization and cytochrome c release are not well characterized. Here, we show that in breast cancer cells, paclitaxel induces a novel displacement mechanism: prodeath BH3-only proteins Bmf and Puma competitively displace prodeath BH3-only protein Bim from antiapoptotic proteins to activate Bax and Bak and commit the cell to apoptotic death. Bim and either Puma or Bmf are required for paclitaxel toxicity. Although prior mechanisms of apoptosis induced by taxol have focused on changes in Bim levels, we find that an increase is not required for paclitaxel killing of breast cancer cells. Rather, competitive displacement of Bim from antiapoptotic proteins is the important step committing the cell to death. This novel mechanism suggests the potential usage of novel therapies targeted at altering BH3-only protein heterodimerization.

Show MeSH
Related in: MedlinePlus