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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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Paclitaxel activates the mitochondrial apoptotic pathway in breast cancer cells. (a) T47D, MDA-MB-468 and BT20 cells were treated with paclitaxel (0, 1, 10, 20, 50, and 100 nM) for 48 h or 100 nM paclitaxel for 0-48 h and apoptosis was evaluated by Annexin V staining (mean ± SEM, n = 4). Bcl-2 family proteins in (b) T47D, (c) MDA-MB-468 and (d) BT20 cells were detected by immunoblot analysis following treatment with paclitaxel (100 nM) for 0-48 h. Actin was probed as a loading control.
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Figure 3: Paclitaxel activates the mitochondrial apoptotic pathway in breast cancer cells. (a) T47D, MDA-MB-468 and BT20 cells were treated with paclitaxel (0, 1, 10, 20, 50, and 100 nM) for 48 h or 100 nM paclitaxel for 0-48 h and apoptosis was evaluated by Annexin V staining (mean ± SEM, n = 4). Bcl-2 family proteins in (b) T47D, (c) MDA-MB-468 and (d) BT20 cells were detected by immunoblot analysis following treatment with paclitaxel (100 nM) for 0-48 h. Actin was probed as a loading control.

Mentions: Above we described a novel mechanism of serial displacement of pro-apoptotic proteins from anti-apoptotic proteins that governs commitment to apoptosis in paclitaxel-treated MCF-7 cells. To explore whether paclitaxel activates a similar proapoptotic pathway in other breast cancer cells, we exposed T47D, MDA-MB-468 and BT20 cells to increasing concentrations of paclitaxel for 48 h and 100 nM paclitaxel for 0-48 h. Using identical methods to those in Figures 1 and 2 for MCF-7, we found that paclitaxel induced mitochondrial apoptosis at similar concentrations with similar kinetics in the T47D, MDA-MB-468, and BT20 breast cancer cell lines (Figure 3a). Cytochrome c was released from the mitochondria and Bax and Bak were activated with similar kinetics in all cases (Supplementary Figure S3a-S3c, S4a-S4c). Treatment of breast cancer cells with paclitaxel for up to 48 hours also revealed the activation of caspases 3 and 9 in breast cancer cells as assessed by immunoblot analysis of cleaved caspase fragments and fluorogenic caspase assays (Supplementary Figure S1d, e and f). Moreover, paclitaxel-induced apoptosis was blocked by pretreating cells with pancaspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK, but not the caspase-8 inhibitor Z-IETD-FMK (Supplementary Figure S1d, e and f). In all breast cancer cell lines studied, therefore, paclitaxel kills in a caspase 9-dependent fashion using the mitochondrial apoptotic pathway.


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)

Paclitaxel activates the mitochondrial apoptotic pathway in breast cancer cells. (a) T47D, MDA-MB-468 and BT20 cells were treated with paclitaxel (0, 1, 10, 20, 50, and 100 nM) for 48 h or 100 nM paclitaxel for 0-48 h and apoptosis was evaluated by Annexin V staining (mean ± SEM, n = 4). Bcl-2 family proteins in (b) T47D, (c) MDA-MB-468 and (d) BT20 cells were detected by immunoblot analysis following treatment with paclitaxel (100 nM) for 0-48 h. Actin was probed as a loading control.
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Related In: Results  -  Collection

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Figure 3: Paclitaxel activates the mitochondrial apoptotic pathway in breast cancer cells. (a) T47D, MDA-MB-468 and BT20 cells were treated with paclitaxel (0, 1, 10, 20, 50, and 100 nM) for 48 h or 100 nM paclitaxel for 0-48 h and apoptosis was evaluated by Annexin V staining (mean ± SEM, n = 4). Bcl-2 family proteins in (b) T47D, (c) MDA-MB-468 and (d) BT20 cells were detected by immunoblot analysis following treatment with paclitaxel (100 nM) for 0-48 h. Actin was probed as a loading control.
Mentions: Above we described a novel mechanism of serial displacement of pro-apoptotic proteins from anti-apoptotic proteins that governs commitment to apoptosis in paclitaxel-treated MCF-7 cells. To explore whether paclitaxel activates a similar proapoptotic pathway in other breast cancer cells, we exposed T47D, MDA-MB-468 and BT20 cells to increasing concentrations of paclitaxel for 48 h and 100 nM paclitaxel for 0-48 h. Using identical methods to those in Figures 1 and 2 for MCF-7, we found that paclitaxel induced mitochondrial apoptosis at similar concentrations with similar kinetics in the T47D, MDA-MB-468, and BT20 breast cancer cell lines (Figure 3a). Cytochrome c was released from the mitochondria and Bax and Bak were activated with similar kinetics in all cases (Supplementary Figure S3a-S3c, S4a-S4c). Treatment of breast cancer cells with paclitaxel for up to 48 hours also revealed the activation of caspases 3 and 9 in breast cancer cells as assessed by immunoblot analysis of cleaved caspase fragments and fluorogenic caspase assays (Supplementary Figure S1d, e and f). Moreover, paclitaxel-induced apoptosis was blocked by pretreating cells with pancaspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK, but not the caspase-8 inhibitor Z-IETD-FMK (Supplementary Figure S1d, e and f). In all breast cancer cell lines studied, therefore, paclitaxel kills in a caspase 9-dependent fashion using the mitochondrial apoptotic pathway.

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