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Resveratrol chemosensitizes HER-2-overexpressing breast cancer cells to docetaxel chemoresistance by inhibiting docetaxel-mediated activation of HER-2 – Akt axis

View Article: PubMed Central - PubMed

ABSTRACT

As breast cancer cells often develop chemoresistance, better therapeutic options are in search to circumvent it. Here we demonstrate that human epidermal growth factor receptor-2 (HER-2)-overexpressing breast cancer cells resist docetaxel-induced cytotoxicity by upregulating HER-2 and its activity downstream, through Akt and mitogen-activated protein kinase (MAPK) pathways. We observed that introducing resveratrol as a chemosensitizer in docetaxel chemotherapy blocks upregulation and activation of HER-2 in addition to blocking downstream signaling pathways such as Akt. Resveratrol and docetaxel combination results in the synergistic induction of cell death in HER-2-overexpressing SK-BR-3 cells, whereas introduction of wild-type HER-2 in MDA-MD-231 cells increased the resistance to docetaxel. Dominant-negative HER-2 sensitizes SK-BR-3 cells to docetaxel. Our study identified a new synergistic therapeutic combination that targets HER-2-induced breast cancer resistance and might help to overcome therapeutic resistance during breast cancer therapy. The synergism of docetaxel and resveratrol was maximum in SK-BR-3, which is unique among the cell lines studied, due to its high expression status of HER-2, a receptor known to dictate the signaling environment of breast cancer cells. Docetaxel could further induce HER-2 activity in these cells, which was downregulated on resveratrol treatment. Transfection of DN-HER-2 in SK-BR-3 cells inhibits the synergism as the transfection itself sensitizes these cells to docetaxel, leaving no role for resveratrol, whereas ectopic expression of HER-2 introduces the synergism in MDA-MB-231, the triple-negative cell line, in which the synergism was minimum, attesting the crucial role of HER-2 in suppressing the sensitivity to docetaxel. Single-agent docetaxel induced HER-2-mediated resistance to cell death, which was blocked by resveratrol. Resveratrol also downregulated docetaxel-induced activation of MAPK and Akt, survival signaling pathways downstream of HER-2. In short, this study, for the first time, establishes the role of HER-2–Akt signaling axis in regulating the synergistic effect of docetaxel and resveratrol in breast cancer cells overexpressing HER-2.

No MeSH data available.


Docetaxel-induced upregulation of XIAP, survivin and Bcl-2 are downregulated by resveratrol in SK-BR-3 cells. (a) Effect of HER-2 inhibition on activation of Akt and downstream target survivin. (b) Effect of HER-2 inhibition on docetaxel-induced upregulation of phospho-Akt. (c) Effect of Akt inhibition on docetaxel-induced activation of HER-2. Western blotting analysis was done using specific antibodies against each molecule and β-actin was used as loading control in all cases. (d) Resveratrol-mediated inhibition of kinase activity of all three classes of Akt. The study was performed using z-lite biochemical assay platform. (e) Kinetics of docetaxel-induced activation of survivin and the inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h) and the whole-cell lysates were immunoblotted against anti-survivin. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for 8 h and immunoblotted against survivin antibody. (f) Kinetics of docetaxel-induced activation of XIAP and inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h). Western blotting analysis was done against anti-XIAP. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for another 16 h and western blot analysis was performed using antibody against anti-XIAP. (g) Kinetics of docetaxel-induced activation of Bcl-2 and inhibition of the same by synergistic combination of resveratrol and docetaxel. Cells were treated with of docetaxel for different time intervals (0–48 h). Western blot analysis was performed against anti-Bcl-2. Cells were pretreated with resveratrol followed by combination of resveratrol and docetaxel for 4 h. Whole-cell lysate was immunoblotted using anti-Bcl-2. β-Actin was used as loading control in all the experiments.
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fig5: Docetaxel-induced upregulation of XIAP, survivin and Bcl-2 are downregulated by resveratrol in SK-BR-3 cells. (a) Effect of HER-2 inhibition on activation of Akt and downstream target survivin. (b) Effect of HER-2 inhibition on docetaxel-induced upregulation of phospho-Akt. (c) Effect of Akt inhibition on docetaxel-induced activation of HER-2. Western blotting analysis was done using specific antibodies against each molecule and β-actin was used as loading control in all cases. (d) Resveratrol-mediated inhibition of kinase activity of all three classes of Akt. The study was performed using z-lite biochemical assay platform. (e) Kinetics of docetaxel-induced activation of survivin and the inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h) and the whole-cell lysates were immunoblotted against anti-survivin. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for 8 h and immunoblotted against survivin antibody. (f) Kinetics of docetaxel-induced activation of XIAP and inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h). Western blotting analysis was done against anti-XIAP. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for another 16 h and western blot analysis was performed using antibody against anti-XIAP. (g) Kinetics of docetaxel-induced activation of Bcl-2 and inhibition of the same by synergistic combination of resveratrol and docetaxel. Cells were treated with of docetaxel for different time intervals (0–48 h). Western blot analysis was performed against anti-Bcl-2. Cells were pretreated with resveratrol followed by combination of resveratrol and docetaxel for 4 h. Whole-cell lysate was immunoblotted using anti-Bcl-2. β-Actin was used as loading control in all the experiments.

Mentions: We have gathered further proof for the existence of HER-2–Akt signaling axis in regulating the synergism. We verified that Akt activity is coupled to HER-2 signaling in SK-BR-3 cells by demonstrating the inhibition of basal Akt signaling in DN-HER-2-transfected cells (Figure 5a). We found that docetaxel could not induce Akt activation in these cells, attesting that arbitration of HER-2 is necessary for docetaxel to induce Akt (Figure 5b). It is interesting to note that Akt inhibition neither blocks HER-2 from being induced in response to docetaxel nor prevent HER-2 from being inhibited in response to the combination of docetaxel and resveratrol, verifying that Akt is downstream of HER-2 (Figure 5c). The independent regulatory role of resveratrol on kinase activity of all three classes of Akt was confirmed by z-lite biochemical assay platform, where isolated Akts (Akt1, Akt2 and Akt3) were screened against resveratrol. Interestingly, the results shows that resveratrol induces maximum inhibition to Akt-2, which is the focus molecule of current study (Figure 5d).


Resveratrol chemosensitizes HER-2-overexpressing breast cancer cells to docetaxel chemoresistance by inhibiting docetaxel-mediated activation of HER-2 – Akt axis
Docetaxel-induced upregulation of XIAP, survivin and Bcl-2 are downregulated by resveratrol in SK-BR-3 cells. (a) Effect of HER-2 inhibition on activation of Akt and downstream target survivin. (b) Effect of HER-2 inhibition on docetaxel-induced upregulation of phospho-Akt. (c) Effect of Akt inhibition on docetaxel-induced activation of HER-2. Western blotting analysis was done using specific antibodies against each molecule and β-actin was used as loading control in all cases. (d) Resveratrol-mediated inhibition of kinase activity of all three classes of Akt. The study was performed using z-lite biochemical assay platform. (e) Kinetics of docetaxel-induced activation of survivin and the inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h) and the whole-cell lysates were immunoblotted against anti-survivin. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for 8 h and immunoblotted against survivin antibody. (f) Kinetics of docetaxel-induced activation of XIAP and inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h). Western blotting analysis was done against anti-XIAP. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for another 16 h and western blot analysis was performed using antibody against anti-XIAP. (g) Kinetics of docetaxel-induced activation of Bcl-2 and inhibition of the same by synergistic combination of resveratrol and docetaxel. Cells were treated with of docetaxel for different time intervals (0–48 h). Western blot analysis was performed against anti-Bcl-2. Cells were pretreated with resveratrol followed by combination of resveratrol and docetaxel for 4 h. Whole-cell lysate was immunoblotted using anti-Bcl-2. β-Actin was used as loading control in all the experiments.
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fig5: Docetaxel-induced upregulation of XIAP, survivin and Bcl-2 are downregulated by resveratrol in SK-BR-3 cells. (a) Effect of HER-2 inhibition on activation of Akt and downstream target survivin. (b) Effect of HER-2 inhibition on docetaxel-induced upregulation of phospho-Akt. (c) Effect of Akt inhibition on docetaxel-induced activation of HER-2. Western blotting analysis was done using specific antibodies against each molecule and β-actin was used as loading control in all cases. (d) Resveratrol-mediated inhibition of kinase activity of all three classes of Akt. The study was performed using z-lite biochemical assay platform. (e) Kinetics of docetaxel-induced activation of survivin and the inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h) and the whole-cell lysates were immunoblotted against anti-survivin. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for 8 h and immunoblotted against survivin antibody. (f) Kinetics of docetaxel-induced activation of XIAP and inhibition of the same by synergistic combination of resveratrol and docetaxel. The cells were treated with docetaxel for different time intervals (0–24 h). Western blotting analysis was done against anti-XIAP. The cells were pretreated with resveratrol for 24 h followed by docetaxel alone or in combination with resveratrol for another 16 h and western blot analysis was performed using antibody against anti-XIAP. (g) Kinetics of docetaxel-induced activation of Bcl-2 and inhibition of the same by synergistic combination of resveratrol and docetaxel. Cells were treated with of docetaxel for different time intervals (0–48 h). Western blot analysis was performed against anti-Bcl-2. Cells were pretreated with resveratrol followed by combination of resveratrol and docetaxel for 4 h. Whole-cell lysate was immunoblotted using anti-Bcl-2. β-Actin was used as loading control in all the experiments.
Mentions: We have gathered further proof for the existence of HER-2–Akt signaling axis in regulating the synergism. We verified that Akt activity is coupled to HER-2 signaling in SK-BR-3 cells by demonstrating the inhibition of basal Akt signaling in DN-HER-2-transfected cells (Figure 5a). We found that docetaxel could not induce Akt activation in these cells, attesting that arbitration of HER-2 is necessary for docetaxel to induce Akt (Figure 5b). It is interesting to note that Akt inhibition neither blocks HER-2 from being induced in response to docetaxel nor prevent HER-2 from being inhibited in response to the combination of docetaxel and resveratrol, verifying that Akt is downstream of HER-2 (Figure 5c). The independent regulatory role of resveratrol on kinase activity of all three classes of Akt was confirmed by z-lite biochemical assay platform, where isolated Akts (Akt1, Akt2 and Akt3) were screened against resveratrol. Interestingly, the results shows that resveratrol induces maximum inhibition to Akt-2, which is the focus molecule of current study (Figure 5d).

View Article: PubMed Central - PubMed

ABSTRACT

As breast cancer cells often develop chemoresistance, better therapeutic options are in search to circumvent it. Here we demonstrate that human epidermal growth factor receptor-2 (HER-2)-overexpressing breast cancer cells resist docetaxel-induced cytotoxicity by upregulating HER-2 and its activity downstream, through Akt and mitogen-activated protein kinase (MAPK) pathways. We observed that introducing resveratrol as a chemosensitizer in docetaxel chemotherapy blocks upregulation and activation of HER-2 in addition to blocking downstream signaling pathways such as Akt. Resveratrol and docetaxel combination results in the synergistic induction of cell death in HER-2-overexpressing SK-BR-3 cells, whereas introduction of wild-type HER-2 in MDA-MD-231 cells increased the resistance to docetaxel. Dominant-negative HER-2 sensitizes SK-BR-3 cells to docetaxel. Our study identified a new synergistic therapeutic combination that targets HER-2-induced breast cancer resistance and might help to overcome therapeutic resistance during breast cancer therapy. The synergism of docetaxel and resveratrol was maximum in SK-BR-3, which is unique among the cell lines studied, due to its high expression status of HER-2, a receptor known to dictate the signaling environment of breast cancer cells. Docetaxel could further induce HER-2 activity in these cells, which was downregulated on resveratrol treatment. Transfection of DN-HER-2 in SK-BR-3 cells inhibits the synergism as the transfection itself sensitizes these cells to docetaxel, leaving no role for resveratrol, whereas ectopic expression of HER-2 introduces the synergism in MDA-MB-231, the triple-negative cell line, in which the synergism was minimum, attesting the crucial role of HER-2 in suppressing the sensitivity to docetaxel. Single-agent docetaxel induced HER-2-mediated resistance to cell death, which was blocked by resveratrol. Resveratrol also downregulated docetaxel-induced activation of MAPK and Akt, survival signaling pathways downstream of HER-2. In short, this study, for the first time, establishes the role of HER-2–Akt signaling axis in regulating the synergistic effect of docetaxel and resveratrol in breast cancer cells overexpressing HER-2.

No MeSH data available.