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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.


Related in: MedlinePlus

Resveratrol enhances docetaxel-induced apoptosis in SK-BR-3 cells. (a) Cells were treated with resveratrol and/or docetaxel for 16 h and stained for Annexin V–propidium iodide (PI) positivity. Annexin V-positive cells in different fields were counted and the average was taken. The green-stained cells are those that have taken only the Annexin V–FITC stain and represent initial stages of apoptosis, and the red-stained cells are those that have taken up both Annexin–FITC and PI, which indicates nuclear membrane damage, and hence represent later stages of apoptosis. Representative histograms indicate percentage of annexin-positive cells. ***P-value ≤0.001.(b–e) Resveratrol-mediated enhancement of docetaxel-induced caspase activation. Whole cell lysate of cells treated with docetaxel and/or resveratrol for 48 h were blotted against caspase antibodies. (f) Resveratrol enhances docetaxel-induced PARP cleavage. Whole-cell extracts were blotted against anti-PARP antibody. (g) Effect of docetaxel and resveratrol, alone or in combination, on cell cycle. Cells were collected 48 h post drug treatment, fixed in alcohol, stained with propidium iodide and assayed for DNA content by flow cytometry. (h) The effect of docetaxel and/or resveratrol on inter-nucleosomal DNA fragmentation. Cells were treated with docetaxel and/or resveratrol for 48 h, DNA was isolated, run on an agarose gel and visualized. All experiments were repeated at least three times to confirm the reproducibility.
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fig2: Resveratrol enhances docetaxel-induced apoptosis in SK-BR-3 cells. (a) Cells were treated with resveratrol and/or docetaxel for 16 h and stained for Annexin V–propidium iodide (PI) positivity. Annexin V-positive cells in different fields were counted and the average was taken. The green-stained cells are those that have taken only the Annexin V–FITC stain and represent initial stages of apoptosis, and the red-stained cells are those that have taken up both Annexin–FITC and PI, which indicates nuclear membrane damage, and hence represent later stages of apoptosis. Representative histograms indicate percentage of annexin-positive cells. ***P-value ≤0.001.(b–e) Resveratrol-mediated enhancement of docetaxel-induced caspase activation. Whole cell lysate of cells treated with docetaxel and/or resveratrol for 48 h were blotted against caspase antibodies. (f) Resveratrol enhances docetaxel-induced PARP cleavage. Whole-cell extracts were blotted against anti-PARP antibody. (g) Effect of docetaxel and resveratrol, alone or in combination, on cell cycle. Cells were collected 48 h post drug treatment, fixed in alcohol, stained with propidium iodide and assayed for DNA content by flow cytometry. (h) The effect of docetaxel and/or resveratrol on inter-nucleosomal DNA fragmentation. Cells were treated with docetaxel and/or resveratrol for 48 h, DNA was isolated, run on an agarose gel and visualized. All experiments were repeated at least three times to confirm the reproducibility.

Mentions: Various apoptotic assays were performed, to confirm the results obtained from the preliminary cytotoxic evaluation of the combination. The results obtained from Annexin V/propidium iodide staining was in concordance with that of MTT assay. SKBR-3 cells treated with the combination exhibited a significant enhancement in externalization of phoshatidyl serine, an early event of apoptosis, compared with that treated with either of these compounds alone (Figure 2a). The combination induced a momentous cleavage of pro-caspase-8 to its active fragment (p18 ) compared with the cells treated with either of the two compounds alone (Figure 2b). The combination also induced the cleavage of procaspase-9, procaspase-3 and procaspase-7 to their active fragments (Figures 2c–e) and a significant enhancement in the cleavage of PARP, the downstream target of caspase cascade (Figure 2f). In addition, treatment with the combination induced a tremendous accumulation of cells in sub-G0 phase (28.1%), confirming the induction of apoptosis by the combination as assessed by PI–FACS analysis. However, resveratrol treatment did not induce a significant enhancement in docetaxel-induced cell cycle arrest (Figure 2g). Moreover, an enhancement in the inter-nucleosomal cleavage of DNA, the biochemical hallmark of apoptosis, was also observed in cells treated with combination (Figure 2h).


Resveratrol chemosensitizes HER-2-overexpressing breast cancer cells to docetaxel chemoresistance by inhibiting docetaxel-mediated activation of HER-2 – Akt axis
Resveratrol enhances docetaxel-induced apoptosis in SK-BR-3 cells. (a) Cells were treated with resveratrol and/or docetaxel for 16 h and stained for Annexin V–propidium iodide (PI) positivity. Annexin V-positive cells in different fields were counted and the average was taken. The green-stained cells are those that have taken only the Annexin V–FITC stain and represent initial stages of apoptosis, and the red-stained cells are those that have taken up both Annexin–FITC and PI, which indicates nuclear membrane damage, and hence represent later stages of apoptosis. Representative histograms indicate percentage of annexin-positive cells. ***P-value ≤0.001.(b–e) Resveratrol-mediated enhancement of docetaxel-induced caspase activation. Whole cell lysate of cells treated with docetaxel and/or resveratrol for 48 h were blotted against caspase antibodies. (f) Resveratrol enhances docetaxel-induced PARP cleavage. Whole-cell extracts were blotted against anti-PARP antibody. (g) Effect of docetaxel and resveratrol, alone or in combination, on cell cycle. Cells were collected 48 h post drug treatment, fixed in alcohol, stained with propidium iodide and assayed for DNA content by flow cytometry. (h) The effect of docetaxel and/or resveratrol on inter-nucleosomal DNA fragmentation. Cells were treated with docetaxel and/or resveratrol for 48 h, DNA was isolated, run on an agarose gel and visualized. All experiments were repeated at least three times to confirm the reproducibility.
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fig2: Resveratrol enhances docetaxel-induced apoptosis in SK-BR-3 cells. (a) Cells were treated with resveratrol and/or docetaxel for 16 h and stained for Annexin V–propidium iodide (PI) positivity. Annexin V-positive cells in different fields were counted and the average was taken. The green-stained cells are those that have taken only the Annexin V–FITC stain and represent initial stages of apoptosis, and the red-stained cells are those that have taken up both Annexin–FITC and PI, which indicates nuclear membrane damage, and hence represent later stages of apoptosis. Representative histograms indicate percentage of annexin-positive cells. ***P-value ≤0.001.(b–e) Resveratrol-mediated enhancement of docetaxel-induced caspase activation. Whole cell lysate of cells treated with docetaxel and/or resveratrol for 48 h were blotted against caspase antibodies. (f) Resveratrol enhances docetaxel-induced PARP cleavage. Whole-cell extracts were blotted against anti-PARP antibody. (g) Effect of docetaxel and resveratrol, alone or in combination, on cell cycle. Cells were collected 48 h post drug treatment, fixed in alcohol, stained with propidium iodide and assayed for DNA content by flow cytometry. (h) The effect of docetaxel and/or resveratrol on inter-nucleosomal DNA fragmentation. Cells were treated with docetaxel and/or resveratrol for 48 h, DNA was isolated, run on an agarose gel and visualized. All experiments were repeated at least three times to confirm the reproducibility.
Mentions: Various apoptotic assays were performed, to confirm the results obtained from the preliminary cytotoxic evaluation of the combination. The results obtained from Annexin V/propidium iodide staining was in concordance with that of MTT assay. SKBR-3 cells treated with the combination exhibited a significant enhancement in externalization of phoshatidyl serine, an early event of apoptosis, compared with that treated with either of these compounds alone (Figure 2a). The combination induced a momentous cleavage of pro-caspase-8 to its active fragment (p18 ) compared with the cells treated with either of the two compounds alone (Figure 2b). The combination also induced the cleavage of procaspase-9, procaspase-3 and procaspase-7 to their active fragments (Figures 2c–e) and a significant enhancement in the cleavage of PARP, the downstream target of caspase cascade (Figure 2f). In addition, treatment with the combination induced a tremendous accumulation of cells in sub-G0 phase (28.1%), confirming the induction of apoptosis by the combination as assessed by PI–FACS analysis. However, resveratrol treatment did not induce a significant enhancement in docetaxel-induced cell cycle arrest (Figure 2g). Moreover, an enhancement in the inter-nucleosomal cleavage of DNA, the biochemical hallmark of apoptosis, was also observed in cells treated with combination (Figure 2h).

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.


Related in: MedlinePlus