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

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.

Figure 1

Resveratrol sensitizes breast cancer cells to docetaxel-induced cytotoxicity, while being non-toxic to normal immortalized breast cells. (a) Dose-dependent cytotoxicity of docetaxel (0.1–10 nM) on breast cancer cells with varied receptor status. (b) Effect of different concentrations of resveratrol (10–25 μM) on different breast cancer cells. (c) Effect of docetaxel (1 nM) and resveratrol (15 μM), alone or in combination on different breast cancer cells. Cells (5×10³) in triplicates were exposed to the indicated concentrations of the docetaxel for 48 h and subjected to MTT assay. Relative cell viability was determined as percentage absorbance over untreated control. (d) Effect of docetaxel and resveratrol, alone or in combination, on SK-BR-3 cells using [³H] thymidine incorporation assay. Cells (5×10³) in triplicates were exposed to the indicated concentrations of the drugs for 24 h and subjected to [³H] thymidine incorporation assay. Relative cell viability was determined as percentage thymidine incorporation over control. (e) The combination is non-toxic to MCF10A as assessed by [³H] thymidine incorporation assay. (f) Inhibitory effect of docetaxel and resveratrol, alone or in combination, on the clonogenic ability of SK-BR-3 cells (scale bar, 1 μm/px).

Figure 2

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.

Figure 3

Resveratrol downregulates docetaxel-induced activation of HER-2. (a Kinetics of docetaxel (1 nM)-induced activation of HER-2 in SK-BR-3 cells (0–48 h). The whole-cell lysate of docetaxel-treated cells at different time intervals was immunoblotted against HER-2 antibody. (b and c) Effect of resveratrol treatment on docetaxel-induced overexpression and activation of HER-2 in SK-BR-3 cells. Cells pretreated with resveratrol for 24 h were further exposed to docetaxel and/or resveratrol for another 24 h and immunoblotted against specific antibodies. β-Tubulin is used as loading control. (d) Effect of HER-2 inhibition on synergism in SK-BR-3 cells. Cells were transiently transfected with vector control and DN-HER-2, respectively. (e) Effect of ectopic expression of HER-2 on synergism in MDA-MB-231 cells. Cells were transfected with vector control and WT-HER-2, respectively. The efficacy of transfection was confirmed using western blotting and cell viability was assessed in the transfected cells using MTT assay after treatment with docetaxel and resveratrol alone, or in combination for 48 h in both cases.

Figure 4

Akt is the regulator of the synergism, although resveratrol downregulates docetaxel-induced upregulation of Akt and MAPK pathways in SK-BR-3 cells. (a) Kinetics of docetaxel-induced activation of Akt. Cells were treated with docetaxel for different time intervals (0–2 h). The whole-cell lysate was immunoblotted against phospho-Akt (ser473) antibody. (b) Resveratrol-mediated downregulation of docetaxel-induced activation of Akt. Western blot analyses were performed with anti-phospho-Akt (ser473) using whole-cell lysates prepared after 30 min exposure to docetaxel. (c) Effect of resveratrol on docetaxel-induced upregulation of phospho-Bad. Western blot analysis was performed against anti-phospho-Bad (ser136). (d) Kinetics of activation of MAPKs by docetaxel (0–2 h). The whole-cell lysate was immunoblotted against phospho-specific antibodies of ERK1/2, JNK and p38. (e) Resveratrol downregulates docetaxel-induced upregulation of various MAPKs. β-Actin was used as loading control in all cases. (f) Inhibition of docetaxel-induced activation of AP-1 by resveratrol. Nuclear extracts prepared after exposing the cells to docetaxel and resveratrol, either alone or in combination for a period of 1 h, were assayed for AP-1 activation by EMSA. (g) Super-shift analysis using anti-c-jun antibody to indicate band specificity. (h) Kinetics of docetaxel-induced activation of NF-κB. Nuclear extracts were prepared after exposing the cells to 1 nM docetaxel for different time intervals (0–3 h) and NF-κB status was assessed by EMSA. (i) Individual and combined effects of docetaxel and resveratrol for a period of 30 min on NF-κB activation. NF-κB activation was assayed by EMSA. (j) Effect of docetaxel and resveratrol, alone or in combination, in cells treated with Akt and MAPKs inhibitors. Cells (5×10³) in triplicates were pretreated with resveratrol, LY294002 (1 μM), U0126 (5 μM), SP600125 (5 μM) and SB203580 (1 μM), followed by docetaxel treatment for 48 h and subjected to MTT assay. Inhibition status of Akt and various MAPKs were shown in inset.

Figure 5

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.

Figure 6

Proposed model for the synergistic effect of docetaxel and resveratrol. Resveratrol downregulates docetaxel-induced upregulation of HER-2, Akt and MAPKs, while that of NF-κB is unaffected. The study postulates that docetaxel-induced upregulation of HER-2–Akt signaling and the downregulation of the same by resveratrol is the main mechanism governing the synergistic effect of docetaxel and resveratrol in HER-2-overexpressing breast cancer cells. Although MAPK pathway does not regulate the synergism, it is getting activated by docetaxel and downregulated by resveratrol. The bold lines indicate the signaling pathways regulating the synergism, whereas the dotted lines represent those that do not have any role in the same.