Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Abstract

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.

Figure 1. Withaferin A (WA) treatment causes reactive oxygen species (ROS) production in human breast cancer cells.

(A) Chemical structure of WA. (B) Flow cytometric analysis for MitoSOX Red fluorescence in DMSO-treated control and WA-treated MDA-MB-231 and MCF-7 cells (4 h treatment). Results are shown as enrichment factor relative to DMSO-treated control (mean ± SD, n = 3). *P<0.05, significantly different compared with DMSO-treated control by one-way ANOVA with Dunnett's adjustment. (C) Fluorescence microscopy for MitoSOX Red fluorescence in MDA-MB-231 and MCF-7 cells following 4 h treatment with DMSO or 2.5 µM WA. (D) Fluorescence microscopy for MitoSOX Red fluorescence in HMEC following 4 h treatment with DMSO or 2.5 µM WA. (E) Representative EPR spectra in HMEC treated for 4 h with DMSO or 5 µM WA. All experiments were repeated at least twice.

Figure 2. Cu,Zn-Superoxide dismutase (Cu,Zn-SOD) overexpression attenuates withaferin A (WA)-induced apoptosis in MDA-MB-231 and MCF-7 cells.

(A) Immunoblotting for Cu,Zn-SOD using lysates from MDA-MB-231 or MCF-7 cells stably transfected with empty vector (lane 1) or vector encoding for Cu,Zn-SOD (lane 2). (B) Representative EPR spectra in MDA-MB-231 and MCF-7 cells stably transfected with empty vector or vector encoding for Cu,Zn-SOD and treated for 4 h with DMSO or 5 µM WA. (C) Quantitation of EPR signal intensity in MDA-MB-231 and MCF-7 cells transfected with empty vector or vector encoding for Cu,Zn-SOD and treated for 4 h with DMSO or 5 µM WA. Results shown are mean ± SD (n = 3). (D) Histone-associated DNA fragment release into the cytosol (a measure of apoptosis) in MDA-MB-231 and MCF-7 cells transfected with empty vector or vector encoding for Cu,Zn-SOD and treated for 24 h with DMSO or WA. Results shown are mean ± SD (n = 3). (E) Immunoblotting for cleaved caspase-3 using lysates from MDA-MB-231 cells stably transfected with empty vector or vector encoding for Cu,Zn-SOD and treated for 24 h with DMSO or WA. (F) Histone-associated DNA fragment release into the cytosol in HMEC treated for 24 h with DMSO or WA. Results shown are mean ± SD (n = 3). Significantly different (P<0.05) compared with ^acontrol (DMSO-treated), and ^b between groups at each dose by one-way ANOVA followed by Bonferroni's adjustment. For data in panels C,D, and F, data are shown as enrichment factor relative to DMSO-treated control. All experiments were repeated at least twice.

Figure 3. Withaferin A (WA) treatment inhibits oxidative phosphorylation (OXPHOS) in MDA-MB-231 and MCF-7 cells.

(A) Pharmacologic profiling of oxygen consumption rate (OCR), an indicator of OXPHOS, in MDA-MB-231 and MCF-7 cells following 4 h treatment with DMSO (control) or the indicated concentrations of WA. After measurement of basal OCR, the cells were treated with a series of metabolic inhibitors, including oligomycin (injection A); FCCP (injection B); 2-DG (injection C); and rotenone (injection D) at the indicated times. (B) Effect of WA treatment on basal OCR in MDA-MB-231 and MCF-7 cells. Basal OCR was calculated using the difference between the mean of time points prior to injection A (#1–#4) and after injection D (#14 to #16; rotenone-sensitive) (). (C) Effect of WA treatment on total reserve respiration capacity in MDA-MB-231 and MCF-7 cells. Total reserve respiration capacity was calculated using the mean of the time points after injection C (#11–#13) minus the mean of time points after injection D (#14–#16) (). Data shown are mean ± SEM of three independent experiments, each performed in triplicate. *P<0.05; **P<0.01; and ***P<0.001, significantly different compared with control by one-way ANOVA with Dunnett's adjustment.

Figure 4. Withaferin A (WA) treatment fails to alter acidification rate (lactate production)and steady-state levels of ATP.

(A) Effect of WA treatment on basal extracellular acidification rate (ECAR), a measure of lactate production, in MDA-MB-231 and MCF-7 cells following 4 h treatment with DMSO (control) or the indicated concentrations of WA. (B) Effect of WA treatment on oligomycin-induced (oligo-induced) ECAR in MDA-MB-231 and MCF-7 cells. Results shown are mean ± SEM of three independent experiments, each performed in triplicate. (C) Steady-state levels of ATP in MDA-MB-231 and MCF-7 cells treated with DMSO (control) or the indicated concentrations of WA in the absence or presence of the metabolic inhibitors. Results shown are mean ± SEM of combination of three independent experiments, each performed in quadruplicate. *P<0.05; **P<0.01; and ***P<0.001, significantly different compared with control by one-way ANOVA with Dunnett's adjustment (Panels A and B) or Student's t-test (panel C).

Figure 5. WA treatment inhibits complex III activity in MDA-MB-231 breast cancer cells.

Mitochondrial complex enzyme activities were determined using lysates from MDA-MB-231 cells treated for 6 h with DMSO or 5 µM WA. Results shown are mean ± SD (n = 3). *P<0.05; **P<0.01, significantly different compared with control by Student's t-test.

Figure 6. Rho-0 variants of MDA-MB-231 and MCF-7 cells are resistant to withaferin A (WA)-mediated apoptosis.

(A) MitoSOX Red fluorescence (a measure of ROS production) in wild-type (WT) and Rho-0 variants of MDA-MB-231 and MCF-7 cells following 4 h treatment with DMSO (control) or WA. Enrichment of MitoSOX Red fluorescence relative to DMSO-treated wild-type cells is shown for both MDA-MB-231 and MCF-7 cells. (B) Histone-associated DNA fragment release into the cytosol in wild-type and Rho-0 variants of MDA-MB-231 and MCF-7 cells following 24 h treatment with DMSO or WA. Enrichment relative to DMSO-treated wild-type cells is shown for both MDA-MB-231 and MCF-7 cells. (C) Analysis of mitochondrial membrane potential (monomeric JC-1-associated green fluorescence) in wild-type and Rho-0 variants of MDA-MB-231 and MCF-7 cells following 24 h treatment with DMSO or WA. Enrichment relative to DMSO-treated wild-type cells is shown for both MDA-MB-231 and MCF-7 cells. Results shown are mean ± SD (n = 3). Significantly different (P<0.05) compared with corresponding ^acontrol (DMSO-treated) and ^bbetween groups at each dose by one-way ANOVA followed by Bonferroni's multiple comparison test. Each experiment was repeated at least twice.

Figure 7. Withaferin A (WA) treatment causes activation of Bak and Bax in breast cancer cells.

Immunofluorescence microscopy for active Bak and Bax in MDA-MB-231 (A), MCF-7 (B), and HMEC (C) following 24 h treatment with DMSO (control) or 2.5 µM WA. Each experiment was repeated at least twice.

Figure 8. Bak and Bax are required for withaferin A (WA)-induced apoptosis.

(A) Immunofluorescence microscopy for active Bak and Bax in MDA-MB-231 cells stably transfected with empty vector or vector encoding for Cu,Zn-SOD and treated for 24 h with DMSO or WA. (B) Immunoblotting for Bak and Bax using lysates from MCF-7 cells transiently transfected with a control nonspecific small interfering RNA (siRNA; lane 1) or Bax- or Bak-targeted siRNA (lane 2). (C) Histone-associated DNA fragment release into the cytosol in siRNA-transfected MCF-7 cells following 24 h treatment with DMSO (control) or the indicated concentrations of WA. Results are shown as enrichment factor relative to DMSO-treated control siRNA transfected cells (mean ± SD, n = 3). (D) Fluorescence microscopic analysis for apoptotic cells with condensed and fragmented DNA (DAPI assay) in SV40 immortalized mouse embryonic fibroblasts (MEF) derived from wild-type (WT) and Bax and Bak double knockout (DKO) mice and treated for 24 h with DMSO (control) or 5 µM WA. (E) Histone-associated DNA fragment release into the cytosol in WT and DKO treated for 24 h with DMSO (control) or the indicated concentrations of WA. Results are shown as enrichment factor relative to DMSO-treated wild-type MEF (mean ± SD, n = 3). Significantly different (P<0.05) compared with ^aDMSO-treated control siRNA-transfected MCF-7 cells (panel C) or DMSO-treated WT MEF (panel E), and ^bbetween groups at each dose by one-way ANOVA followed by Bonferroni's test. Similar results were observed in two independent experiments.