Autophagy fails to alter withaferin A-mediated lethality in human breast cancer cells

Abstract

We have shown previously that withaferin A (WA), which is a highly promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits viability of cultured breast cancer cells in association with reactive oxygen species (ROS)-dependent apoptosis induction. Because ROS production is implicated in induction of autophagy, which is an evolutionary conserved process for bulk degradation of cellular components including organelles (e.g., mitochondria) and considered a valid cancer chemotherapeutic target, we questioned whether WA treatment resulted in autophagy induction. Indeed exposure of MDA-MB-231 and MCF-7 human breast cancer cells as well as a spontaneously immortalized and non-tumorigenic normal human mammary epithelial cell line (MCF-10A) to pharmacologic concentration of WA resulted in autophagy as evidenced by transmission electron microscopy, processing of microtubuleassociated protein 1 light chain 3 isoform B, and/or acridine orange staining. Inhibition of MDA-MB-231 xenograft growth in vivo by WA administration was also associated with a significant increase in level of LC3 protein in the tumor. However, WA-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was not compromised either by pharmacological suppression of autophagy using 3-methyl adenine or genetic repression of autophagy by RNA interference of Atg5, a critical component of the autophagic machinery. Finally, Beclin1 was dispensable for WA-mediated autophagy as well as inhibition of MDA-MB-231 cell viability. Based on these observations we conclude that autophagy induction fails to have any meaningful impact on WA-mediated lethality in breast cancer cells, which may be a therapeutic advantage because autophagy serves to protect against apoptosis by several anticancer agents.

Fig. (1). Withaferin A treatment caused autophagy in human breast cancer cells

Effect of WA treatment (12 hour) on ultrastructure (enlarged in inset) of MDA-MB-231 (A) and MCF-7 (B) cells at the indicated magnifications (scale bar, 2 microns). Autophagosome-like structures are identified by red arrows. C, Representative images depicting acridine orange staining in MDA-MB-231 and MCF-7 cells treated with DMSO or 2 µM of WA for the specified time periods. Consistent results were obtained from independent experiments.

Fig. (2). Withaferin A treatment caused LC3 cleavage in human breast cancer cells

Representative immunofluorescence images and quantitation for LC3 puncta (indicated by the white arrow) from MDA-MB-231 (A) and MCF-7 (B) cells treated with DMSO (control) or 2 µM of WA for specified time periods (100×objective magnification). Results shown are mean ± SD (n=3). ^*Statistical significance of difference (P<0.05) was analyzed by a two-sided student’s t-test. (C) Immunoblotting for LC3B-I and LC3B-II (cleaved form identified by an arrow) using lysates from MDA-MB-231 (left panel) and MCF-7 (right panel) cells treated with DMSO (control) or 2 µM of WA for specified time periods. Numbers on the top of bands represent the relative expression compared to corresponding DMSO-treated control. All the experiments were done at least twice, and consistent results were observed.

Fig. (3). Withaferin A treatment caused autophagy in MCF-10A normal human mammary cells

A, Immunoblotting for LC3B-I and LC3B-II (cleaved form identified by an arrow) using lysates from MCF-10A cells treated with DMSO (control) or 2 µM of WA for specified time periods. Numbers on the top of bands represent the relative expression level compared to corresponding DMSO-treated control. B, Representative images for LC3 localization in MCF-10A cells treated for specified time periods with DMSO or 2 µM of WA. LC3 puncta are indicated by arrows (100×objective magnification). C, Representative images depicting acridine orange staining in MCF-10A cells treated with DMSO or 2 µM of WA for specified time periods. D, Representative immunohistochemical images and quantitative analysis for LC3 expression from MDA-MB-231 xenografts from control or 4 mg/kg WA-treated mice (magnification 200×, Scale bar 100 µm). Results are presented as mean H-score ± SD (n=7). ^*Statistical significance of difference between groups (P<0.05) was analyzed by a two-sided student’s t-test. All the experiments were repeated with similar results.

Fig. (4). Autophagy inhibition by 3-methyl adenine (3-MA) did not influence WA-mediated inhibition of cell viability

Effect of 3-MA treatment on WA-mediated growth inhibition of MDA-MB-231 (A) and MCF-7 (B) cells. Cells were pretreated with 4 mM 3-MA for 2 hours and then exposed to DMSO or 2 µM of WA for 6 or 12 hours. Cell viability was determined by trypan blue dye exclusion assay. Results are presented as mean ± SD (n=3). ^aSignificantly different (P<0.05) compared with DMSO-treated control by one way ANOVA followed by Bonferroni’s multiple comparison test. Similar results were obtained from replicate experiments.

Fig. (5). RNA interference of ATG5 did not affect WA-mediated inhibition of cell viability

A, Immunoblot for Atg5–12 using lysates from MDA-MB-231 and MCF-7 cells transiently transfected with a control siRNA- or Atg5-specific siRNA. B, Immunoblot for LC3B using lysates from MDA-MB-231 and MCF-7 cells transiently transfected with a control siRNA or Atg5-targeted siRNA and treated for 12 hours with DMSO or 2 µM of WA. C, Viability of MDA-MB-231 and MCF-7 cells transiently transfected with a control siRNA or Atg5-targeted siRNA and treated for 12 hours with DMSO or 2 µM of WA. Cell viability was determined by trypan blue dye exclusion assay. Results are shown as mean ± SD (n=3). ^aSignificantly different (P<0.05) compared with DMSO-treated control by one way ANOVA followed by Bonferroni’s multiple comparison test. D, Immunoblot for PARP using lysates from MDA-MB-231 and MCF-7 cells transiently transfected with a control siRNA or Atg5-targeted siRNA and treated for 12 hours with DMSO or 2 µM of WA. Similar results were obtained from replicate experiments.

Fig. (6). Beclin1 was dispensable for WA-mediated cleavage of LC3 and growth inhibition in MDA-MB-231 cells

A, Immunoblot for Beclin1 using lysates from MDA-MB-231 cells transiently transfected with a control siRNA- or a Beclin1-specific siRNA. B, Immunoblot for LC3B using lysates from MDA-MB-231 cells transiently transfected with a control siRNA or a Beclin1-targeted siRNA and treated for 12 hours with DMSO or 2 µM of WA. C, Viability of MDA-MB-231 cells transiently transfected with a control siRNA or a Beclin1-targeted siRNA and treated for 12 hours with DMSO or 2 µM of WA. Cell viability was determined by trypan blue dye exclusion assay. Results are shown as mean ± SD (n=3). ^aSignificantly different (P<0.05) compared with DMSO-treated control by one way ANOVA followed by Bonferroni’s multiple comparison test. Similar results were obtained from replicate experiments.