Abstract
Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.
Highlights
Breast cancer continues to be a leading cause of cancer-related deaths in women worldwide even after remarkable progress towards targeted therapies [1]
Autophagic response to benzyl isothiocyanate (BITC) treatment was confirmed by analysis of acidic vesicular organelles (AVOs) and cleavage of microtubule-associated protein 1 light chain 3 (LC3), which are hallmarks of autophagy [19,20,21]
The present study shows that BITC treatment causes autophagy in breast cancer cells that is not influenced by the estrogen receptor expression
Summary
Breast cancer continues to be a leading cause of cancer-related deaths in women worldwide even after remarkable progress towards targeted therapies [1]. Novel approaches for chemoprevention of breast cancer are clinically attractive because many of the known risk factors associated with this devastating disease (e.g., family history and delayed menopause) are beyond human control and currently available chemopreventive options, such as selective estrogen receptor modulators (e.g., tamoxifen) and aromatase inhibitors, are sub-optimal [2,3,4,5]. Even though the mechanisms underlying growth inhibitory effect of BITC against breast cancer are not fully understood, we have shown previously that BITC treatment inhibits complex III of the mitochondrial respiratory chain leading to production of reactive oxygen species (ROS), activation of c-Jun N-terminal kinase-Bax axis, and apoptotic cell death in MDA-MB231 and MCF-7 cells [9,14]. We showed further that while p53 tumor suppressor is dispensable for BITC-
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