Critical Role of p53 Upregulated Modulator of Apoptosis in Benzyl Isothiocyanate-Induced Apoptotic Cell Death Marie Lue Antony, Su-Hyeong Kim, Shivendra V. Singh* Department of Pharmacology & Chemical Biology, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America Abstract Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC- mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl- 2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA. Citation: Antony ML, Kim S-H, Singh SV (2012) Critical Role of p53 Upregulated Modulator of Apoptosis in Benzyl Isothiocyanate-Induced Apoptotic Cell Death. PLoS ONE 7(2): e32267. doi:10.1371/journal.pone.0032267 Editor: Aamir Ahmad, Wayne State University School of Medicine, United States of America Received December 8, 2011; Accepted January 25, 2012; Published February 16, 2012 Copyright: 2012 Antony et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This investigation was supported by a research grant from the National Cancer Institute at the National Institutes of Health [RO1 CA129347-05 (SVS)]. This research project used the Tissue and Research Pathology Facility supported by a grant from the National Cancer Institute at the National Institutes of Health (P30 CA047904). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: singhs@upmc.edu Introduction Bioactive compounds from dietary sources continue to draw attention for possible use to prevent breast cancer [1–3], which is a leading cause of cancer-related mortality in American women [4]. Cruciferous vegetable constituent benzyl isothiocyanate (BITC) is one such compound with compelling preclinical evidence for preventive efficacy against breast cancer in experimental rodents. Mammary cancer prevention using BITC was first demonstrated by Wattenberg in a rat model of chemically-induced cancer [5]. BITC administration prior to the carcinogen challenge inhibited 7,12-dimethylbenz[a]anthracene-induced mammary tumor devel- opment in female Sprague-Dawley rats [5]. Studies from our laboratory have revealed that BITC administration in the diet confers significant protection against mammary cancer develop- ment in MMTV-neu transgenic mice [6]. The BITC-mediated inhibition of breast cancer xenograft growth in vivo has also been documented [7,8]. We have shown previously that BITC-mediated prevention of mammary cancer development in MMTV-neu mice is associated with inhibition of cell proliferation and increased apoptosis [6]. In agreement with our findings [6], BITC-mediated inhibition of 4T1 murine breast cancer xenograft growth in BALB/c mice was accompanied by increased apoptosis [8]. In cellular models of human breast cancer (MDA-MB-231 and MCF-7), BITC treatment causes G2/M phase cell cycle arrest and apoptosis induction [9–12]. A spontaneously immortalized and non- tumorigenic human mammary epithelial cell line (MCF-10A), originally isolated from a fibrocystic breast disease, is signifi- cantly more resistant to BITC-induced apoptosis compared with breast cancer cells [11]. The mechanism by which BITC causes cell death is not fully understood, but proapoptotic response to this agent in human breast cancer cells is inti- mately linked to production of reactive oxygen species (ROS) because of inhibition of complex III of the mitochondrial respiratory chain [12]. Activation of caspases and suppression of X-linked inhibitor of apoptosis protein are other mechanistic events associated with BITC-induced apoptosis in breast cancer cells [11–13]. We have also observed other novel pharmacological responses for BITC, including inhibition of oncogenic actions of leptin and suppression of epithelial-mesen- chymal transition [14,15]. PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32267