Figure 2. PUMA knockout HCT-116 cells are partially resistant to BITC-induced apoptosis. (A) Immunoblotting for PUMA protein using lysates from wild-type HCT-116 cells (WT) and PUMA knockout HCT-116 cells (PUMA KO) following 24 h treatment with DMSO or BITC (2.5 or 5 mM). (B) Quantitation of histone-associated DNA fragment release into the cytosol in WT and PUMA KO HCT-116 cells after 24 h treatment with DMSO or BITC (2.5 or 5 mM). Results are expressed as enrichment relative to corresponding DMSO-treated control. Data represent mean 6 SD (n = 4). Significantly different (P,0.05) compared PUMA Regulates BITC-Induced Apoptosis with arespective DMSO-treated control and bbetween WT and PUMA KO HCT-116 cells by one-way ANOVA followed by Bonferroni’s multiple comparison test. (C) Visualization of apoptotic nuclei (DAPI assay) with condensed and fragmented DNA (identified by arrows) in WT HCT-116 cells after 24-hour treatment with DMSO or 5 mM BITC. (D) Quantitation of apoptotic nuclei in WT and PUMA KO HCT-116 cells following 24 h treatment with DMSO or BITC (2.5 or 5 mM). Data represent mean 6 SD (n = 3). Significantly different (P,0.05) compared with arespective DMSO-treated control and bbetween WT and PUMA KO cells by one- way ANOVA followed by Bonferroni’s multiple comparison test. Each experiment was repeated at least twice. doi:10.1371/journal.pone.0032267.g002 (Fig. 4C), overexpression of Bcl-xL protein was protective against BITC-mediated apoptosis in MDA-MB-231 cells (Fig. 5C). Because the expression of Bcl-2 protein was increased after BITC treatment in MDA-MB-231 cells, we determined the effect of its knockdown on BITC-induced apoptosis. Level of Bcl-2 protein was decreased by 80% in MDA-MB-231 cells transfected with the Bcl-2-targeted siRNA compared with control siRNA transfected cells (Fig. 5D). Knockdown of Bcl-2 alone increased apoptosis in MDA-MB-231 cells (Fig. 5E). In addition, the BITC-induced apoptosis was increased significantly in MDA-MB-231 cells upon knockdown of Bcl-2 when compared with cells transfected with the control siRNA (Fig. 5E). Collectively, these results indicated that MCF-7 and MDA-MB-231 cells responded differentially to BITC- mediated alterations in Bcl-2 protein expression. p53 is dispensable for BITC-mediated downregulation of Bcl-2 protein in MCF-7 cells Next, we questioned whether differential behavior of MCF-7 versus MDA-MB-231 cells to BITC-mediated change in Bcl-2 protein expression was related to difference in p53 status. Twenty- four hour treatment of control siRNA transfected MCF-7 cells to 5 mM BITC resulted in .10-fold increase in level of p53 protein (Fig. 5F). This effect was not observed in MCF-7 cells transfected with the p53-targeted siRNA. However, the BITC-mediated suppression of Bcl-2 protein level was observed in MCF-7 cells transfected with both control siRNA and p53-targetd siRNA (Fig. 5F). These results indicated that BITC-mediated downreg- ulation of Bcl-2 was not influenced by the p53 status at least in MCF-7 cells. Role of p53 in BITC-mediated induction of PUMA We sought to determine the role of p53 in BITC-mediated induction of PUMA using MCF-7 cells. As can be seen in Fig. 5G, MCF-7 cells transfected with the p53-targeted siRNA showed a 60% decrease in the level of p53 protein in comparison with cells transfected with the control siRNA. In addition, the BITC- mediated induction of PUMA was partially reversed upon RNA interference of p53. Two possibilities exist to explain these results: (a) the BITC-mediated induction of PUMA in MCF-7 cells is only partially regulated by p53, and (b) PUMA induction after treatment with BITC in MCF-7 cells transfected with the p53- targeted siRNA is simply a consequence of incomplete knockdown of the p53 protein. Nevertheless, based on data in MDA-MB-231 cells, it is likely that both p53-dependent and -independent mechanisms are responsible for BITC-mediated induction of PUMA at least in breast cancer cells. BITC administration causes in vivo induction of PUMA in MDA-MB-231 xenografts We used tumor specimens from our previously published MDA- MB-231 xenograft study [7] to determine the in vivo effect of BITC PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32267