PUMA Regulates BITC-Induced Apoptosis Figure 3. BITC treatment causes G2/M phase cell cycle arrest in PUMA knockout HCT-116 cells. (A) Representative flow histograms depicting cell cycle distribution in wild-type HCT-116 cells (WT) and PUMA knockout HCT-116 cells (PUMA KO) after 24 h treatment with DMSO or 5 mM BITC. Quantitation of (B) sub-G0/G1 and (C) G2/M fraction in WT and PUMA KO HCT-116 cells after 24 h treatment with DMSO or 5 mM BITC. The experiment was repeated twice and merged data from both the experiments are shown. Data represent mean 6 SD (n = 4). aSignificantly different (P,0.05) compared with DMSO-treated control by one-way ANOVA followed by Bonferroni’s multiple comparison test. (D) Immunoblotting for p21 and cdk1 using lysates from WT and PUMA KO HCT-116 cells treated for 24 h with DMSO or the indicated concentrations of BITC. Each experiment was repeated at least twice. doi:10.1371/journal.pone.0032267.g003 administration on the expression of PUMA and Bcl-xL proteins. The MDA-MB-231 tumors from BITC-treated mice exhibited induction of PUMA protein when compared with tumors from vehicle-treated control mice as judged by immunohistochemistry (Fig. 6A) and western blotting (Fig. 6B). Even though BITC treatment resulted in in vivo induction of Bcl-xL as well (Fig. 6C), the difference was not significant (Fig. 6D). These results provided in vivo evidence for BITC-mediated induction of PUMA protein in MDA-MB-231 xenografts. Discussion The results presented herein indicate that PUMA plays an important role in BITC-induced apoptosis. This conclusion is based on the following observations: (a) the BITC treatment increases the level of PUMA protein in breast cancer cells, which is not a cell line- specific response but relatively more pronounced in cells with wild- type p53 (MCF-7); although RNA interference of p53 alone has minimal impact on BITC-induced apoptosis in MCF-7 cells [13], PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32267