cell-cycle regulators [37] is evident in cells resistant to DNA- damage-induced apoptosis. We have shown previously that ROS generation is a critical event in BITC-induced apoptosis in MDA-MB-231 and MCF-7 cells [12]. Attenuation of apoptosis by ectopic expression of catalase and superoxide dismutase reinforces the notion that ROS provide initial signal for BITC-induced apoptosis at least in breast cancer cells [12]. The BITC-mediated ROS production is associated with inhibition of complex III of the mitochondrial respiratory chain [12]. Notably, PUMA can be induced by oxidative stress in neuronal and colon cancer cells [38,39]. The PUMA-deficient neurons are resistant to apoptosis induction by oxidative stress [38]. At the same time, Liu et al [40] have shown that PUMA overexpression itself causes ROS generation in colon cancer cells. It would be interesting to determine if ROS function upstream of PUMA induction or PUMA induction is partly responsible for the pro-oxidant effect of BITC in addition to inhibition of mitochondrial electron transport chain. Nevertheless, both ROS production and PUMA induction seem important for BITC-induced apoptosis in breast cancer cells [12, and present study]. The multidomain proapoptotic protein Bax seems necessary for PUMA-mediated apoptosis. For example, the Bax knockout HCT-116 cells are fully resistant to apoptosis induction by PUMA overexpression as well as stimuli leading to PUMA-dependent apoptosis [41]. Moreover, PUMA overexpression has been shown to cause conformational change, polymerization, and mitochon- drial translocation of Bax [41,42]. We have also shown previously that BITC treatment causes ROS-dependent activation (confor- mational change) and mitochondrial translocation of Bax in MDA-MB-231 cells [12]. Moreover, SV40 immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice are partially but significantly more resistant to BITC-induced apoptosis compared with mouse embryonic fibroblasts derived from wild-type mice [11]. We propose a working mechanistic model involving ROS-dependent induction of PUMA and Bax activation in BITC-induced apoptosis. PUMA contributes to apoptosis by directly interacting with anti- apoptotic Bcl-2 family members [20]. Direct interaction between anti-apoptotic proteins and PUMA is dependent on its BH3 domain as this interaction is disrupted by deletion or mutations in BH3 domain [28]. The BH3 peptide of PUMA is capable of interacting with anti-apoptotic proteins [43,44]. Overexpression of PUMA has been shown to cause dissociation of Bax from Bcl- xL [42]. Interestingly, BITC treatment not only causes the induction of PUMA, which is likely to relieve inhibitory effects of Bcl-xL and Bcl-2, but also causes downregulation of Bcl-xL in both MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells. It is possible that Bcl-xL and Bcl-2 downregulation itself contributes to PUMA-independent apoptosis in our model. However, the molecular basis for differential effect of BITC treatment on the level of Bcl-2 protein in MCF-7 (downregula- tion) versus MDA-MB-231 cells (upregulation) remains elusive. We have already ruled out involvement of p53 in this differential response. In summary, the present study indicates that: (a) the Bim protein is dispensable for proapoptotic response to BITC in breast cancer cells; (b) PUMA induction and downregulation of Bcl-xL protein contribute to BITC-induced apoptosis, which is not a cell line- specific phenomenon; (c) BITC treatment differentially affects the levels of Bcl-2 protein in MCF-7 versus MDA-MB-231 cells; and (d) PUMA deficiency in HCT-116 human colon cancer cell line increases its sensitivity to BITC-induced G2/M phase cell cycle arrest. PUMA Regulates BITC-Induced Apoptosis Methods Ethics statement The MDA-MB-231 tumor xenografts from control and BITC- treated mice archived from our previously published study [7] were used in the present study to determine the effect of BITC administration on expression of PUMA and Bcl-xL proteins. Use of mice was approved by the Institutional Animal Care and Use Committee (protocol number 1004983A-4). Reagents BITC was purchased from LKT Laboratories (St. Paul, MN). Stock solution of BITC was prepared in DMSO and an equal volume of DMSO (,0.05%) was added to the controls. Reagents for cell culture including medium, fetal bovine serum, antibiotics and Alexa Fluor 568-conjugated donkey anti-rabbit antibody were purchased from Invitrogen-Life Technologies (Carlsbad, CA). Antibody against Bcl-2 was from DAKO Cytomation (Carpin- teria, CA); antibody against Bim was from Cell Signaling Technology (Beverly, MA); anti-p53 antibody was from Calbio- chem-EMD Chemicals (Gibbstown, NJ); anti-actin antibody, 49,6- diamidino-2-phenylindole (DAPI) and propidium iodide were from Sigma-Aldrich (St. Louis, MO). The siRNA targeted against p53, Bcl-2, Bim, and PUMA, and antibodies against Bcl-xL, PUMA, and cdk1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti p21 antibody was from BD Biosciences (San Diego, CA). A nonspecific control siRNA was from Qiagen (Germantown, MD). A kit for quantification of histone-associated DNA fragment release into the cytosol was purchased from Roche Applied Science (Indianapolis, IN). Cell lines MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture collection (Manassas, VA), and main- tained as described by us previously [11,12]. Wild-type HCT-116 human colon cancer cell line and its isogenic PUMA knockout variant were generously provided by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD) and cultured in McCoy’s 5A modified medium supplemented with 10% fetal bovine serum and penicillin/streptomycin antibiotic mixture. Western blotting Control and BITC-treated cells and tumor tissues from mice treated with vehicle control and BITC were processed for immunoblotting as described by us previously [45–47]. Proteins from cell lysates and tumor supernatants were resolved by sodium- dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto membrane. The membrane was incubated with the desired primary antibody for overnight at 4uC after blocking in 5% non- fat dry milk. Immunoreactive bands were visualized by enhanced chemiluminescence method. Densitometric quantitation was done using UN-SCAN-IT software version 5.1 (Silk Scientific Corpo- ration, Orem, Utah, USA). RNA interference MDA-MB-231 and MCF-7 cells were transfected at ,50% confluency with 100 nM of target specific siRNA or a control siRNA using OligoFECTAMINE (Invitrogen-Life Technologies). Twenty-four hours post transfection, cells were treated with DMSO (control) or BITC for specified time period. Cells were collected and processed for immunoblotting and measurement for histone-associated DNA fragment release into the cytosol. PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e32267