Mitochondria-mediated apoptosis downstream of ROS produc- tion and upstream of caspase activation is regulated by Bcl-2 family proteins, which function to either inhibit (e.g., Bcl-2, Bcl-xL etc.) or facilitate (e.g., Bak, Bax, and Bim) apoptosis [16–20]. We have already established that SV40 immortalized mouse embry- onic fibroblasts derived from Bax and Bak double knockout mice are significantly more resistant to BITC-induced apoptosis compared with those derived from the wild-type mice [11]. However, the role of other members of the Bcl-2 family pro- teins in regulation of BITC-induced apoptosis remains elusive. The present study logically extends our previous findings [11–13] to systematically investigate the role of B-cell lymphoma 2 interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (PUMA), Bcl-xL, and Bcl-2 proteins in regulation of BITC-induced apoptosis using MCF-7 (breast), MDA-MB-231 (breast), and HCT-116 (colon) human cancer cells as a model. Results Bim is dispensable for BITC-induced apoptosis in MCF-7 and MDA-MB-231 cells The c-Jun N-terminal kinase (JNK) is often implicated in ROS-dependent apoptosis by different stimuli, including certain natural agents [21–24]. The JNK activation results in phosphor- ylation of the BH3-only protein Bim leading to activation of multidomain proapoptotic protein Bax [25,26]. Our previous studies have indicated that BITC treatment causes ROS/JNK- dependent activation of Bax in breast cancer cells [12]. Thus, it was logical to test whether BITC-induced apoptosis was mediated by Bim. The BITC-induced apoptosis, as judged by analysis of histone-associated DNA fragment release into the cytosol, was not attenuated by RNA interference of Bim in either MCF-7 or MDA-MB-231 cells (results not shown). These results indicated that Bim protein was dispensable for proapoptotic response to BITC at least in MCF-7 and MDA-MB- 231 cells. BITC treatment increases PUMA protein expression in MCF-7 and MDA-MB-231 cells PUMA is another BH3-only member of the Bcl-2 family that facilitates apoptosis by different stimuli [20]. For example, PUMA has been shown to indirectly activate Bax in the absence of Bim and Bid [27]. There was a marked increase in the levels of PUMA protein after treatment with BITC in both MCF-7 and MDA-MB- 231 cells (Fig. 1A). However, this response was relatively more pronounced in the wild-type p53 expressing MCF-7 cells than in the MDA-MB-231 cells, which express mutant p53 (Fig. 1A). Immunofluorescence microscopy confirmed BITC-mediated in- duction of PUMA in MCF-7 cells (Fig. 1B). Because BITC- mediated induction of PUMA was relatively more pronounced in MCF-7 cells, we used this cell line for functional assays involving RNA interference. Level of PUMA protein was decreased by .90% upon transient transfection of MCF-7 cells with the PUMA-targeted small interfering RNA (siRNA) in comparison with cells transfected with a control siRNA (Fig. 1C). Furthermore, the BITC-mediated induction of PUMA was fully abolished by its knockdown. Moreover, knockdown of PUMA protein conferred partial but statistically significant protection against BITC- mediated increase in histone-associated DNA fragment release into the cytosol (Fig. 1D). These results indicated that PUMA induction potentially contributed to BITC-induced apoptosis in MCF-7 cells. PUMA Regulates BITC-Induced Apoptosis Next, we questioned if PUMA-dependence of BITC-induced apoptosis was unique to the MCF-7 cells. We used wild-type and PUMA knockout variant of HCT-116 cells to address this question. As can be seen in Fig. 2A, level of PUMA protein was modestly increased after 24 h treatment of wild-type HCT-116 cells with 5 mM BITC. The BITC treatment resulted in a dose- dependent and significant increase in histone-associated DNA fragment release into the cytosol over DMSO-treated control in wild-type HCT-116 cells (Fig. 2B). The PUMA knockout HCT- 116 cells were relatively more resistant to BITC-induced apoptosis compared with wild-type cells (Fig. 2B). Representative micro- scopic images depicting apoptotic cells with condensed and fragmented DNA in wild-type HCT-116 cells after 24 h treatment with DMSO (control) and 5 mM BITC are shown in Fig. 2C (apoptotic nuclei are marked with arrows). In agreement with results shown in Fig. 2B, PUMA knockout HCT-116 cells were significantly more resistant to BITC-induced apoptosis in comparison with wild-type HCT-116 cells at least at the 5 mM concentration (Fig. 2D). Collectively, these results indicated that PUMA-dependence of BITC-induced apoptosis was not a cell line-specific response. PUMA deficiency increases BITC-mediated G2/M phase cell cycle arrest in HCT-116 cells We have shown previously that BITC treatment causes G2/M phase cell cycle arrest in breast cancer cells [11]. We designed experiments using wild-type and PUMA knockout HCT-116 cells to determine if PUMA deficiency affected BITC-mediated cell cycle arrest. Fig. 3A shows representative flow histograms for cell cycle distribution in wild-type and PUMA knockout HCT-116 cells after 24 h treatment with DMSO (control) or 5 mM BITC. As can be seen in Fig. 3B, BITC treatment resulted in a significant increase in fraction of sub-diploid (apoptotic) cells in both wild- type and PUMA knockout HCT-116 cells. Consistent with data shown in Fig. 2 (B,D), the BITC-mediated enrichment of sub- diploid fraction was relatively more pronounced in the wild-type HCT-116 cells than in its PUMA knockout variant (Fig. 3B). Furthermore, the BITC-induced G2/M phase cell cycle arrest was relatively more pronounced in the PUMA knockout cells compared with wild-type HCT-116 cells (Fig. 3C). The BITC- treated PUMA knockout cells exhibited an increase in protein level of p21, but this effect was not evident in the wild-type HCT- 116 cells (Fig. 3D). Finally, BITC treatment caused a modest increase in protein level of cyclin-dependent kinase 1 (cdk1) (30– 50% increase over DMSO-treated control) in wild-type HCT-116 cells (Fig. 3D). In contrast, expression of cdk1 protein was decreased markedly upon treatment of PUMA knockout HCT- 116 cells with BITC (Fig. 3D). These results indicated that attenuation of BITC-induced apoptosis in PUMA knockout HCT- 116 cells was accompanied by an increase in G2/M phase cell cycle arrest due to induction of p21 and downregulation of cdk1 protein. BITC treatment decreases levels of Bcl-xL and Bcl-2 proteins in MCF-7 cells The PUMA protein serves to activate Bax by relieving inhibition by the anti-apoptotic Bcl-2 family members, including Bcl-2, Bcl-xL, and Mcl-1 [20]. We have shown previously that BITC-induced apoptosis in MCF-7 and MDA-MB-231 cells is associated with suppression of Bcl-xL and/or Bcl-2 [11]. However, functional studies to test the role of these proteins in the context of BITC-induced apoptosis in breast cancer cells are lacking. As shown in Fig. 4A, BITC treatment resulted in suppression of both PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32267