Figure 4. Protein expression in HCC cells treated with Lexa or the combination of Lexa and CHX. LH86 cells were treated with Lexa (1 mg/ml) or pre-treated with CHX (10 mg/ml) followed by Lexa (1 mg/ml) for up to 6 h. Cells were harvested and cell lysates were prepared for Western blotting. Bcl-xL, survivin, Bim, Bad, Bak, Bax, DR4, and DR5 were detected with specific antibodies respectively. b-actin was detected with anti-b-actin mouse antibody for an equal protein loading control. doi:10.1371/journal.pone.0016966.g004 DR5 showed no significant changes (Fig. 4). These results suggest that the inhibitor CHX could not completely block protein synthesis stimulated by Lexa in HCC cells and that apoptosis induced by combination treatment of Lexa and CHX may be mediated by Bax/Bak activation. Combination treatment of Lexa and CHX triggers the activation of Bax It is well known that under various cellular stresses, pro- apoptotic Bax/Bak can be activated through oligomerization and conformation change, resulting in mitochondria-dependent apop- tosis [32–35]. Therefore, we examined whether Lexa and CHX co-treatment can activate Bax through oligomerization in HCC cells. Our results revealed that in the presence of CHX (10 mg/ml), Bax dimerization was up-regulated by Lexa in HCC cells (Fig. 5A, 5B, and 5C). To further test whether ROS was involved in Bax dimerization, cells were pre-incubated with NAC before the addition of Lexa and CHX. As shown in Fig. 5C, pre-treatment with NAC resulted in significant inhibition of Bax dimerization induced by combination treatment of Lexa and CHX. Next we attempted to test the possibility that intracellular ROS could induce Bax to undergo conformation change in HCC cells combination-treated with Lexa and CHX. To this end, a Bax monoclonal antibody 6A7 that specifically recognizes conforma- tionally changed Bax was used to do immunoprecipitation. As shown in Fig. 5D, combination treatment of Lexa and CHX could induce Bax conformation change in both LH86 and Huh7 HCC cells. As expected, with the addition of antioxidants NAC to block ROS, conformationally changed Bax was significant decreased. After oligomerization and conformational change, the activated Bax can translocate to mitochondria, which results in cytochrome CHX and Lexa Combination-Induced Apoptosis in HCC c release. In our study, we observed transiently transfected GFP- Bax translocation to mitochondria in cells treated with Lexa and CHX for 6 h (Fig. 6A). Next, we studied cytochrome c release from mitochondria in cells with combinational treatment of Lexa and CHX. Immunofluorescence staining results indicated that in cells treated with Lexa and CHX, cytochrome c was released and diffused into the cytosol (Fig. 6B, center), but this process was blocked by NAC (Fig. 6B, right panel). These observations were consistent with the results presented above: Lexa and CHX co- treatment-induced apoptosis can be negated by NAC (Fig. 3C, 3D, 3E, and 3F). Meanwhile, subcellular fractionation assay also showed Lexa and CHX combination treatment induced cyto- chrome c release in HCC cells, whereas, NAC addition significantly could block this process (Fig. 6C). Taken together, these results suggest that Lexa and CHX combination treatment- induced ROS generation results in apoptosis via the activation of Bax. HSP90 is involved in Lexa and CHX combination treatment-induced ROS and apoptosis Heat shock protein 90 (HSP90) may confer a survival advantage to tumor cells through association with several regulators of apoptosis [36–38]. HSP90 binding to its client protein may protect against physiological oxidative stress [39,40]. Here, our data demonstrated that CHX specifically decreased HSP90 protein expression (Fig. 7A). Then, we asked whether HSP90 plays a key role in regulating Lexa and CHX combination-induced apoptosis. Using DMAG-17, a specific HSP90 inhibitor to block its activity, we observed that HSP90 inhibition significantly enhanced Lexa treatment-induced apoptotic cell death (Fig. 7B and 7D) and ROS production (Fig. 7C and 7D) in HCC cells, suggesting HSP90 may be involved in Lexa-induced ROS generation. It has also been reported that DMAG-17 can induce apoptosis in tumor cells through down-regulating IKKs, Mcl-1, and survivin expression [41]. However, in this study, apoptosis assays indicated that HCC cells are insensitive to DMAG-17 single treatment (Fig.7B and 7D). Further Western blotting results showed that protein levels of IKK-b were decreased; IkB-a and the NF-kB targeted proteins survivin were increased; Mcl-1, another anti-apoptotic protein, was not affected by DMAG-17 treatment (Fig. 7E). Finally, to further confirm that HSP90 inactivation is critical for Lexa- induced apoptosis, HCC cells were transiently transfected with HSP90 specific siRNA to knockdown HSP90 expression and were treated with Lexa. Apoptosis assays indicated that HSP90 knockdown (Fig. 7F) significantly sensitized Lexa-induced apop- tosis in HCC cells (Fig. 7G, and 7H). These results suggest that HSP90 can be targeted by CHX to regulate cell sensitivity to Lexa-induced apoptosis. Lexa and CHX combination treatment has no apoptotic effects on normal human hepatocytes A major barrier for the clinical use of TNF-a or TRAIL is their potential toxicity to normal human cells [42–45]. In this study, we examined whether Lexa and CHX combination treatment can selectively induce apoptosis in HCC cells. As shown in Fig. 8A, combination treatment with Lexa (1 mg/ml) and CHX (10 mg/ml) induced caspase 8 activation in HCC cells, but not in normal human hepatocytes. To further evaluate why normal hepatocytes are insensitive to CHX and Lexa co-treatment, we examined the expression of anti-apoptotic and pro-apoptotic proteins in HCC cells and normal human hepatocytes. In comparison with HCC cells, levels of pro-apoptotic Bax and Bak were undetectable in normal human hepatocytes, anti-apoptotic Bcl-xL levels were PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e16966