Figure 6. Lexa and CHX co-treatment induces Bax translocation and cytochrome c release in HCC cells. A, Huh7 cells were transfected with GFP-N1-Bax plasmid (0.25 mg/ml). After 10 h post- transfection, cells were treated with Lexa (1 mg/ml) in the presence of CHX (10 mg/ml) or not. GFP-N1-Bax translocation (green) was observed under fluorescent microscope. Mitochondria were stained with Mito Tracker (red) CMXRos (con, control; mito, mitochondria). Nuclear was stained with DAPI (blue). B, Huh7 cells cultured on cover slips were treated with Lexa (1 mg/ml) and CHX (10 mg/ml) in the presence (right panel) or absence (middle panel) of NAC (10 mM) for 6 h. Fluorescence immunostaining was performed to label cytochrome c with anti- cytochrome c primary antibody and FITC conjugated goat anti-mouse secondary antibody (green). C, Huh7 cells were exposed to different conditions as indicated and subjected to subcellular fractionation. Then, the cytosolic fractions were analyzed by Western blotting with antibody specific for cytochrome c. b-actin was detected with anti-b-actin mouse antibody for equal protein loading control. doi:10.1371/journal.pone.0016966.g006 HCC cells as a model to examine the apoptotic effects of Lexa. Our results showed that first, compared with positive control TNF-a, IkB-a phosphorylation could not be detected in HCC cells after treatment with Lexa, suggesting that specific binding to DR5 avoids activation of the NF-kB signaling pathway in HCC cells. This differs from the other two cytokines (TNF-a and TRAIL) that can induce activation of NF-kB to produce anti- apoptotic signals through binding to membrane [46–48]. Never- theless, we did observe Lexa could reduce IkB-a expression in HCC cells, of which the significance remains to be clarified in the CHX and Lexa Combination-Induced Apoptosis in HCC future. Second, single treatment of Lexa showed no lethal toxicity to HCC cells, which is consistent with previous studies reporting that HCC cells are resistant to TRAIL [49–51]. Third, we observed that pre-incubation with CHX could sensitize HCC cells to Lexa-induced apoptosis. It is therefore likely that besides inhibition of NF-kB, there are some other mechanisms through which CHX can sensitize Lexa to induce apoptosis in HCC cells. Several groups have reported that activation of DR5 could signal non-apoptotic pathways through IKK, MAPK, P38, and JNK signaling pathways to neutralize TRAIL-induced apoptosis [52–54]. Our data demonstrated that specific inhibitors, including NBD, PD98059, SB203580, and SP600125, that respectively block the above mentioned anti-apoptotic signaling pathways, failed to reverse the resistance of HCC cells to apoptosis induced by Lexa, suggesting that CHX sensitizing cells to Lexa lethal toxicity does not require blocking anti-apoptotic effects from the activation of these signaling pathways. As a pan-protein synthesis inhibitor, CHX may inhibit the activity of some apoptosis associated proteins by decreasing their expressions. However, our results demonstrated that CHX could not completely block protein synthesis because under CHX and Lexa co-treatment, Bak protein was significantly increased, suggesting that Bax/Bak may be activated to result in apoptotic cell death. As expected, our studies showed that Lexa and CHX co-treatment induced Bax oligomerization, conformational change, and translocation, which resulted in cytochrome c release and apoptosis. It has been reported that Bax/Bak can be activated by intracellular ROS to induce cell death in human colorectal cancer cells [55,56]. Similarly in this study, when HCC cells were co-treated with CHX and Lexa, we observed ROS increase in apoptotic cells, but not in non-apoptotic ones. Moreover, the blockade of ROS with NAC not only prevented Bax oligomer- ization and conformational changes, but also decreased apoptosis significantly. These results suggest that generation of ROS plays a major role in CHX and Lexa co-treatment-induced apoptosis. Our findings are in agreement with a study that reported that the generation of ROS induced mitochondria-dependent apoptosis in colon cancer cells through Bax activation [57]. In comparison to treatment with either agent alone, Lexa- induced ROS accumulation occurred only in the presence of CHX, which suggests that CHX may modify some intracellular signaling pathway(s) or molecule(s) that act as a switch to control Lexa to induce ROS generation Our results based on this hypothesis indicated that pretreatment of cells with CHX decreased HSP90 expression significantly, and HSP90 inactivation by specific inhibitor DMAG-17 not only resulted in a Lexa- induced ROS increase, but also sensitized Lexa-induced apoptosis. Moreover, inhibition of HSP90 activity through gene silencing by specific siRNA significantly sensitized Lexa-induced apoptosis. These findings are also in agreement with a report showing that blockade of HSP90 sensitized prostate cancer cells to TRAIL [58]. Meanwhile, we have observed that DMAG-17 single treatment could not induce HCC cells to undergo apoptosis. Further analysis indicated that although IKK-b was decreased by DMAG-17, the NF-kB targeted proteins including survivin and Mcl-1 were not down-regulated. This is in contrast to a previous study which reported that DMAG-17 induced apoptosis in chronic lympho- cytic leukemia (CLL) cells by decreasing NF-kB targeted gene transcription [59]. In order to bring the death receptor agonists closer to clinical therapy, we evaluated the safety of CHX and Lexa combination treatment in normal human hepatocytes. Our data indicated that normal human hepatocytes are resistant to this combination treatment. Comparison of DR5 expression levels and Lexa PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e16966