CHX and Lexa Combination-Induced Apoptosis in HCC Figure 5. Lexa and CHX co-treatment-induced ROS increase triggers Bax activation. A, 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 then treated with the cross-linking agent DSS. Bax proteins were resolved by SDS-PAGE and detected by Western blotting using a Bax polyclonal antibody. A 23 kDa band represents Bax monomers, and bands at 46 kDa, detected only in the presence of DSS, represent Bax homodimers. Relative intensity of Bax dimer bands was analyzed by Image J software. B, Huh7 cells were treated with a combination of Lexa (1 mg/ml) and CHX(10 mg/ml) for up to 6 h. Cell lysates were prepared and incubated with cross linker BMH as described in Materials and Methods. Cell lysates and cross linker mixture was boiled with 16SDS loading buffer and Bax oligomerization was analyzed by Western blotting. The Bax dimer was recognized by Bax polyclonal antibody. Relative intensity of Bax dimer bands was quantified by Image J software. C, Huh7 cells treated with conditions as indicated and were harvested and extracts were prepared for Western blotting analysis to analyze Bax dimer formation. Relative intensity of Bax dimer bands was qualified by Image J software. D, LH86 (right panel) or Huh 7 (left panel) cells treated with various stimuli as indicated were harvested and lysed in Chaps lysis buffer and subjected to immunoprecipitation with anti-Bax 6A7 antibody for detection of conformationally changed Bax protein. Cell lysate obtained by NP-40 lysis was used as positive control. In addition, the total lysates were applied directly to SDS-PAGE/Western blotting analysis with specific anti-Bax polyclonal antibody and severed as loading control. doi:10.1371/journal.pone.0016966.g005 similar in all the cell lines tested, and Bid expression levels were much higher in HCC cells than those in normal human hepatocytes. However, DR5 protein in normal human hepatocytes showed much greater expression compared with that in HCC cells (Fig. 8B). These observations suggest that the insensitivity of human normal liver cells to Lexa and CHX combination treatment is not controlled by death receptor 5 (DR5) expression level on the cellular membrane, but rather determined by the expression levels of intracellular pro-apoptotic proteins, including Bax, Bak, and Bid. Taken together, these results suggest that CHX can selectively enhance Lexa to induce apoptosis in HCC cells. Discussion In the present study, we investigated apoptosis induced by combination treatment with Lexa and CHX in resistant HCC cells. Our results demonstrated that pre-incubation with CHX could render resistant HCC cells sensitive to monoclonal antibody Lexa-induced apoptosis. Lexa and CHX combination treatment- induced apoptosis was triggered by intracellular ROS production. ROS accumulation in cells resulted in pro-apoptotic Bax/Bak activation through oligomerization and conformation change. HSP90 may act as a switch controlled by CHX to regulate intracellular ROS levels upon cell death stimulation. More interestingly, the combination treatment did not induce apoptosis in normal human hepatocytes comparing with the HCC cells under identical conditions. These findings are promising for the clinical use of CHX and Lexa as a novel chemotherapeutic option in the treatment of patients with HCC. Lexa, a humanized monoclonal antibody, specifically binds to DR5 to activate death signals and has a longer half life in vivo rendering it as a potential killer of tumor cells. Here, we used PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e16966