CHX and Lexa Combination-Induced Apoptosis in HCC Figure 7. HSP90 is involved in Lexa and CHX co-treatment-induced ROS increase and apoptosis. A, Huh7 cells were treated with CHX (10 mg/ml) or not. Cell lysates were prepared for Western blotting. Anti-HSP90, anti-Bax, anti-Bcl-xL polyclonal antibodies were used to detect HSP90, Bax and Bcl-xL protein levels respectively. b-actin was used as an equal protein loading control. Relative intensities of bands in Fig. 7A were analyzed by Image J software. B, Huh7 cells were treated with DMSO (Con), Lexa (1 mg/ml), CHX (10 mg/ml), the combination of Lexa (1 mg/ml) and CHX (10 mg/ml), DMAG (0.5 mg/ml), or the combination of Lexa (1 mg/ml) and DMAG (0.5 mg/ml) for 6 h (Cells were treated with combination of Lexa and CHX as positive control). Apoptosis was assessed by nuclear morphological changes. C, Huh7 cells were treated as in Fig. 7B and intracellular ROS level was detected by fluorescent staining with DFHD (Cells were treated with combination of Lexa and CHX as positive control). D, Huh7 cells were treated with various conditions as indicated (Ctrl, control; L, Lexa (1 mg/ml); C, CHX (10 mg/ml), D, DMAG-17 (0.5 mg/ml); L+D, the combination of Lexa (1 mg/ml) and DMAG-17 (0.5 mg/ml); L+C, the combination of Lexa (1 mg/ml) and CHX (10 mg/ml). Cells with both ROS positive and DNA fragmentation were counted. Data represent the mean values of three independent experiments (*p,0.05; **p,0.05). E, Huh7 cells were treated with different dosage of DMAG-17 for 24 h. Cells were harvested and cell lysates were prepared for Western blotting. IKK-b, IkB-a, survivin and Mcl-1 protein levels were detected using specific antibodies respectively. b-actin was detected by anti-b-actin mouse antibody for an equal protein loading control. F, Huh7 cells were transfected with synthesized random siRNA (as control) and HSP90 specific siRNA, and after 48 h post-transfection cells were subjected to Western blotting analysis with HSP90 polyclonal antibody. b-actin was used as an equal protein loading control. The intensity of immunoblot bands Fig. 7F was analyzed by Image J software. G, Huh7 cells were transfected with synthesized random control siRNA and HSP90 siRNA, and 48 h post-transfection, cells were treated with Lexa or not and then subjected to Hoechst staining for apoptosis analysis (RC, random siRNA control; RS+L, random siRNA cells treated with Lexa (1 mg/ml); HSC, HSP90 siRNA control; HS+L, HSP90 siRNA cells treated with Lexa (1 mg/ml)). H, Huh7 cells treated as in Fig. 7G were harvested and subjected to Western blotting analysis with caspase 3 (for cleaved bands only) polyclonal antibody. b-actin was detected with anti-b-actin mouse antibody for an equal protein loading control. doi:10.1371/journal.pone.0016966.g007 sensitivity of the normal hepatocytes and HCC cells used in this study did not reveal a consistent relationship, suggesting that Lexa sensitivity is not dependent on death receptor expression levels and that other intracellular mechanisms may control death signal transduction in resistant cells. As death receptor-mediated cell death can be regulated by Bcl-2 family BH3 only proteins, such as Bax, Bak, and Bid. Here, we found that expression levels of the BH3 only proteins Bax and Bak were undetectable in normal human hepatocytes, while the HCC cells showed clear high expression of these proteins. This obvious difference may be a key element in protecting normal cells against the toxicity of Lexa and CHX combination treatment. In addition, to extend the use of Lexa to treat HCC, we tested the effects of combination of the death receptor agonists with other chemotherapeutic reagents such as cisplatin or doxorubicin. Our data showed that only doxorubicin could sensitize Lexa-induced apoptosis in HCC cells (see Figure S1 and Figure S2). The underlying mechanism remains to be investigated. In summary, we have found that HCC cells are generally resistant to Lexa, and CHX can sensitize HCC cells to Lexa- PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e16966