CHX and Lexa Combination-Induced Apoptosis in HCC Figure 8. Lexa and CHX co-treatment has no lethal effects on normal human hepatocytes. A, Freshly prepared normal human primary hepatocytes were seeded on six-well plates and treated after two days with Lexa (1 mg/ml) and CHX (10 mg/ml) for up to 6 h. Huh7 cells treated under the same conditions were used as a positive control. Caspase 8 activation was measured through detecting the cleaved bands by Western blotting. B, Cell lysates from normal human hepatocytes and HCC cells LH86 and Huh7 were prepared and Western blotting was performed to detect Bcl-xL, Bax, Bak, Bim, Bid, and DR5 with specific antibodies. doi:10.1371/journal.pone.0016966.g008 induced apoptosis. The underlying mechanism of action for combination treatment is related to ROS generation and Bax activation. Our study also demonstrates that normal hepatocytes are resistant to the combination therapy. We suggest that administration of CHX followed by Lexa should be considered for further investigation for the treatment of patients with HCC. Supporting Information Figure S1 Cisplatin (Cis) could not sensitize Lexatumu- mab (Lexa)-induced apoptosis in HCC cells. Huh7 cells were treated with DMSO (control), Lexa (1 mg/ml), different doses of cisplatin (1-20 mg/ml), or a combination of Lexa and cisplatin as indicated. Apoptosis was measured by nuclear dye Hoechst 33258 staining to label DNA fragmentation (nuclear morphological changes). Note that Lexa, cisplatin, or the combination treatment of Lexa and cisplatin had no apoptotic toxicity to HCC cells. (TIF) Figure S2 Doxorubicin (Dox) sensitizes Lexatumumab (Lexa)-induced apoptosis in HCC cells. Huh7 cells were References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2009) Cancer statistics, 2009. CA Cancer J Clin 59: 225–249. 2. Deuffic S, Poynard T, Buffat L, Valleron AJ (1998) Trends in primary liver cancer. Lancet 351: 214–215. 3. Russo M, Mupo A, Spagnuolo C, Russo GL (2010) Exploring death receptor pathways as selective targets in cancer therapy. Biochem Pharmacol 80: 674–682. 4. Wiezorek J, Holland P, Graves J (2010) Death receptor agonists as a targeted therapy for cancer. Clin Cancer Res 16: 1701–1708. 5. Camidge DR, Herbst RS, Gordon MS, Eckhardt SG, Kurzrock R, et al. (2010) A phase I safety and pharmacokinetic study of the death receptor 5 agonistic antibody PRO95780 in patients with advanced malignancies. Clin Cancer Res 16: 1256–1263. 6. Forero-Torres A, Shah J, Wood T, Posey J, Carlisle R, et al. (2010) Phase I trial of weekly tigatuzumab, an agonistic humanized monoclonal antibody targeting death receptor 5 (DR5). Cancer Biother Radiopharm 25: 13–19. treated with DMSO (control), Lexa (1 mg/ml), different doses of doxorubicin (0.25-2 mM), or a combination of Lexa and doxorubicin as indicated. Apoptosis was measured by nuclear dye Hoechst 33258 staining to label DNA fragmentation (nuclear morphological changes). Note that either Lexa or doxorubicin could not induce apoptosis in HCC cells. However, the combination treatment of Lexa and doxorubicin induced significant apoptosis in HCC cells. (TIF) Acknowledgments We give thanks to Dr. Robin Humphreys and Dr. Mani Subramanian of Human Genome Science for providing Lexa and Ton Wang for editing the paper. Author Contributions Conceived and designed the experiments: XXZ CL. Performed the experiments: XXZ. Analyzed the data: XXZ CL. Contributed reagents/ materials/analysis tools: XXZ CL. Wrote the paper: XXZ CL. 7. Lee SH, Park DW, Sung ES, Park HR, Kim JK, et al. (2010) Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity. Mol Immunol 47: 816–824. 8. Sung ES, Park KJ, Lee SH, Jang YS, Park SK, et al. (2009) A novel agonistic antibody to human death receptor 4 induces apoptotic cell death in various tumor cells without cytotoxicity in hepatocytes. Mol Cancer Ther 8: 2276– 2285. 9. Zhang L, Zhang X, Barrisford GW, Olumi AF (2007) Lexatumumab (TRAIL- receptor 2 mAb) induces expression of DR5 and promotes apoptosis in primary and metastatic renal cell carcinoma in a mouse orthotopic model. Cancer Lett 251: 146–157. 10. Menoret E, Gomez-Bougie P, Geffroy-Luseau A, Daniels S, Moreau P, et al. (2006) Mcl-1L cleavage is involved in TRAIL-R1- and TRAIL-R2-mediated PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e16966