Reactive Oxygen Species Is Essential for Cycloheximide to Sensitize Lexatumumab-Induced Apoptosis in Hepatocellular Carcinoma Cells Xiangxuan Zhao1, Mengde Cao2, Joy J. Liu1, Haizhen Zhu1¤, David R. Nelson 2 , Chen Liu1* 1 Departments of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, United States of America, 2 Department of Medicine, University of Florida, Gainesville, Florida, United States of America Abstract This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa- induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC. Citation: Zhao X, Cao M, Liu JJ, Zhu H, Nelson DR, et al. (2011) Reactive Oxygen Species Is Essential for Cycloheximide to Sensitize Lexatumumab-Induced Apoptosis in Hepatocellular Carcinoma Cells. PLoS ONE 6(2): e16966. doi:10.1371/journal.pone.0016966 Editor: Wael El-Rifai, Vanderbilt University Medical Center, United States of America Received September 22, 2010; Accepted January 18, 2011; Published February 10, 2011 Copyright: 2011 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from the National Institutes of Health: R01CA133086 to CL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. Competing Interests: The authors have declared that no competing interests exist. * E-mail: liu@pathology.ufl.edu ¤ Current address: State Key Laboratory of Chemo/Biosensing and Chemometrics, School of Biology, Hunan University, Changsha, China Introduction Hepatocellular cancer is one of the five most common cancers worldwide and is fatal in more than 90% of patients [1]. Currently, there are no effective therapies for liver cancer other than surgical resection or liver transplantation in the early stages of tumor development. Such treatments only apply to a small percentage of patients, while the majority die within 6 months of diagnosis [2]. Therefore, new therapeutic strategies are urgently needed. Targeting death receptor activation-mediated cell death is quickly becoming one of the most promising strategies for anti- cancer therapy [3,4]. An overwhelming number of studies have demonstrated that the administration of death receptor agonists can selectively induce apoptosis in tumor cells and significantly inhibit xenograft human tumor growth [5–8]. Among the death receptor agonists, lexatumumab (Lexa) was developed as a potential humanized anti-death receptor 5 (DR5) monoclonal antibody. It has been shown that Lexa specifically binds to death receptor 5 and induces apoptosis in a number of tumor cell lines, including renal cell carcinoma (RCC) [9], human myeloma cell lines (HMCL) [10], and malignant pleural mesothelioma (MPM) [11]. Different researchers have also reported that combination treatment with agonistic death receptor 5 mAbs and chemother- apeutic drugs exert a synergistic apoptotic effect in some tumor cell lines, such as lymphoma [12–14], breast cancer [15], colorectal cancer [16], and malignant mesothelioma [11]. Nevertheless, it remains unknown whether Lexa can induce apoptosis in hepatocellular carcinoma (HCC) cells or whether it has apoptotic toxicity to normal hepatocytes. In the present study, we are the first to show data indicating that Lexa can significantly induce apoptosis in resistant HCC cell lines in the presence of cycloheximide (CHX). We provide evidence to demonstrate that treatment combining Lexa and CHX induces caspase-dependent apoptosis in HCC cells. Intracellular reactive oxygen species (ROS) generation, Bax/Bak activation, and heat shock protein 90 (HSP90) inactivation are involved in killing the HCC cells. More importantly, we found that Lexa and CHX combination treatment has no apparent apoptotic toxicity to normal human hepatocytes. Materials and Methods Cell culture and reagents Human hepatocellular carcinoma cell lines, Huh7 and LH86 were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (Sigma, St. Louis, MO) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) at 37uC in 5% CO2. Normal primary human hepatocytes were PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e16966