title: Hepatitis B Virus X Protein (HBx) Is Responsible for Resistance to Targeted Therapies in Hepatocellular Carcinoma: Ex Vivo Culture Evidence.
abstract: OBJECTIVE
  Molecular targeted therapy is an important approach for advanced hepatocellular carcinoma (HCC). Hepatitis B virus-related HCC (HBV-HCC) accounts for approximately 50% of all HCC cases. Bortezomib, a proteasome inhibitor (PI), is used extensively for the treatment of hematologic malignancies, but its application in HCC, particularly in HBV-HCC, has not been fully explored.
  
  
  METHODS
  The effects of bortezomib on HCC tissues were evaluated by TUNEL assays. The growth inhibitory activity was measured using cell viability assays, and apoptosis was measured using flow cytometry. The levels of HBx, P-Raf/Raf, and P-Erk/Erk expression were measured by Western blot analysis. The ability of the MEK inhibitor PD98059 to enhance the cell killing activity of bortezomib was evaluated using ex vivo and in vivo methods.
  
  
  RESULTS
  The potency of bortezomib varied among HCC samples and cell lines, and HBV/HBx expression was associated with resistance to bortezomib. Bortezomib increased the levels of P-Raf and P-Erk in HBV/HBx-positive cells but not in HBV/HBx-negative HCC cells or in breast cancer or glioblastoma multiform cells. HBx was also upregulated after exposure to bortezomib, which was associated with the inhibition of proteasome activity. P-Erk upregulation mediated by bortezomib was effectively suppressed by the addition of the MEK inhibitor PD98059. Moreover, bortezomib and PD98059 synergistically inhibited HCC cell proliferation, as measured using both ex vivo and in vivo models.
  
  
  CONCLUSIONS
  Our studies demonstrate for the first time that HBx causes resistance to bortezomib in HCC, and this resistance can be antagonized by a MEK signaling inhibitor, providing a novel therapeutic approach.
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Pinbo
authors.initials: P
authors.lastname: Huang
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Baoxiong
authors.initials: B
authors.lastname: Zhuang
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Heyun
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Jianlong
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Qibin
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Kaishun
authors.initials: K
authors.lastname: Hu
authors.affiliation: Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California. National University of Singapore (CSI, NCIS), Singapore, Singapore.
authors.forename: H Phillip
authors.initials: HP
authors.lastname: Koeffler
authors.affiliation: Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. yin_dong@yahoo.com sumsjw1957@163.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.affiliation: Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. yin_dong@yahoo.com sumsjw1957@163.com.
authors.forename: Dong
authors.initials: D
authors.lastname: Yin
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D000069286:Bortezomib / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism / Q000821:virology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D019008:Drug Resistance, Neoplasm; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000821:virology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D005419:Flavonoids; D061988:Proteasome Inhibitors; D015534:Trans-Activators; D014452:Ubiquitins; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D000069286:Bortezomib; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one