title: Interferon-α sensitizes HBx-expressing hepatocarcinoma cells to chemotherapeutic drugs through inhibition of HBx-mediated NF-κB activation.
abstract: BACKGROUND
  Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) is characterized by high chemotherapy resistance; however, the underlying mechanism has not been fully clarified. In addition, HBx protein has been reported to play a key role in virus-mediated hepatocarcinogenesis. Therefore, the present study aims to investigate the role of HBx in the drug-resistance of HBV-related HCC and examine whether such drug-resistance can be reversed by IFN-α treatment.
  
  
  METHODS
  We established HBx-expressing cells by liposome-mediated transfection of HBx into the Huh7 cell line. MTT, Annexin V/PI, and cell cycle assay were used for determining the cellular growth inhibition, apoptosis, and growth arrest, respectively, after treatment with chemical drug. We further used tumor-bearing mice model to compare the tumor growth inhibition efficacy of ADM and 5-FU between the Huh7-HBx group and the control group, as well as the ADM + IFN-α or ADM + IMD treated group and the ADM treated group. SQ-Real time-PCR was performed to analyze the expression of MDR-associated genes and anti-apoptotic genes. Moreover, immunofluorescence and Western blotting were used to determine the subcellular localization of p65 and the phosphorylation of IκBα.
  
  
  RESULTS
  The IC₅₀ values of Huh7-HBx cells against ADM and Amn were 2.317 and 1.828-folds higher than those of Huh7-3.1 cells, respectively. The apoptosis ratio and growth arrest was significantly lower in Huh7-HBx cells after treatment with ADM. The in vivo experiment also confirmed that the Huh7-HBx group was much more resistant to ADM or 5-FU than the control. Furthermore, the expression of MDR-associated genes, such as MDR1, MRP1, LRP1, and ABCG2, were significantly up-regulated in Huh7-HBx cells, and the NF-κB pathway was activated after HBx gene transfection in Huh7 cells. However, combined with IFN-α in ADM treatment, the HBx induced drug-resistance in Huh7-HBx cells can be partly abolished in in vitro and in vivo models. Moreover, we found that the NF-κB canonical pathway was affected by IFN-α treatment, and the expression of anti-apoptotic genes, such as Gadd45β, Survivin, and c-IAP-1 was down-regulated by IFN-α treatment in a dose-dependent manner.
  
  
  CONCLUSIONS
  HBx protein can induce MDR of HBV-related HCC by activating the NF-κB pathway, which can be partly abolished by IFN-α treatment.
authors.affiliation: State Key Laboratory of Infectious Disease Diagnosis and Treatment, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
authors.forename: Yanning
authors.initials: Y
authors.lastname: Liu
authors.forename: Guohua
authors.initials: G
authors.lastname: Lou
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Yu
authors.initials: Y
authors.lastname: Shi
authors.forename: Min
authors.initials: M
authors.lastname: Zheng
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D004351:Drug Resistance; D006515:Hepatitis B virus / Q000276:immunology*; D022781:Hepatocytes / Q000187:drug effects / Q000821:virology; D006801:Humans; D020128:Inhibitory Concentration 50; D016898:Interferon-alpha / Q000276:immunology*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D015534:Trans-Activators / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D000970:Antineoplastic Agents; D016898:Interferon-alpha; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein