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.