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. 2013 May 29:10:168.
doi: 10.1186/1743-422X-10-168.

Interferon-α sensitizes HBx-expressing hepatocarcinoma cells to chemotherapeutic drugs through inhibition of HBx-mediated NF-κB activation

Yanning Liu  1 , Guohua Lou, Wei Wu, Yu Shi, Min Zheng, Zhi Chen
Affiliations

Interferon-α sensitizes HBx-expressing hepatocarcinoma cells to chemotherapeutic drugs through inhibition of HBx-mediated NF-κB activation

Yanning Liu et al. Virol J. .

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.

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Figures

Figure 1
Figure 1
Increase in drugs resistance induced by HBx. A) The IC50 value of ADM and Amn against Huh7, Huh7-3.1, and Huh7-HBx cells respectively. Huh7-HBx cells represent a higher resistance of 2.317 and 1.828 times, respectively, than Huh7-3.1 cells against these two chemotherapeutic drugs. B) After treatment with ADM for 24 h, the Annexin V/PI assay was used for the analysis of cell apoptosis. A significantly less degree of apoptosis was found in Huh7-HBx cell than in Huh7-3.1 cell (P < 0.05). C) Cell cycle analysis of Huh7, Huh7-3.1 and Huh7-HBx cells after 24 h of treatment with ADM, as determined by flow cytometry for DNA content.
Figure 2
Figure 2
HBx induces NF-κB pathway activation. A) Confocal immunofluorescence analysis of Huh7-3.1, Huh7-HBx, and Huh7-HBx treated with IMD-0354 or IFN-α, using p65 antibody (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red). P65 is mainly localized in the cytoplasm of Huh7-3.1 cells, while was partly translocated into the nucleus of Huh7-HBx cells. However, the level of p65 in the nucleus of Huh7-HBx cells decreased to some extent after treatment with IMD-0354 or IFN-α. B) The expression level of phosphorylated IκBα was detected by Western blotting (lane 1, Huh7 cells; lane 2, Huh7-3.1 cells; lane 3, Huh7-HBx; lane 4, Huh7-HBx treated with IMD-0354; lane 5, Huh7-HBx treated with IFN-α).
Figure 3
Figure 3
HBx up-regulated the expression of MDR-associated genes and anti-apoptosis genes mediated by the NF-κB pathway. A) Apoptosis index of Huh7, Huh7-3.1, and Huh7-HBx cells after treatment with ADM. The apoptosis ratio in Huh7-HBx cells was significantly lower than that of the Huh7-3.1 cells (P < 0.01). However the level of apoptosis index in the Huh7-HBx cells increased to some extent after co-treatment with the NF-κB pathway inhibitor, IMD-0354 (P<0.05). B) The expression of MDR associated genes of Huh7, Huh7-3.1 and Huh7-HBx cells was detected by real-time RT-PCR, with β-actin as internal control. The gene expression levels of Huh7 cells were set as calibrator to compare with others. C) The transcript level of anti-apoptosis genes in Huh7, Huh7-3.1, and Huh7-HBx cells was detected by real-time RT-PCR analysis with β-actin as internal control. Logarithmic scale expression values were normalized to Huh7 (ref =1, n = 3).
Figure 4
Figure 4
Interferon-α diminishes Huh7-HBx cells resistance to ADM. A) Apoptotic index quantitated by FACS. Compared with Huh7-HBx cells treated with either IFN-α or ADM, the cells treated with both IFN-α and ADM clearly showed an increase in annexin+/PI- & annexin+PI+ cell population. Data are showed as mean ± SD, and statistically analyzed with Student’s t test assay. * P < 0.05, ** P < 0.01, each cells v.s. controle (non-treated Huh7-HBx cells). B) The transcript level of the anti-apoptosis genes in Huh7-HBx cells (control) and IFN-α or IMD-0354 pretreated Huh7-HBx cells were detected by real-time RT-PCR analysis, with β-actin as internal control. Logarithmic scale expression values were normalized to Huh7-HBx (ref =1, n = 3).
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