干扰素-α通过抑制 HBx 介导的 NF-κB 激活使 HBx 表达的肝癌细胞对化疗药物敏感。
Interferon-α sensitizes HBx-expressing hepatocarcinoma cells to chemotherapeutic drugs through inhibition of HBx-mediated NF-κB activation.
机构信息
State Key Laboratory of Infectious Disease Diagnosis and Treatment, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
出版信息
Virol J. 2013 May 29;10:168. doi: 10.1186/1743-422X-10-168.
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.
背景
乙型肝炎病毒（HBV）相关肝细胞癌（HCC）的特点是化疗耐药性高；然而，其潜在机制尚未完全阐明。此外，已有研究报道 HBx 蛋白在病毒介导的肝癌发生中起关键作用。因此，本研究旨在探讨 HBx 在 HBV 相关 HCC 耐药中的作用，并研究 IFN-α 治疗是否可以逆转这种耐药性。
方法
通过脂质体介导的 HBx 转染将 HBx 导入 Huh7 细胞系，建立 HBx 表达细胞。用 MTT、Annexin V/PI 和细胞周期检测分别测定化学药物处理后细胞生长抑制、凋亡和生长阻滞。我们进一步使用荷瘤小鼠模型比较 ADM 和 5-FU 在 Huh7-HBx 组和对照组、ADM+IFN-α 或 ADM+IMD 处理组和 ADM 处理组中的肿瘤生长抑制作用。通过 SQ-Real time-PCR 分析多药耐药相关基因和抗凋亡基因的表达。此外，免疫荧光和 Western blot 用于确定 p65 的亚细胞定位和 IκBα 的磷酸化。
结果
Huh7-HBx 细胞对 ADM 和 Amn 的 IC₅₀ 值分别是 Huh7-3.1 细胞的 2.317 倍和 1.828 倍。ADM 处理后 Huh7-HBx 细胞的凋亡率和生长阻滞明显降低。体内实验也证实，Huh7-HBx 组对 ADM 或 5-FU 的耐药性明显高于对照组。此外，在 Huh7-HBx 细胞中，MDR 相关基因如 MDR1、MRP1、LRP1 和 ABCG2 的表达明显上调，HBx 基因转染后 NF-κB 通路在 Huh7 细胞中被激活。然而，在体外和体内模型中，与 IFN-α 联合 ADM 治疗相结合，HBx 诱导的 Huh7-HBx 细胞耐药性可部分被消除。此外，我们发现 NF-κB 经典途径受 IFN-α 治疗的影响，IFN-α 以剂量依赖性方式下调抗凋亡基因如 Gadd45β、Survivin 和 c-IAP-1 的表达。
结论
HBx 蛋白可通过激活 NF-κB 通路诱导 HBV 相关 HCC 的多药耐药，IFN-α 治疗可部分抑制该通路。
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