Liver Disease Department, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No.528. Zhangheng Road, Pudong New District, Shanghai, People's Republic of China.
Department of pharmacology, School of Pharmacy, Harbin Medical University, Harbin, People's Republic of China.
Cell Oncol (Dordr). 2020 Dec;43(6):1129-1145. doi: 10.1007/s13402-020-00546-0. Epub 2020 Jul 4.
Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), which has a male predominance, lacks effective therapeutic options. Previously, the cardiac glycoside analogue bufalin has been found to inhibit HBV infection and HCC development. As yet, however, its molecular role in HBV-associated HCC has remained obscure.
Colony formation and soft agar assays, xenograft and orthotopic mouse models and HBV X protein (HBx) transgenic mice with exposure to diethylnitrosamine were used to evaluate the effect of bufalin on HBV-associated HCC growth and tumorigenicity. HBx-induced oncogenic signaling regulated by bufalin was assessed using PCR array, chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, transcription and protein expression assays. Synergistic HCC therapeutic effects were examined using combinations of bufalin and sorafenib.
We found that bufalin exerted a more profound effect on inhibiting the proliferation of HBV-associated HCC cells than of non HBV-associated HCC cells. Bufalin significantly inhibited HBx-induced malignant transfromation in vitro and tumorigenicity in vivo. Androgen receptor (AR) signaling was found to be a target of bufalin resistance to HBV-associated hepatocarcinogenesis. We also found that bufalin induced both AR dephosphorylation and cell cycle-related kinase (CCRK) degradation to inhibit β-catenin/TCF signaling, which subsequently led to cell cycle arrest via cyclin D1 down-regulation and p21 up-regulation, resulting in HCC regression. Furthermore, we found that bufalin reduced > 60% diethylnitrosamine-induced hepatocarcinogenesis in HBx transgenic mice, and improved the sensitivity of refractory HBV-associated HCC cells to sorafenib treatment.
Our results indicate that bufalin acts as a potential anti-HCC therapeutic candidate to block HBx-induced AR/CCRK/β-catenin signaling by targeting AR and CCRK, which may provide a novel strategy for the treatment of HBV-associated HCC.
乙型肝炎病毒（HBV）相关肝细胞癌（HCC）在男性中更为常见，缺乏有效的治疗方法。先前，已发现强心苷类似物蟾毒灵可抑制 HBV 感染和 HCC 的发展。然而，其在 HBV 相关 HCC 中的分子作用仍然不清楚。
采用集落形成和软琼脂测定、异种移植和原位小鼠模型以及用二乙基亚硝胺处理的 HBV X 蛋白（HBx）转基因小鼠，评估蟾毒灵对 HBV 相关 HCC 生长和致瘤性的影响。使用 PCR 阵列、染色质免疫沉淀、定点突变、荧光素酶报告基因、转录和蛋白表达测定评估蟾毒灵调节的 HBx 诱导的致癌信号。使用蟾毒灵和索拉非尼联合治疗来检查协同 HCC 治疗效果。
我们发现蟾毒灵对抑制 HBV 相关 HCC 细胞的增殖作用比非 HBV 相关 HCC 细胞更显著。蟾毒灵显著抑制 HBx 诱导的体外恶性转化和体内致瘤性。雄激素受体（AR）信号被发现是蟾毒灵抵抗 HBV 相关肝癌发生的靶标。我们还发现，蟾毒灵诱导 AR 去磷酸化和细胞周期相关激酶（CCRK）降解，以抑制 β-连环蛋白/TCF 信号，随后通过下调细胞周期蛋白 D1 和上调 p21 导致细胞周期停滞，从而导致 HCC 消退。此外，我们发现蟾毒灵降低了 >60%二乙基亚硝胺诱导的 HBx 转基因小鼠肝癌的发生，并提高了难治性 HBV 相关 HCC 细胞对索拉非尼治疗的敏感性。
我们的结果表明，蟾毒灵作为一种潜在的抗 HCC 治疗候选物，通过靶向 AR 和 CCRK 阻断 HBx 诱导的 AR/CCRK/β-连环蛋白信号，可能为治疗 HBV 相关 HCC 提供新的策略。
