蟾蜍灵通过雄激素受体去磷酸化和细胞周期相关的激酶降解

StephenW

Bufalin Inhibits Hepatitis B Virus-Associated Hepatocellular Carcinoma Development Through Androgen Receptor Dephosphorylation and Cell Cycle-Related Kinase Degradation
Zhuo Yu  1 , Hai Feng  2 , Yunhui Zhuo  3 , Man Li  4 , Xiaojun Zhu  3 , Lingying Huang  3 , Xin Zhang  4 , Zhenhua Zhou  4 , Chao Zheng  3 , Yun Jiang  3 , Fan Le  3 , Dae-Yeul Yu  5 , Alfred Szelok Cheng  6 , Xuehua Sun  7 , Yueqiu Gao  8   9
Affiliations
Affiliations

    1
    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. [email protected].
    2
    Department of pharmacology, School of Pharmacy, Harbin Medical University, Harbin, People's Republic of China.
    3
    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.
    4
    Laboratory of Cellular Immunity, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, People's Republic of China.
    5
    Disease Model Research Laboratory, Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Republic of Korea.
    6
    School of Biomedical Sciences, State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, SAR, People's Republic of China.
    7
    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. [email protected].
    8
    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. [email protected].
    9
    Laboratory of Cellular Immunity, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, People's Republic of China. [email protected].

    PMID: 32623699 DOI: 10.1007/s13402-020-00546-0

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Androgen receptor; Bufalin; Cell cycle-related kinase; HBV X protein; HBV-associated HCC; HBx transgenic mice.

StephenW

蟾蜍灵通过雄激素受体去磷酸化和细胞周期相关的激酶降解抑制乙型肝炎病毒相关的肝细胞癌的发展。
卓宇1，海丰2，云辉卓3，曼丽4，朱晓军3，黄凌颖3，张新4，周振华4，超正3，运江3，范乐3，大邱裕5，郑秀文6，孙雪华7，高岳秋8 9
隶属关系
隶属关系

    1个
    上海中医药大学附属曙光医院肝病科，编号528。中华人民共和国上海市浦东新区张横路。 [email protected]。
    2
    哈尔滨医科大学药学院药理学系，哈尔滨。
    3
    上海中医药大学附属曙光医院肝病科，编号528。中华人民共和国上海市浦东新区张横路。
    4
    上海中医药大学附属曙光医院细胞免疫实验室，中华人民共和国上海。
    5
    韩国生物科学与生物技术研究所，基因组编辑研究中心，疾病模型研究实验室，大田，305-806。
    6
    香港中文大学生物医学学院，消化系统疾病国家重点实验室，中国香港特别行政区。
    7
    上海中医药大学附属曙光医院肝病科，编号528。中华人民共和国上海市浦东新区张横路。 [email protected]。
    8
    上海中医药大学附属曙光医院肝病科，编号528。中华人民共和国上海市浦东新区张横路。 [email protected]。
    9
    上海中医药大学附属曙光医院细胞免疫实验室，中华人民共和国上海。 [email protected]。

    PMID：32623699 DOI：10.1007 / s13402-020-00546-0

抽象

目的：以男性为主的乙型肝炎病毒（HBV）相关的肝细胞癌（HCC）缺乏有效的治疗选择。以前，已发现强心苷类似物蟾蜍灵可抑制HBV感染和HCC的发展。然而，迄今为止，其在HBV相关肝癌中的分子作用仍然不清楚。

方法：采用菌落形成和软琼脂试验，异种移植和原位小鼠模型以及暴露于二乙基亚硝胺的HBV X蛋白（HBx）转基因小鼠，评估蟾蜍灵对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，定点诱变，萤光素酶报告基因，转录和蛋白质表达分析评估了由bufalin调节的HBx诱导的致癌信号。使用蟾蜍灵和索拉非尼的组合检查了HCC的协同治疗作用。

结果：我们发现蟾蜍灵在抑制HBV相关HCC细胞的增殖方面比非HBV相关HCC细胞发挥更深远的作用。蟾蜍灵在体外和体内均显着抑制HBx诱导的恶性转化。发现雄激素受体（AR）信号传导是蟾蜍灵对HBV相关肝癌发生抵抗的靶标。我们还发现bufalin诱导AR去磷酸化和细胞周期相关激酶（CCRK）降解以抑制β-catenin/ TCF信号传导，随后通过cyclin D1下调和p21上调导致细胞周期停滞，从而导致肝癌回归。此外，我们发现蟾蜍灵在HBx转基因小鼠中减少了60％以上的二乙基亚硝胺诱导的肝癌发生，并提高了难治性HBV相关HCC细胞对索拉非尼治疗的敏感性。

结论：我们的结果表明，蟾蜍灵可作为靶向AR和CCRK阻断HBx诱导的AR / CCRK /β-catenin信号传导的潜在抗HCC治疗候选药物，这可能为治疗HBV相关HCC提供了新的策略。

关键词：雄激素受体布法林;细胞周期相关激酶； HBV X蛋白；乙肝病毒相关肝癌； HBx转基因小鼠。