华蟾素具抗乙肝及肝癌双重作用，临床用于乙肝相关性HCC取得良好疗效，但具体机制尚不明晰。我们前期发表结果已证实新激酶CCRK介导的促癌信号通路能促进乙肝相关性HCC的发生发展，进一步实验显示华蟾素能抑制乙肝相关性肝癌细胞增殖，并能减少细胞中CCRK及HBx的表达。由此，我们首次提出：华蟾素能通过抑制CCRK介导的促癌信号通路，抑制细胞增殖及恶性转化，从而抗乙肝相关性HCC的发生与发展。本项目拟在体外两种乙肝背景肝癌细胞系，HBx诱导的正常肝细胞及体内裸鼠异种，原位移植模型中，运用细胞集落形成，软胶集落形成，Western Blot，RT-PCR，CHIP，Luciferase等实验探索证明华蟾素对CCRK介导的分子信号通路的作用，并在HBx转基因小鼠模型中加以证实。本项目将阐明华蟾素抗乙肝相关性HCC的新机制，揭示华蟾素“以毒攻毒”理论的实质内涵，对优化及发展华蟾素的临床运用具有重要意义。

Cinobufacini which show both anti-HBV and anti-HCC effect achieve ideal therapeutic effect in clinic, but the mechanism still needs further investigation. Our previously published study has verified CCRK-driven oncogenic signaling pathway played a significant role during HBV-associated hepatocarcinogenesis. Further study also showed that cinobufacini inhibited cell proliferation in HBV-associated hepatoma cells. Meanwhile, the expression of CCRK and HBx were suppressed. Based on our previous study and existing evidence, our hypothesis is cinobufacini may inhibit CCRK-driven oncogenic signaling pathway to suppress the cell proliferation and malignant transformation and show good effect during HBV-associated hepatocarcinogenesis. In two HBV-associated HCC cell lines , HBx-induced human immortal cell line (in vitro)and in xenograft, orthotropic nude mice models(in vivo), our project plan to demonstrate the mechanism of cinobufacini on CCRK-driven oncogenic signaling pathway using colony formation, soft agar, Western Blot, RT-PCR, CHIP and Luciferase essays. We will further verify the mechanism in a HBx transgenic mice model. The present proposal hence aims to clarify the new mechanism of cinobufacini in anti-HBV associated HCC function and disclose the theory connotation of “combat poison with poison” of cinobufacini. This study may provide insights into the optimization and development of cinobufacini-based clinical use for HCC therapy.

蟾毒灵是中药华蟾素的生物学活性单体，能够抑制乙肝病毒感染和肝癌发生。细胞周期相关性激酶CCRK通过调控促癌分子信号通路，在乙肝相关性肝癌发展中起到重要作用。本课题评价蟾毒灵对乙肝相关性肝癌的抑制作用，揭示其阻断CCRK介导的乙肝病毒蛋白HBx诱导肝癌发生的分子机制。体外实验中，应用CCK8法，细胞粘附性和非粘附性生长检测法，鉴定蟾毒灵对HBx诱导的细胞增殖和恶性转化的影响。采用PCR阵列分析方法鉴别蟾毒灵作用的HBx调控的信号通路。利用基因敲除和过表达技术，结合启动子募集，免疫荧光染色，基因蛋白表达分析等多种分子生物学方法，检测蟾毒灵对信号通路上细胞分子的调控作用。在肿瘤移植小鼠和HBx转基因小鼠模型中，应用肿瘤生长监测以及Ki67免疫染色，蛋白表达分析，验证蟾毒灵抑制体内肝癌发生，以及对信号通路的调控作用。结果表明蟾毒灵以剂量和时间依赖的方式，有效抑制细胞增殖和肿瘤转化。重要的，蟾毒灵对乙肝相关性肝癌细胞的抑制效果强于非乙肝相关性肝癌细胞，提示HBx在蟾毒灵发挥抗癌效应中具有重要意义。体内实验中，蟾毒灵降低肝内肿瘤发生率，减小肿瘤体积和重量，从而抑制肝癌发生。机制上，我们证明CCRK是蟾毒灵阻断HBx分子信号通路抑制肝癌发生的重要靶点。蟾毒灵通过阻断雄激素受体与CCRK启动子结合，抑制CCRK转录和表达，妨碍β-catenin激活，减少下游基因CCND1和EGFR表达。我们的研究首次提出CCRK是蟾毒灵的作用靶点，详细阐述蟾毒灵阻断CCRK介导HBx调控信号通路，抑制肿瘤发生的分子机制。证实蟾毒灵作为小分子抑制剂，有效抑制乙肝相关性肝癌发生的潜在效力，为临床肝癌治疗的药物选择提供更加有益的策略。