蟾皮活性成分沙蟾毒精经EpCAM通路调控肝癌干细胞生物学特性的机制研究

Liver cancer is the third leading cause of cancer mortality in the world. Liver cancer stem cells (LCSCs) with infinite proliferation and multiple differentiation potential are responsible for the recurrence and metastasis, and current existing therapies cannot completely eliminate the LCSCs. Our recent results found that the traditional Chinese medicine toad skin toad skin bioactive component arenobufagin (ABF) inhibited the proliferation, self-renewal and tumorigenicity of LCSCs. We also found that the EpCAM pathway is involved in ABF regulating the features of LCSCs in vitro and in vivo. Therefore, we construct multiple models of LCSCs (derived from HuH7 cells, Hep3B cells and primary liver cancer cells) to screen the anti-tumor active ingredients of toad skin, and clarify the pharmacodynamic components of toad skin regulating the features of LCSCs. We also establish HuH7-sphere cells transplantation tumors and liver cancer PDX models to confirm the anti-tumor effect of ABF in vivo. Furthermore, we utilize plasmid transfection, siRNA interference and the inhibitors of EpCAM pathway to reveal the mechanisms of ABF regulating LCSCs through EpCAM pathway in vitro and in vivo. This project aims to illustrate the effective substances and the underlying mechanisms of toad skin’s actions on regulating LCSCs, which will lead to a significant benefit for the preclinical development and clinical usage of toad skin as anti-cancer agent.

肝癌是全球死亡率排名第三的癌症。当前的治疗方法不能彻底杀死具有无限增殖和多向分化潜能的肿瘤干细胞是其复发和转移的主要原因。在前期研究中，我们发现中药蟾皮活性成分沙蟾毒精（Arenobufagin, ABF）可抑制细胞系和原代肝癌干细胞的增殖、自我更新和致瘤性，信号通路研究显示EpCAM通路参与ABF调控肝癌干细胞。本项目拟从细胞系和临床标本构建肝癌干细胞模型，明确中药蟾皮调控肝癌干细胞的药效成分；构建HuH7-Sphere细胞移植瘤和肝癌PDX模型，体内验证ABF的抗肝癌效果；基于EpCAM通路，联合质粒转染、siRNA干扰以及EpCAM信号通路阻断剂揭示ABF调控肝癌干细胞的体内外机制。旨在阐明中药蟾皮调控肝癌干细胞的药效物质和作用机制，为临床上蟾皮治疗肝癌提供现代药理学依据，也为中药来源的抗肿瘤药物研发提供可借鉴的范例。

肝癌是全球第五大常见癌症，也是全球死亡率排名第三的癌症，我国肝癌死亡人数占全球肝癌死人数的25%以上。中医认为“癌毒传舍”和“痰毒流注”是肿瘤转移的病机，而“余毒未消，伏邪未尽”是肿瘤复发的前提，因此，“癌毒”是导致肿瘤发生、恶变、复发以及转移的的根源，故巧除“癌毒”是肿瘤治疗的关键。现代研究表明，EpCAM信号通路是维持和调控肝癌干细胞生物学特性的关键通路。本项目利用72例肝癌及癌旁组织芯片检测EpCAM蛋白的表达。研究发现EpCAM高表达HCC组织，并促进HCC干细胞增殖、自我更新、侵袭转移能力以及致瘤性。ABF，作为一种来源于传统中药蟾酥的主要活性成分之一，可以杀伤肝癌干细胞，并且抑制EpCAM介导的肿瘤干性，进而抑制和杀伤肝癌细胞。本研究阐明了EpCAM在促进HCC干性中的关键作用和分子机制，并且提出，靶向抑制EpCAM可以作为临床治疗HCC的重要策略，为ABF发展成为治疗肝癌的药物提供坚实的理论基础和实验依据。