天然产物鸦胆子苷D抗肝癌的分子靶标发现与验证

Our team has screened more than 6000 species of Chinese herbal drugs and successfully found yadanzioside D with good anti-hepatoma activity. This is the first time to discover this type of compound has anti-hepatoma activity. In order to understand the mechanism of action of this compound, the mechanism of action and molecular target of yadanzioside D was studied. Signaling pathway study revealed yadanzioside D as a Wnt pathway inhibitor. Then affinity chromatography indicated that β-catenin may be the potential target of yadanzioside D. But the unresolved issue is: it need more direct evidence to prove yadanzioside D interacting with β-catenin. This is the major research plan of this subject, including: a) The binding property of yadanzioside D with β-catenin by LC/MS/MS analysis b) kinetic and thermodynamics binding property of yadanzioside D with β-catenin; c) The inhibitory effect of yadanzioside D on nuclear β-catenin content in the cytoplasmic and nucleus fraction of HCC cells by western blot analysis and immunocytochemistry. The completion of this project will provide detailed target-drug interaction mode data for Wnt/β-catenin inhibitors, speed up the development process of new anti-hepatoma drugs.

本课题组从6000多种中草药中成功筛选发现了具有较好肝癌治疗活性的化合物“鸦胆子苷D”，其特色在于体内抑瘤活性显著且安全性高，并具有潜在的肝癌肿瘤干细胞抑制活性，这是首次发现该类型化合物具有肝癌治疗活性。信号通路研究和亲和色谱实验发现Wnt通路中的关键蛋白“β-catenin”（β-连环蛋白）可能是其抗肿瘤靶标。为了验证上述靶标，本课题拟开展相关研究获取鸦胆子苷D靶向β-catenin并影响其入核导致下游抗肿瘤功效的直接证据，具体内容包括：①鸦胆子苷D与β-catenin结合的质谱学验证；②鸦胆子苷D与β-catenin结合的动力学和热力学验证；③通过核蛋白分离和免疫荧光实验确证鸦胆子苷D减少β-catenin入核，发挥肝癌抑制活性。本课题的完成将首次清晰地阐明鸦胆子苷D这一新型肝癌治疗药物的分子靶标，为肝癌治疗药物研发提供一个机理明确、特色突出、靶标清楚的新候选分子。

原发性肝癌是全球常见的恶性肿瘤之一。目前认为，晚期肝癌药物开发策略主要为寻找靶向新通路的分子或寻找索拉非尼增敏的分子。Notch和Wnt通路是介导肝癌发生发展的重要通路，其中Wnt通路的重要转导蛋白β-catenin在肝癌中的突变率达到20-40%。本实验室前期通过RBP-Jκ荧光素酶报告基因系统筛选到一个Notch通路的抑制剂鸦胆子苦素D (BD)，其能明显抑制Notch通路配体Jagged1的表达，同时对肝癌细胞有明显的抑制作用。本研究表明BD在体内亦能抑制原位肝肿瘤的生长，同时能明显增强索拉非尼的抗肝癌作用，可作为目前肝癌一线用药索拉非尼的协同剂。机制研究方面，发现Jagged1在肝癌细胞中受β-catenin调控，BD能通过诱导β-catenin的蛋白酶体降解，抑制β-catenin的核内聚集水平，从而下调Wnt/TCF4的转录水平。综上，本研究发现了一个靶向β-catenin/Jagged1通路转导的抗肝癌小分子，其还能作为索拉非尼抗肝癌的协同增效剂，为肝癌药物开发提供一定的临床前证据和先导化合物。