内质网应激与天然产物诱导肿瘤细胞自噬的关系及其抗肿瘤活性研究

Autophagy is an essencial celluar stress response to maintain homoeostasis by degrading excessive or unnecessary proteins and dysfunctional cellular organelles. It is revealed multiple natural products could induce cellular autophagy, and the exact molecular mechanism and how to regulate autophagic function beneficially for anti-tumor therapy have attracted increasing interests of oncologists. Although it is becoming increasingly clear that ER stress can lead to the induction of autophagy, the role of PERK/eIF2/ATF4 and Ca2+/CaMKKβ pathway in natural products mediated-autophagy has not been systematically examined. Take cardiac glycosides for example, our previous work have shown that AMPK and TSC2 mediated down-regulation of mTOR pathway is found to be included in the autophagy induction. On this basis, this project to study the connection between ER stress and autophagy induced by cardiac glycosides, regulation of AMPK/TSC2 by ER stress signal pathways as well as the impact by autophagy on drug's anti-tumor activity. Then it is extended to research on other natural products as flavones, alkaloids and polyphenols for finding the upstream molecular mechanism of anti-tumor function through autophagy and discussing possibility for ER stress as a potential druggable targets.

自噬是以降解多余或非必需蛋白质和异常细胞器来维持细胞内稳态的重要途径，有研究表明多类天然产物均能诱导肿瘤细胞自噬，因此天然产物诱导自噬的分子机制和如何调控自噬有利于抗肿瘤治疗成为研究者关注的问题。内质网应激是引起细胞自噬作用的重要诱因之一，其中PERK/eIF2/ATF4和Ca2+/CaMKKβ通路与天然产物诱导自噬的关系研究甚少。以强心甙类为例，我们前期工作发现该类药物激活AMPK/TSC2通路，抑制mTOR从而诱导自噬。在此基础上，本课题拟利用多种生物学手段，先研究内质网应激与强心甙类诱导自噬发生的关系、内质网应激信号通路对AMPK/TSC2的调控作用以及内质网引起的自噬对药物抗肿瘤机制的影响。然后将研究扩展到已知诱导细胞自噬的黄酮、生物碱以及多酚类代表药物，阐明天然产物通过自噬发挥抗肿瘤活性的上游分子机制并分析以内质网应激作为抗肿瘤药物作用靶点的可行性。

本研究按照计划顺利完成了研究内容，研究结果显示，哇巴因在肺癌细胞和乳腺癌细胞中都能诱导细胞自噬发生，且自噬作用发挥抗肿瘤作用。机制研究表明，Src通路的活化、AMPK活性的升高可能参与自噬的调节。从调控肿瘤细胞代谢的角度探索了哇巴因抗肿瘤活性，发现哇巴因时间依赖性地降低MCF7细胞内ATP水平，剂量依赖性地降低线粒体呼吸作用，同时能够促进葡萄糖吸收。此外，哇巴因还能上调内质网应激相关分子PERK、eIF2α和CHOP的表达。本研究还通过模型筛选获得了化合物巨大戟醇-5,20-缩丙酮-3-当归酸酯（IAA），机制研究发现，IAA能够诱导肿瘤细胞增殖抑制，引起G2/M期细胞周期阻滞，诱导较弱的细胞凋亡效应。在蛋白水平上，IAA上调凋亡通路、自噬及内质网应激相关蛋白的表达。通过全面细致的研究，我们进一步揭示了强心甙类药物的多种抗肿瘤机制，并且将这种研究思路，扩展用于其他天然产物的研究中，也发现了IAA的抗肿瘤活性涉及多方面，提示天然化合物抗肿瘤作用机制的多样化，也为抗肿瘤治疗策略提供实验依据。