3′-epi-12β-hydroxyfroside诱导自噬抑制肿瘤细胞免疫原性死亡机制研究

3’-epi-12β-hydroxyfroside (HyFS) is a new cardenolide compound isolated from Calotropis gigantea in tropical Hainan Island. Its structure was elucidated by our research group. Our study results showed that HyFS treatment induced autophagy, stronger inhibition of cell proliferation and induction of cell apoptosis were shown when HyFS-mediated autophagy was blocked. The Hsp90/Akt/mTOR axis was found to be involved in the activation of HyFS-mediated autophagy. Combination treatment with HyFS and chloroquine showed remarkably increased tumor inhibition in mice, suggesting that HyFS induced cytoprotective autophagy through ubiquitin-mediated degradation of Hsp90, which further blocked the Akt/mTOR pathway. In addition, we also found that HyFS also induced stronger immunogenic cell death (ICD) when HyFS induced autophagy was blocked by autophagic inhibitor. However, there are not any other mechanism research done till now except for our group. The detail molecular mechanism of autophagy and ICD，the relationship between autophagy and ICD, the shared molecular signaling pathway and the target protein moleculs between autophagy and ICD in the tumor cells induced by HyFS remains unknown, it is thus need further more studies. In this study, we will in vitro culture cancer cells and establish in vivo tumor models to investigate whether autophagy and ICD are surely existed in cancer cells induced by HyFS. In addition, we will use molecular methods to study the autophagy and ICD signaling pathways and the relationship between autophagy and ICD induced by HyFS. Moreover, we will also use new techniques, such as quantum dot (QD) and cellular thermal shift assay, to locate the HyFS’s binding-site and screen its target protein molecules in the cancer cells. At last, we will also use RNA interference, molecular blockers or even gene knockout technique to confirm the target protein molecules to ultimately prove the detail autophagic and ICD mechanism induced by HyFS. Our study will offer experimental data for HyFS to be developed as a novel anti-tumor compound.

3′-epi-12β-hydroxyfroside（HyFS）是我们合作团队从热带植物牛角瓜分离的一种新的强心苷化合物。我们前期研究发现HyFS能诱导肿瘤细胞产生保护性自噬，抑制自噬促使肿瘤细胞产生免疫原性死亡（ICD），但自噬和ICD之间的关系、自噬降解的靶分子、HyFS直接作用的分子通路等更详细的分子机理仍需要进行深入的研究。因此，本项目拟从体外细胞培养和肿瘤小鼠体内模型两个层面进行研究，利用免疫学、分子生物学等技术明确HyFS诱导肿瘤细胞自噬和ICD的关系，确定自噬降解的靶分子及其信号通路。应用磁性量子点标记、CETSA等技术对HyFS进行细胞定位，筛选并确定HyFS结合的靶蛋白分子，同时利用Western blotting、RNA干扰和功能阻断剂等方法证明靶分子相关通路，争取最终阐明HyFS诱导肿瘤细胞自噬和ICD的分子机制，为HyFS的临床应用转化提供实验及理论依据。

越来越多的研究表明，某些强心苷类化物，比如临床常用的地高辛（digoxin, DIG）和洋地黄（digitoxin），可以诱导肿瘤产生免疫原性细胞死亡（immunogenic cell death，ICD）。前期研究发现，我们小组分离的一种新型强心苷类化合物3'-epi-12β-羟基糖苷（3′-epi-12β-hydroxyfroside，HyFS）可以通过抑制Akt/mTOR通路来诱导细胞产生保护性自噬，但是HyFS是否能够诱导肿瘤细胞产生ICD尚不清楚。我们在前期研究的基础上，在本项目中进一步系统深入研究HyFS是否能同时诱导产生自噬和ICD，并在三个三阴性乳腺癌（TNBC）细胞系中研究了自噬和ICD之间的关系。在这项研究中，我们利用传统临床药物DIG作为对照，了解DIG和HyFS两个药物在诱导TNBC细胞自噬和ICD的差异。结果与预期明显不同，在研究中，我们发现DIG处理的TNBC细胞可以有效诱导产生完整的自噬流和明显的ICD，但是HyFS处理的三个TNBC细胞系（MDA-MB-231、MDA-MB-436和HCC38）中都发现可以产生完整的自噬流，但却不能诱导产生ICD。用自噬抑制剂和RNA干扰阻断HyFS诱导产生的自噬则可使TNBC 细胞和DIG处理的细胞一样，能够诱导产生ICD。进一步的机制研究表明，我们发现在DIG处理的TNBC细胞中，RIPK1/RIPK3坏死体的形成是诱导产生ICD是重要环节，但在HyFS处理细胞中则没有检测到RIPK1/RIPK3坏死体的生成，后继也不能导致RIPK1/RIPK3坏死体下游信号分子MLKL的磷酸化激活从而诱导坏死性凋亡；但是HyFS诱导的自噬导致降解RIPK1/RIPK3，致使MLKL不能被磷酸化，不能诱导产生坏死性凋亡，因而不能诱导产生ICD。此外，体内肿瘤模型试验利用自噬抑制剂氯喹来抑制HyFS诱导产生的自噬，发现TNBC细胞可以重新产生ICD，同时也能改变TNBC肿瘤免疫抑制的微环境，利用免疫缺陷小鼠进行比交较研究，发现联合HyFS和CQ治疗在免疫正常小鼠中抗肿瘤疗效更加显著。本项这些研究结果表明，HyFS诱导自噬降解RIPK1/RIPK3导致了TNBC细胞不能产生ICD；HyFS和自噬抑制剂联合治疗可增强抗肿瘤活性，有一定的转化应用前景。