深裂叶阿魏中萜酯类化合物诱导宫颈癌HeLa细胞paraptosis作用机制及构效关系研究

Paraptosis is found in recent years a new type of programmed cell death.Previous study found that the new compounds of terpenoid benzoates in Ferula dissecta (Ledeb.) Ledeb of Xinjiang could induced cervical cancer HeLa paraptosis. And this kind of compounds could induce cervical cancer HeLa cell line Paraptosis. In this study, the subsequent research work intends to systematically investigate the mechanisms and structure-activity relationship of terpenoid benzoates against cervical cancer. Firstly, the overall expression difference of proteins participating in the process of the paraptosis induced by Syreiteate A, one of the terpenoid benzoates, would be examined by means of two-dimensional gel electrophoresis and mass spectrometry technology. On this basis, enzyme specificity inhibitor, siRNA, and so on methods would be used to further confirm these differences and systematically investigate the related signal transduction pathway. Secondly, the HeLa cell BALB/c nude mouse xenograft model in combination with the immunohistochemistry, etc methods would be applied to prove the molecular regulation mechanism and analyze correlationship of the mechanisms between in vitro and in vivo. Again, based on these results in vitro and in vivo, the global PPI network and the bioinformatics calculation software and method would put in use to establish the paraptosis-related protein interaction network in order to comprehensively elaborate the molecular mechanism of the paraptosis in HeLa triggered by the terpenoid benzoates. Finally, on the base of the above investigation in the molecular mechanism, the gene and protein playing the key roles in regulation of paraptosis would be determined. Further, the deep work would be carried out to demonstrate the structure-activity relationship.

Paraptosis是近几年发现的一种细胞程序性死亡。前期发现，深裂叶阿魏中一系列萜酯类新化合物能够诱导HeLa发生Paraptosis。本课题拟对该类化合物抗宫颈癌的分子机制及构效关系进行深入研究。 首先，运用二维凝胶电泳-质谱技术，系统鉴定参与萜酯类化合物Syreiteate A诱导HeLa细胞发生Paraptosis的相关差异表达蛋白。在此基础上，采用酶专属性抑制剂和siRNA等方法对差异表达蛋白信号途径进行确证性研究。其次，采用HeLa细胞BALB/c裸鼠移植模型，结合免疫组化等方法，确证相应蛋白在体内表达。再次，根据上述研究结果，利用生物信息模拟等方法构建该类化合物诱导HeLa细胞发生Paraptosis的蛋白相互作用网络，系统阐述其分子作用机制。最后，根据体内、外研究和蛋白相互作用网络构建结果，确证该类化合物诱导Paraptosis的关键调控基因和蛋白，并进行构-效关系研究。

本课题为了系统的研究深裂叶阿魏 (Ferula dissecta (Ledeb.) Ledeb.) 抗肿瘤的作用物质基础，作用机制及其构效关系。首先，对深裂叶阿魏的95%乙醇提取物的化学成分进行了研究，从中分离鉴定了20个化合物，萜酯类化合物16个，其中未见文献报道的新化合物14个。其次，对从深裂叶阿魏中分离得到的16个萜酯类单体化合物进行了体外抗肿瘤活性测试，发现该类化合物对宫颈癌HeLa细胞表现出很好的生长抑制活性，且活性最好的为新化合物1(命名为TAW，Syreiteate A)。同时，体内HeLa细胞裸鼠异种移植实验结果表明，TAW在体内对肿瘤的生长亦具有一定的抑制作用。再次，本课题以活性最好的TAW为代表，对该类化合物诱导HeLa细胞死亡作用机制进行了研究。结果表明TAW通过ERS（内质网应激）和UPR（未折叠蛋白反应）中IRE1-XBP1和PERK-eIF2α途径诱导细胞发生paraptosis（一种新型死亡方式）。此外，随着TAW作用时间的延长，还发现TAW可诱导细胞发生autophagy，且autophagy可抑制ERS，从一定程度上拮抗paraptosis了产生。最后，在上述工作的基础上，我们利用生物信息学建立了paraptosis和autophagy蛋白相互作用网络，并对16个阿魏萜酯类化合物的作用靶点进行了预测分析。以上结果为寻找和开发新型抗宫颈癌药物提供重要的理论依据。