摘要
目的:研究从天名精中分离得到的萜类化合物Telekin诱导的胃癌细胞HGC-27自噬并探讨其分子作用机制。方法:MTT法研究Telekin对不同肿瘤细胞的细胞毒活性;透射电镜观察Telekin诱导的HGC-27细胞自噬;用mRFP-GFP-LC3点状聚集物的形成,评价HGC-27细胞内自噬流的变化;MTT法研究用不同自噬抑制剂氯喹(CQ)、3-甲基腺嘌呤(3-MA)和mTOR抑制剂雷帕霉素(Rapamycin)预处理时,Telekin对HGC-27细胞的细胞毒活性,研究自噬与细胞死亡的关系。Western blot检测PI3K/Akt/mTOR信号通路关键蛋白PI3K、Akt、mTOR、p-PI3K、p-Akt、p-mTOR、p-ULK1、ULK1、Beclin1、p-Beclin1、p62、LC3Ⅰ、LC3Ⅱ蛋白的表达。结果:MTT实验结果表明Telekin对胃癌细胞HGC-27具有较好的细胞毒活性和一定的选择性;透射电镜和激光共聚焦显微镜观察到Telekin诱导HGC-27细胞发生了明显的自噬和显著的自噬流变化;用Rapamycin预处理时增强了Telekin的细胞毒性,IC_(50)值由22.78μM降为14.4μM。用3-MA和CQ预处理后降低了Telekin的细胞毒性,IC_(50)值从22.78μM增加为30.06μM和29.46μM,说明自噬促进了细胞死亡;Western blot结果显示Telekin降低了p-PI3K、p-Akt、p-mTOR、p62、LC3Ⅰ的表达,增强了p-ULK1、p-Beclin1、LC3Ⅱ的表达,而对PI3K、Akt、mTOR、ULK1、Beclin1总蛋白没有明显影响。结论:Telekin通过降低p-PI3K、p-Akt、p-mTOR、p62、LC3Ⅰ的表达水平,增强p-ULK1、p-Beclin1、LC3Ⅱ的表达水平,来诱导HGC-27细胞发生自噬,从而促进细胞死亡。
Objective: To investigate telekin,a terpenoid compound isolated from Carpesium abrotanoides L. induced autophagy in human gastric carcinoma HGC-27 cells,and explore its mechanism. Methods: The anti-tumor activity of telekin against different cancer cells was assessed by MTT assay. Autophagy induction was detected by transmission electron microscopy( TEM) observation. Meanwhile,changes of autophagy flux in HGC-27 cell was assessed by the formation of mRFP-GFP-LC3 puncta. To detect the role of autophagy in cells death,telekin toxicity activity on HGC-27 cell was determined by MTT,pretreated with autophagy inhibitors( 3-MA,CQ) or mT OR inhibitor rapamycin. The protein expressions of PI3 K,Akt,mT OR,p-PI3 K,p-Akt,p-mTOR,p-ULK1,ULK1,Beclin1,p-Beclin1,p62,and LC3 were detected by Western blot. Results: The results showed that telekin had potential anti-tumor activity against different human cancer cells,especially on HGC-27 cells. Induced by telekin,the autophagy and acceleration of autophagic flux were observed by TEM. After pretreated with rapamycin,the cytotoxicity of telekin was enhanced and the IC50 value was decreased from 22. 78μM to 14. 40μM. When pretreated with 3-MA or CQ,the cytotoxicity of telekin was weakened and the IC50 value was increased from 22. 78μM to 30. 06μM and 29. 46μM respectively,which indicated that autophagy could promote HGC-27 cell death. Western blot result indicated the telekin significant down-regulated protein expressions of p-PI3 K,p-Akt,p-mTOR,p62,LC3Ⅰand up regulated p-ULK1,p-Beclin1,LC3Ⅱ,and showed no obvious influence on total protein expressions of PI3 K,Akt,mT OR,ULK1 and Beclin1. Conclusion: Telekin can induce autophagy in HGC-27 cells by down-regulating the levels of p-PI3 K,p-Akt,p-mTOR and p62,increasing the protein expressions of p-ULK1、p-Beclin1 and LC3Ⅱ,to promote cell death.
作者
田丽
向卫
汪蕾
程凡
邹坤
陈剑锋
Tian Li;Xiang Wei;Wang Lei;Cheng Fan;Zou Kun;Chen Jianfeng(Hubei Key Laboratory of Natural Products Research and Development, College of Biological and Pharmaceutical Sciences,China Three Gorges University,Yichang 443002)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2019年第1期67-71,共5页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然科学基金面上项目(81773952)
宜昌市科技局研究项目(A16-302-A04)
