姜黄素类似物EF24诱导A549细胞自噬及凋亡关系的研究

Study on the Relationship between Autophagy and Apoptosis in A549 Cells Induced by Curcumin Analogue EF24

从细胞自噬及凋亡关系角度探讨姜黄素类似物EF24对人肺腺癌细胞（A549）的杀伤机理。选用不同浓度的EF24对体外培养的A549处理,采用MTT方法检查细胞存活率,吖啶橙染色观察细胞形态,蛋白质免疫印迹（Western blot）方法检测与细胞自噬及凋亡相关蛋白的表达及对AMPK-mTOR-S6K信号通路的影响。结果显示,EF24作用24 h的IC50为8.5μmol/L,对A549细胞生长抑制作用优于姜黄素,而接近顺铂。自噬及凋亡蛋白检测显示,在4μmol/L、8μmol/L时A549细胞以自噬为主,在16μmol/L时以凋亡为主;加入100 nmol/L自噬抑制剂渥曼青霉素（wortmannin）后,细胞存活率同比升高。同时还发现,随着EF24浓度的增加,细胞内AMPK-Thr172磷酸化水平上升,mTOR-Ser2481、S6K-Thr389磷酸化水平的下调。由此可见,EF24可通过AMPK的激活下调mTOR-S6K途径抑制细胞生长,在EF24浓度4~8μmol/L范围内,自噬对凋亡起到促进作用。

This paper studies the cytotoxicity mechanism of curcumin analogue EF24 in A549 cell line（human lung adenocarcinoma cells） through the relationship between autophagy and apoptosis.After treatment with different concentrations of EF24 in A549 cell line,we used MTT assay to check cell liviability,acridine orange staining to observe cell morphology change,and Western blot to detect cell autophagy and apoptosis-related protein in AMPK-mTOR-S6K signaling pathway.These results showed that the half lethal dose IC50 of EF24 in A549 cell line at 24 h was 8.5 μmol/L,which was more less than curcumin and similar to cisplatin.With the detection of autophagy and apoptosis-related protein,we found that autophagy was a mainly phenomenon at the concentration of 4 μmol/L and 8 μmol/L,however,the apoptosis played an important role at the concentration of 16 μmol/L;Autophagy inhibitor wortmannin（100 nmol/L） could partly increase the cell survival rate.Meanwhile,we also found the increasing phosphorylation level of AMPK-Thr172 and decreasing phosphorylation level of mTOR-Ser2481 and S6K-Thr389 after treatment with EF24.Our results indicate that EF24 inhibited cell growth in A549 cell line through activing AMPK kinase and attenuate the mTOR-S6K pathway.Autophagy promoted cell apoptosis in the concentration range of 4~8 μmol/L EF24.