京大戟提取物pekinenin D通过PI3K/AKT/mTOR信号通路诱导人肝癌SMMC-7721细胞凋亡

Apoptosis of human hepatocellular carcinoma SMMC-7721 cells induced by pekinenin D of Euphorbia pekinensis Rupr through PI3K/AKT/m TOR signal pathway.

目的:通过观察京大戟提取物pekinenin D对肝癌SMMC-7721细胞体外生长的抑制作用,初步探讨其诱导凋亡的可能机制。方法:采用MTT法分析不同剂量的pekinenin D对体外培养SMMC-7721细胞增殖的影响;原位末端标记法(TUNEL)和Annexin V/PI双染法检测对细胞凋亡的作用;应用流式细胞术分析对细胞周期的影响;采用RT-PCR检测pekinenin D对PI3K/AKT/m TOR mRNA的影响;应用生物膜干涉技术(BLI)分析pekinenin D与PI3K启动子是否存在相互作用。结果:MTT结果显示3.988ug/ml的pekinenin D可显著抑制SMMC-7721细胞的增殖,抑制率过半,并且呈剂量依赖性。Annexin V/PI双染法结果显示,随着pekinenin D剂量的增加,SMMC-7721细胞的凋亡率明显增加,与对照组比较,差异具有明显统计学意义。TUNEL法检测结果表明,0.249、0.997、3.988 ug/ml的pekinenin D作用于SMMC-7721细胞后均可诱导肝癌细胞凋亡,凋亡指数(AI)随着药物浓度的升高明显增加。流式细胞术分析显示,0.249、0.997、3.988 ug/ml的pekinenin D均可使细胞被阻滞于S期,RT-PCR显示pekinenin D显著下调SMMC-7721细胞中PI3K,AKT,m TOR的mRNA表达量。采用BLI技术分析发现pekinenin D与PI3K的启动子存在一定亲和力。结论:pekinenin D具有抑制肝癌细胞增殖及诱导凋亡的作用,显著下调SMMC-7721细胞中PI3K,AKT,m TOR的mRNA表达,其机制可能是与PI3K的启动子相结合从而抑制PI3K/AKT/m TOR信号通路有关。

Objective： TO investigate the inhibitory effect of pekinenin D on the growth of hepatoma SMMC-7721 cells in vitro and observe the possible mechanism of apoptosis induced by diterpenoids. Methods： MTT assay was used to investigate the effect of pekinenin D on proliferation of SMMC-7721 cells; TUNEL method and Annexin V/PI staining were employed to observe the cell apoptosis; Flow cytometry was applied to analyzing the distribution of cell cycle. The effect of pekinenin D on PI3 K/AKT/m TOR mRNA was detected by RT-PCR. Analysis of the interaction between pekinenin D and PI3 K promoter was performed by biofilm interference technique（ BLI）. Results ： MTT results showed that 3. 988 ug/ml of pekinenin D significantly inhibited the proliferation of SMMC-7721 cells in a dose-dependent manner,and the difference was statistically significant（ P〈0. 01） compared with the control group.; Annexin V/PI staining showed that with the increase of pekinenin D concentration,the apoptosis rate of SMMC-7721 cells significantly increased,compared with the control group,the difference was statistically significant. TUNEL method test results showed that 0. 249 ug/ml,0. 997 ug/ml and 3. 988 ug/ml of pekinenin D induced liver cancer cell apoptosis and the apoptosis index（ AI） with the increase of drug concentration significantly increased. Flow cytometry analysis showed that 0. 249 ug/ml,0. 997 ug/ml and 3. 988 ug/ml of pekinenin D all blocked cells in S phase. RT-PCR showed that pekinenin D significantly down-regulated mRNA expressions of PI3 K,AKT and m TOR in SMMC-7721 cells. Using BLI technique,it was found that pekinenin D had a certain affinity for PI3 K promoter. Conclusion： Pekinenin D could inhibit the proliferation and induce apoptosis of hepatoma cells,and down-regulate mRNA expressions of PI3 K,AKT and m TOR in SMMC-7721 cells. The mechanism may be related to the PI3 K promoter which inhibits the PI3 K/AKT/m TOR signaling pathway.