黄芪甲苷通过PI3K/Akt/mTOR通路诱导鼻咽癌细胞自噬和凋亡

AstragalosideⅣInduces Autophagy and Apoptosis in Nasopharyngeal Carcinoma Cells via PI3K/Akt/mTOR Pathway

目的:探讨黄芪甲苷对鼻咽癌细胞自噬与凋亡的影响及潜在分子机制。方法:在体外实验中,通过单丹磺酰戊二胺(MDC)染色和透射电镜观察黄芪甲苷(AS-Ⅳ)对鼻咽癌细胞自噬的影响。在体内实验中,建立裸鼠移植瘤模型后采用免疫荧光法(IF)及蛋白免疫印迹法(Western blot)检测各组别中自噬和凋亡情况的变化及磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路关键蛋白的表达变化。结果:与空白组比较,不同浓度AS-Ⅳ(5、10、20μmol·L^(-1))作用于5-8F细胞后,各AS-Ⅳ组的自噬荧光强度均明显增强,其中,干预24 h,各AS-Ⅳ组的自噬荧光表达最强,10μmol·L^(-1)AS-Ⅳ组的荧光表达最明显;与空白组比较,透射电镜下自噬激活剂雷帕霉素(RAPA)诱导5-8F细胞内出现较多自噬体;3-甲基腺嘌呤(3-MA)作为自噬抑制剂,透射电镜下并未观察到5-8F细胞内有自噬体的形成;AS-Ⅳ10μmol·L^(-1)组细胞内结构和细胞膜完整、清楚,可见自噬体形成;与空白组比较,AS-Ⅳ各组的肿瘤体积明显受到抑制(P<0.05,P<0.01),微管相关蛋白l轻链3Ⅱ/微管相关蛋白l轻链3Ⅰ(LC3Ⅱ/Ⅰ)和活化的胱天蛋白酶-3(cleaved Caspase-3)荧光信号明显增强(P<0.05,P<0.01),自噬相关蛋白酵母ATG6同源物(Beclin-1)、LC3Ⅱ/Ⅰ和凋亡相关蛋白cleaved Caspase-3、活化的多腺苷二磷酸多聚酶(cleaved PARP)的表达量均明显升高(P<0.05,P<0.01),而自噬相关蛋白泛素结合蛋白(p62)的表达明显下调(P<0.05,P<0.01);磷酸化(p)-PI3K、p-Akt、p-mTOR蛋白表达量明显降低(P<0.05,P<0.01)。结论:黄芪甲苷可诱导鼻咽癌细胞发生自噬和凋亡,其机制可能是通过激活PI3K/Akt/mTOR信号通路。

Objective:To investigate the effect and underlying molecular mechanism of astragaloside-Ⅳ(AS-Ⅳ)on autophagy and apoptosis of nasopharyngeal carcinoma cells.Method:In experiments in vitro,the effect of AS-Ⅳon the autophagy of nasopharyngeal carcinoma cells was observed by monodansylcadaverine(MDC)staining and transmission electron microscopy(TEM).In experiments in vivo,immunofluorescence(IF)and Western blot were used to detect the changes in autophagy and apoptosis and the expression of key proteins in the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway after the establishment of a xenograft tumor model in nude mice.Result:After 5-8F cells were treated with AS-Ⅳof different doses(5,10,20μmol·L^(-1)),the fluorescence intensity of autophagy in AS-Ⅳgroups significantly increased as compared with that in the blank group.The fluorescence expression of autophagy in AS-Ⅳgroups was the strongest after intervention for 24 hours,and the fluorescence expression in the 10μmol·L^(-1)AS-Ⅳgroup was the most obvious.The autophagy activator rapamycin(RAPA)induced more autophagosomes in 5-8F cells under the transmission electron microscope,and 3-methyladenine(3-MA),an autophagy inhibitor,did not induce autophagosome formation in 5-8F cells under the transmission electron microscope as compared with the results in the blank group.In the 10μmol·L^(-1)AS-Ⅳgroup,the intracellular structure and cell membrane were intact and clear,and autophagosome formation was observed.Compared with the blank group,the AS-Ⅳgroups showed inhibited tumor volume(P<0.05,P<0.01),potentiated fluorescence signals of microtubule-associated protein l light chain 3 typeⅡ/microtubule-associated protein l light chain 3 typeⅠ(LC3Ⅱ/Ⅰ)and cleaved Caspase-3(P<0.05,P<0.01),increased expression levels of the mammalian homolog of yeast ATG6(Beclin-1),LC3Ⅱ/Ⅰ,cleaved Caspase-3,and cleaved PARP(P<0.05,P<0.01),down-regulated expression of ubiquitin-binding protein(p62)(P<0.05,P<0.01),and reduced protein expression levels of phosphorylated PI3K(p-PI3K),phosphorylated Akt(p-Akt),and phosphorylated mTOR(p-mTOR)(P<0.05,P<0.01).Conclusion:AS-Ⅳcan induce autophagy and apoptosis of nasopharyngeal carcinoma cells,and the mechanism is presumably attributed to the activation of the PI3K/Akt/mTOR signaling pathway.