去氢木香内酯通过PI3K/Akt/mTOR通路对鼻咽癌细胞自噬和凋亡的影响及机制

Effects and mechanism of dehydrocostuslactone on autophagy and apoptosis of nasopharyngeal carcinoma cells via PI3K/Akt/mTOR pathway

目的 探讨去氢木香内酯(DCL)对鼻咽癌细胞CNE1自噬和凋亡的影响及可能机制。方法 含不同浓度DCL培养液(0、5、10、20、40μmol/L)培养对数生长期CNE1细胞48 h,CCK-8法检测细胞存活率;Hoechst 33258荧光染色法观察细胞凋亡形态;流式细胞术检测细胞凋亡率;透射电子显微镜检测CNE1细胞自噬体形成情况;Western blotting法检测CNE1细胞中磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)活性水平及微管相关蛋白1A/1B轻链3(LC3)、p62、B细胞淋巴瘤/白血病-2基因(Bcl2)、Bcl2相关X蛋白(Bax)、cleaved caspase-3蛋白相对表达水平。结果 CNE1细胞存活率、PI3K、Akt和mTOR蛋白活性水平及p62、Bcl2蛋白相对表达水平均随DCL作用浓度增大而降低,CNE1细胞凋亡率、LC3Ⅱ/LC3Ⅰ比值、Bax和cleaved caspase-3蛋白相对表达水平随DCL作用浓度增大而升高(P<0.05),同时DCL给药可显著促进CNE1细胞凋亡小体及自噬体的形成;DCL作用效果呈剂量依赖性(P<0.05)。结论 DCL可促进鼻咽癌细胞发生凋亡,其作用机制可能与抑制PI3K/Akt/mTOR通路激活有关。

Objective To investigate the effects and possible mechanism of dehydrocostuslactone(DCL) on autophagy and apoptosis of nasopharyngeal carcinoma cell line CNE1. Methods DCL culture medium with different concentrations(0, 5, 10, 20, 40 μmol/L) cultured CNE1 cells in logarithmic growth phase for 48 hours,and the cell survival rate was detected by CCK-8 method. Hoechst 33258 fluorescent staining was used to observe the morphology of apoptosis. Apoptosis rate was detected by flow cytometry. The autophage formation of CNE1cells was detected by transmission electron microscopy. The activities of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt) and mammalian rapamycin target protein(mTOR) in CNE1 cells and the relative expression levels of microtubule associated protein 1A/1B light chain 3(LC3), p62, B-cell lymphoma/leukemia 2gene(Bcl2), Bcl2 associated X protein(Bax), and cleared caspase-3 protein were detected by western blotting.Results CNE1 cell survival rate, PI3K, Akt and mTOR protein activity levels and the relative expression levels of p62 and Bcl2 protein decreased with the increase of DCL concentration. CNE1 cell apoptosis rate, LC3 Ⅱ/LC3I ratio, relative expression levels of Bax and cleaved caspase-3 protein increased with the increase of DCL concentration(P < 0.05). At the same time, DCL administration significantly promoted the formation of CNE1 cell apoptotic bodies and autophagic vesicles. The effects of DCL were dose dependent(P < 0.05). Conclusion DCL could promote apoptosis of nasopharyngeal carcinoma cells, and its mechanism may be related to inhibition of PI3K/Akt/mTOR pathway.