木犀草素促进鼻咽癌细胞凋亡和自噬的机制

Mechanism of luteolin promoting apoptosis and autophagy in nasopharyngeal carcinoma cells

目的:探讨木犀草素对鼻咽癌细胞增殖、凋亡和自噬的影响及可能的分子机制。方法:四氮唑蓝盐化合物(MTS法)检测木犀草素对人鼻咽癌细胞CNE-1增殖的作用。流式细胞法检测细胞凋亡,透射电镜观察细胞自噬小体,Western印迹检测木犀草素对CNE-1细胞凋亡抑制蛋白(Bcl-2)和自噬相关蛋白[自噬效应蛋白(Beclin-1)、微管相关蛋白轻链3蛋白(LC3B)、泛素结合蛋白(p62)]表达的变化并检测磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、雷帕霉素靶蛋白(mTOR)蛋白磷酸化水平,检测PI3K/Akt/mTOR信号通路相关蛋白。结果:木犀草素可抑制CNE-1细胞活力,呈浓度和时间依赖性(P<0.05),同时木犀草素与对照组相比凋亡率增加(P<0.05)。透射电镜结果显示,木犀草素干预CNE-1细胞后,随着药物浓度的增加,细胞内自噬体出现不同程度的增加。Western印迹法结果显示,与对照组比较,木犀草素处理组细胞Beclin-1和LC3-Ⅱ蛋白表达上调(P<0.05),Bcl-2、p62、磷酸化磷脂酰肌醇(p-PI3K)、磷酸化蛋白激酶B(p-Akt)和磷酸化雷帕霉素靶蛋白(p-mTOR)表达明显降低,差异均有统计学意义(P<0.05)。结论:木犀草素通过调节PI3K/Akt/mTOR级联反应,抑制CNE-1细胞生长,启动凋亡和自噬介导的细胞死亡。木犀草素可能成为未来治疗鼻咽癌的有效候选药物。

Objective To investigate the effects of luteolin on proliferation, apoptosis and autophagy in nasopharyngeal carcinoma CNE-1 cells and its possible molecular mechanisms.Method The inhibitory effect of luteolin on CNE-1 cell proliferation was assessed by MTS method.Annexin V-FITC/PI double-staining method detected apoptosis rate.Autophagosomes were observed with transmission electron microscope.The levels of apoptosis and autophagy-related protein Bcl-2, Beclin-1, LC3 B, p62 were examined by western blot.Additionally, the phosphorylation protein levels of PhosphoInositide-3 Kinase(PI3 K), Protein Kinase B(PKB/Akt), Reapamycin target protein(mTOR) associate with PI3 K/Akt/mTOR pathway were examined using western blot analysis too.Results Luteolin inhibited the proliferation of CNE-1 cell in a dose and time dependent manner(P<0.05).Results of flow cytometry showed that compared with the control group, the total apoptosis rate of luteolin treatment group increased(P<0.05).The results of transmission electron microscopy showed that after the intervention of luteolin on CNE-1 cells, the intracellular autophagosomes increased to varying degrees with the increase of drug concentration.The analysis of western blot revealed that the expression of Beclin-1 and LC3 Ⅱ protein increased(P<0.05), the expression of Bcl-2、p62 and the expression of p-PI3 K, p-Akt and p-mTOR decreased compared with the control group, the difference was statistically significant(P<0.05).Conclusion Luteolin inhibits CNE-1 cell growth and initiates apoptosis and autophagy-mediated cell death by regulating PI3 K/Akt/mTOR cascade reaction.Luteolin may become an effective candidate for the treatment of nasopharyngeal carcinoma in the future.