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目的:研究自噬抑制剂3-甲基腺苷(3-MA)对姜黄素诱导喉癌Hep-2细胞系自噬和凋亡的影响。方法:常规培养人喉癌Hep-2细胞,MTT法检测不同浓度姜黄素对细胞的增殖抑制率;AO吖啶橙染色法观察细胞的自噬水平,Annexin VFITC/PI双染法流式细胞仪检测各组细胞凋亡率;Western blot检测LC3、Beclin1、Bcl-2以及Bax蛋白的表达水平。结果:MTT法结果显示姜黄素呈时间-剂量依赖性抑制Hep-2细胞的增殖;姜黄素联合使用3-MA后可使凋亡率增多(P<0.05)。吖啶橙染色结果显示3-MA联合姜黄素能明显降低姜黄素引起的喉癌Hep-2细胞的自噬水平。Western blot结果显示联合使用3-MA后,蛋白Bcl-2以及Bclin-1表达降低,Bax蛋白表达水平升高,LC3Ⅱ表达减少(P<0.05)。结论:姜黄素能诱导喉癌Hep-2细胞凋亡,同时也诱导了保护性自噬的发生。自噬抑制剂3-MA抑制自噬后,能明显增强姜黄素对喉癌Hep-2细胞系的凋亡诱导效应。
Objective: To study the effect of autophagy inhibitor 3-methyladenosine (3-MA) on the autophagy and apoptosis induced by curcumin in Hep-2 laryngeal carcinoma cell line. Methods: Human laryngeal carcinoma Hep-2 cells were cultured routinely. MTT assay was used to detect the inhibitory effect of curcumin on cell proliferation. AO acridine orange staining was used to observe the autophagy. Annexin VFITC / PI double staining flow cytometry The apoptosis rate of each group was detected. The expression of LC3, Beclin1, Bcl-2 and Bax protein were detected by Western blot. Results: MTT assay showed that curcumin inhibited the proliferation of Hep-2 cells in a dose-dependent manner. Curcumin combined with 3-MA increased the apoptosis rate (P <0.05). Acridine orange staining results show that 3-MA combined with curcumin can significantly reduce curcumin-induced laryngeal Hep-2 cells autophagy levels. Western blot results showed that Bcl-2 and Bclin-1 protein expression decreased, Bax protein expression increased, and LC3Ⅱ protein expression decreased (P <0.05). Conclusion: Curcumin can induce the apoptosis of laryngeal carcinoma Hep-2 cells and induce the occurrence of protective autophagy. Autophagy inhibitor 3-MA inhibited autophagy and significantly enhanced the apoptosis-inducing effect of curcumin on Hep-2 cell line.