细胞自噬在褪黑素诱导的肝癌细胞株HepG2死亡中的作用

The role of autophagy in human hepatocarcinoma cell line HepG2 induced by melatonin

目的研究褪黑素(MT)对人肝癌HepG2细胞自噬功能的影响,并初步探讨自噬在MT诱导细胞死亡中的作用。方法体外培养肝癌细胞株HepG2,用不同浓度的MT(10-7、10-5、10-3mol/L)作用于HepG2细胞,Western blot法检测HepG2细胞自噬相关蛋白LC3(LC3-Ⅰ/LC3-Ⅱ)的表达,并用单丹磺酰尸胺(MDC)染色在激光共聚焦显微镜下观察HepG2细胞内自噬体形成情况;在给予MT的同时使用3-甲基腺嘌呤(3-MA)或氯喹(CQ)对细胞自噬进行抑制,利用MTT和Western blot法分别测定HepG2细胞的细胞活力和LC3蛋白的表达变化。结果 MT作用后可诱导HepG2细胞内自噬小体形成,自噬相关蛋白LC3-Ⅱ表达增多且呈剂量依赖关系。MT作用后,HepG2细胞生长受到抑制,加入3-MA或CQ抑制自噬后,细胞生长受抑现象更加显著。MT+3-MA组LC3-Ⅱ/β-actin比值较MT单药组明显减小,MT+CQ组则比值增大。结论 MT可激活肝癌细胞HepG2保护性自噬反应,通过3-MA或CQ阻断MT诱导的自噬通路可显著增强MT对HepG2细胞的生长抑制作用,提示自噬可能是肝癌细胞HepG2逃避MT肿瘤杀伤作用的一种有效机制。

Objective To investigate the change in function of autophagy in hepatocarcinoma cell line HepG2 induced by melatonin （MT）,and to explore the role of autophagy in MT-induced cell death.Methods Hepatocarcinoma cancer cell line HepG2 was treated with different concentrations of MT（10-7,10-5,10-3 mol/L）.The expression of autophagy-related protein LC3 （LC3-Ⅰ/LC3-Ⅱ） during MT-induced cell death was analysed by Western blot assay,autophgosomes and autolysosomes in HepG2 cells were detected by Monodansylcadaverin （MDC）staining under laser scanning confocal microscope （LSCM）.The viability of HepG2 cells was examined by MTT assay after treated either with different concentrations of MT alone or in combination with the autophagy inhibitor 3-methyladenine（3-MA） or chloroquine（CQ）.The expression of LC3 in MT-treated HepG2 cells was analysed by Western blot assay before and after 3-MA or CQ treatment.Results Western blot assay demonstrated that MT could dose-dependently increased LC3-Ⅱ expression,and the formation of autophagosomes in HepG2 cells after MT treatment could be observed under LSCM.Compared with the control group,MT treatment significantly inhibited cell growth which was further enhanced by combined use of autolysosome inhibitor 3-MA or CQ,the ratio of LC3-Ⅱ/β-actin was decreased markedly after 3-MA treatment,while after CQ treatment,the ratio of LC3-Ⅱ/β-actin was increased markedly.Conclusion MT can enhance the function of autophagy in HepG2 cells and induce an increased formation of autophagosomes.The autolysosome inhibitor 3-MA or CQ can significantly enhance the effect of MT-induced proliferative inhibition in HepG2 cells by blocking MT-induced autophagy pathway,indicating autophagy is likely to be a mechanism for HepG2 cells to avoid MT-induced cell death.