丹参酮ⅡA通过自噬诱导肺癌A549细胞周期阻滞的作用研究

Cell Cycle Arrest in A549 Cells Caused by TanshinoneⅡA Induced Autophagy

目的观察丹参酮ⅡA(TanshinoneⅡA,TⅡA)对人非小细胞肺癌A549细胞增殖的影响,探讨TⅡA诱导A549细胞自噬与细胞周期阻滞的关系。方法采用CCK8法检测TⅡA(0.5、1、2、4、8μmol·L^-1)对A549细胞增殖的抑制作用;利用流式细胞术检测TⅡA(1、2、4μmol·L^-1)对A549细胞周期分布的影响;采用Western Blot法检测TⅡA(1、2、4μmol·L^-1)对A549细胞周期相关蛋白Cyclin D1和自噬相关蛋白p62、LC3B表达的影响;分别采用自噬激动剂雷帕霉素(Rapamycin,RAPA;0.25μmol·L^-1)+TⅡA(2μmol·L^-1)或自噬抑制剂3-甲基腺嘌呤(3-MA,5 mmol·L^-1)+TⅡA(2μmol·L^-1)培养处理A549细胞,然后采用Western Blot法检测细胞Cyclin D1、p62、LC3B蛋白的表达,并通过流式细胞术检测细胞周期分布。结果与对照组比较,TⅡA不同浓度组对A549细胞均具有明显的增殖抑制作用(P<0.05,P<0.01),且呈时间-剂量依赖关系;药物作用12、24 h后,TⅡA的IC50值分别为7.82、2.04μmol·L^-1。与对照组比较,TⅡA能降低A549细胞Cyclin D1的表达量(P<0.01),使细胞周期阻滞于G0/G1期(P<0.05,P<0.01),且呈剂量依赖性;TⅡA能够升高A549细胞LC3-Ⅱ/LC3-Ⅰ比值(P<0.05,P<0.01),降低p62表达(P<0.01),诱导细胞发生自噬。与TⅡA组比较,3-MA+TⅡA组A549细胞中的LC3-Ⅱ/LC3-I比值明显降低(P<0.01),p62及Cyclin D1表达水平明显上调(P<0.01),停滞于G0/G1期的细胞比例明显减少(P<0.05)。结论TⅡA可能通过上调人非小细胞肺癌A549细胞自噬诱导细胞周期阻滞,从而抑制细胞增殖,发挥抗肿瘤作用。

Objective To observe the effect of tanshinoneⅡA(TⅡA)on the proliferation of human non-small cell lung cancer A549 cells,and to explore the interrelationship between autophagy and cell cycle arrest induced by TⅡA.Methods The inhibitory effect of TⅡA(0.5,1,2,4,8μmol·L^-1)on the proliferation of A549 cells was detected by CCK 8 method.The effect of TⅡA(1,2,4μmol·L^-1)on A549 cell cycle distribution was detected by flow cytometry.Western Blot was used to detect the effects of TⅡA(1,2,4μmol·L^-1)on the expression of Cyclin D1 and autophagy related proteins p62 and LC3 B in A549 cells.The A549 cells were treated separately with autophagy activator Rapamycin(RAPA,0.25μmol·L^-1)+TⅡA(2μmol·L^-1)or autophagy inhibitor 3-methyladenine(3-MA,5 mmol·L^-1)+TⅡA(2μmol·L^-1).The expressions of Cyclin D1,p62 and LC3 B were detected by Western Blot,and the cell cycle distribution was detected by flow cytometry.Results Compared with the control group,TⅡA at different concentrations had significant inhibitory effect on proliferation of A549 cells in a time-dose dependent manner(P<0.05,P<0.01).After 12 and 24 hours of treatment,the IC50 values of TⅡA were 7.82 and 2.04μmol·L^-1,respectively.Compared with the control group,the content of Cyclin D1 in A549 cells was reduced(P<0.01)and the cell cycle in G0/G1 phase was arrested by TⅡA in a dose-dependent manner(P<0.05,P<0.01).TⅡA could induce autophagy by increasing the ratio of LC3-Ⅱ/LC3-I(P<0.05,P<0.01)and reducing the expression of p62(P<0.01)in A549 cells.Compared with TⅡA group,the ratio of LC3-Ⅱ/LC3-I in A549 cells in 3-MA+TⅡA group was significantly reduced(P<0.01).Meanwhile,the level of p62 was significantly increased(P<0.01),and the expression of Cyclin D1 was significantly up-regulated(P<0.01),while the proportion of cells in G0/G1 phase was significantly decreased(P<0.05).Conclusion TⅡA might induce cell cycle arrest by up-regulating autophagy in A549 cells,thereby inhibit cell proliferation and play an anti-tumor effect.