丹参酮ⅡA对人喉癌Hep-2细胞增殖和细胞周期的影响及机制研究

Effects of Tanshinone ⅡA on proliferation and cell cycle of human laryngeal carcinoma Hep-2 cells and its mechanism

目的:研究丹参酮ⅡA对人喉癌Hep-2细胞增殖和周期的影响及机制。方法:以不同浓度丹参酮ⅡA干预对数生长期人喉癌Hep-2细胞48 h,计算半抑制浓度(IC_(50))作为后续实验药物浓度。取对数生长期Hep-2细胞,设空白对照组、丹参酮ⅡA组、丹参酮ⅡA+IGF-1(PI3K激活剂)组、丹参酮ⅡA+LY294002(PI3K抑制剂)组,分别给药干预48 h后,平板克隆实验检测细胞克隆形成数,CCK-8法、PI单染流式细胞术检测细胞增殖抑制率和细胞周期分布,Western blot法检测cyclin B1、cdc2、p-cdc2、p27、磷脂酰肌醇3激酶(PI3K)、p-PI3K、蛋白激酶B(Akt)、p-Akt蛋白表达。结果:丹参酮ⅡA对Hep-2细胞IC_(50)值为9.6μmol/L。与空白对照组比较,丹参酮ⅡA组Hep-2细胞克隆形成数降低,细胞增殖抑制率升高,G_(2)/M期细胞比例升高,cyclin B1、cdc2、p-cdc2、p-PI3K、p-Akt表达量及p-cdc2/cdc2、p-PI3K/PI3K、p-Akt/Akt比值降低,p27表达量升高(均P<0.05)。IGF-1能够明显逆转丹参酮ⅡA对Hep-2细胞的上述调控作用,LY294002能够明显加强丹参酮ⅡA的上述作用。结论:丹参酮ⅡA可抑制人喉癌Hep-2细胞增殖、诱导细胞G_(2)/M期阻滞,作用机制可能与抑制PI3K/Akt信号通路活化有关。

Objective:To investigate the effect and mechanism of TanshinoneⅡA on the proliferation and cycle of human laryngeal carcinoma Hep-2 cells.Methods:Different concentrations of TanshinoneⅡA were used to intervene the human laryngeal carcinoma Hep-2 cells in logarithmic growth phase for 48 hours,and the half-inhibitory concentration(IC 50)was calculated as the follow-up experimental drug concentration.The Hep-2 cells in logarithmic growth phase were divided into blank control group,TanshinoneⅡA group,TanshinoneⅡA+IGF-1(PI3K activator)group and TanshinoneⅡA+LY294002(PI3K inhibitor)group.After 48 hours of drug intervention,the number of cell clones formation was detected by plate cloning assay,cell proliferation inhibition rate and cell cycle distribution were detected by CCK-8 method and PI single staining flow cytometry.The expressions of cyclin B1,cdc2,p-cdc2,p27,phosphatidylinositol 3 kinase(PI3K),p-PI3K,protein kinase B(Akt),p-Akt were detected by Western blot.Results:The IC 50 of TanshinoneⅡA on Hep-2 cell was 9.6μmol/L.Compared with the blank control group,the number of cell clones formation of Hep-2 cells in TanshinoneⅡA group was decreased,the proliferation inhibition rate was increased,the cell ratio in G 2/M phase was increased,the expressions of cyclin B1,cdc2,p-cdc2,p-PI3K,p-Akt and the ratio of p-cdc2/cdc2,p-PI3K/PI3K,p-Akt/Akt were decreased,the expression of p27 was decreased(all P<0.05).IGF-1 could obviously reverse the above-mentioned regulatory effect of TanshinoneⅡA on Hep-2 cells,and LY294002 could enhance the above-mentioned effect of TanshinoneⅡA.Conclusion:TanshinoneⅡA can inhibit the proliferation of human laryngeal carcinoma Hep-2 cells and induce cell G 2/M phase arrest,and the mechanism may be related to the inhibition of PI3K/Akt signaling pathway activation.