丹参酮ⅡA体外促进黑素瘤A375细胞自噬及信号通路的实验研究

In vitro effects of tanshinone II A on autophagy of A375 melanoma cells and related signaling pathway

目的探讨丹参酮ⅡA体外对黑素瘤细胞A375细胞自噬的影响及其信号通路研究。方法0．5、1、2、4mg／L丹参酮ⅡA作用黑素瘤A375细胞24、48、72h后，噻唑蓝（MTT）比色法检测A375细胞的增殖活性。1、2、4mg／L丹参酮ⅡA作用黑素瘤A375细胞48h，采用流式细胞仪检测细胞自噬小体的数量，Western印迹检测自噬相关蛋白Beelin。1和微管相关蛋白1轻链3（LC3）．Ⅱ蛋白及磷酸肌醇3激酶（P13K）、蛋白激酶B（Akt）、雷帕霉素靶蛋白（mTOR）、p70S6激酶1（p70S6K1）蛋白的表达水平。结果MTT分析显示，0．5、1、2和4mg／L丹参酮ⅡA分别作用黑素瘤A375细胞24、48、72h，均能抑制A375细胞的增殖能力，且抑制作用呈剂量和时间依赖性（F=2564．12、1235．25，均P〈0．05）。流式细胞仪显示，1、2和4mg／L丹参酮ⅡA作用A375细胞48h后，细胞内自噬小体比例分别为6．91％±0．35％、13．11％±0．73％、25．51％±0．83％，均明显高于对照组（0．41％±0．02％），各组间差异有统计学意义（均P〈0．05）。Western印迹显示，1、2和4mg／L丹参酮ⅡA作用A375细胞48h后，细胞自噬相关蛋白Beelin-1和LC3-Ⅱ表达水平随丹参酮ⅡA浓度增加而升高，各丹参酮ⅡA组间差异有统计学意义，且高于对照组（均P〈0．05）。而P13K．Akt-mTOR—p70S6K1信号通路中P13K、p-Akt、p-mTOR和P—p70S6K1蛋白表达随丹参酮ⅡA浓度增加而下降，各丹参酮ⅡA组间差异有统计学意义，且低于对照组（均P〈0．05）。结论丹参酮ⅡA可通过抑制P13K-Akt-mTOR-p70S6K1信号通路，促进黑素瘤细胞发生自噬。

Objective To investigate in vitro effects of tanshinone Ⅱ A on the autophagy of A375 melanoma cells and related signaling pathway. Methods Some cultured A375 cells were divided into 5 groups to be treated with tanshinone Ⅱ A at concentrations of 0.5, 1, 2 and 4 mg/L, and DMEM containing 0.1% dimethyl sulfoxide （DMSO）, respectively, for 24, 48, 72 hours. Methyl thiazol tetrazolium （MTT） assay was performed to estimate the proliferative activity of A375 cells. Some cultured A375 cells were divided into 4 groups to be treated with 1, 2 and 4 mg/L tanshinone Ⅱ A （1-, 2- and 4-mg/L tanshinone group）, and DMEM containing 0.1% DMSO （control group）, respectively, for 48 hours. Then, flow cytometry was conducted to count autophagosome-positive cells, and Western blot analysis to determine protein expression of autophagy-associated proteins Beclin-1, microtubule-associated protein 1 light chain 3 （LC3） - Ⅱ, phosphatidylinositol 3-kinase （PI3K）, protein kinase B （Akt）, mammalian target of rapamycin （mTOR） and p70 ribosomal protein S6 kinase 1 （p70S6K1）. Results MTF assay showed that 24-, 48-, 72-hour treatments with tanshinone Ⅱ A at concentrations of 0.5, 1, 2 and 4 mg/L all could inhibit the proliferative activity of A375 cells, and the inhibitory effects increased in a dose- and time-dependent manner （F = 2 564.12, 1 235.25, both P 〈 0.05）. The percentage of autophagosome-positive cells and protein expression of Beclin- 1 and LC3- 11 increased gradually and significantly in the 1-, 2- and 4-mg/L tanshinone groups （autophagosome-positive cells： 6.91% ± 0.35%, 13.11% ± 0.73%, 25.51% ± 0.83%, respectively; Beclin- 1：0.33 ± 0.01, 0.53 ± 0.04, 0.63 ± 0.02, respectively; LC3-Ⅱ ： 0.41 ± 0.01, 0.52 ± 0.02, 0.64 ± 0.02, respectively）, after 48-hour treatment, which were significantly different between the tanshinone groups （all P 〈 0.05）, and higher in the tanshinone groups than in the control group （0.41% ± 0.02%; 0.09 ± 0.02; 0.21 ± 0.01, all P 〈 0.05）. However, the protein expression of PI3K, phosphorylated Akt （p- Akt）, p- mTOR and p- p70S6K 1 in the PI3K- Akt- roTOR- p70S6K 1 signaling pathway decreased gradually and significantly with the increase in tanshinone concentrations after 48-hour treatment, and were significantly lower in all the tanshinone groups than in the control group （all P 〈 0.05）. Conclusion Tanshinonc Ⅱ A can promote the auophagy of A375 cells, likely by blocking the PI3K-Akt-mtTOR- p70S6K1 signaling pathway.