丹参酮ⅡA诱导体外胃癌细胞的自噬

Tanshinone ⅡA induces autophagy of human gastric cancer SGC7901 cells line in vitro

目的探讨丹参酮ⅡA体外对人胃癌SGC7901细胞自噬的影响,初步探讨其抑制肿瘤的机制。方法 MTT法检测不同质量浓度(0.5、1、2和4μg/mL)丹参酮ⅡA作用体外培养的胃癌SGC7901细胞共孵24、48、72 h后,细胞增殖活力的改变;1、2和4μg/mL丹参酮ⅡA作用SGC7901细胞48 h后,流式细胞术检测细胞凋亡率和细胞自噬小体的水平;实时定量RT-PCR和Western blot分别测定自噬相关蛋白Beclin 1、磷酸酯酶与张力蛋白同源物(PTEN)、LC3-Ⅱ和LC3-I的mRNA和蛋白的表达水平。结果 0.5μg/mL丹参酮ⅡA对胃癌SGC7901细胞没有明显的生长抑制作用,1μg/mL以上各质量浓度丹参酮ⅡA对胃癌SGC7901细胞则有明显的生长抑制作用;流式细胞分析结果显示,与对照组相比,丹参酮ⅡA诱导细胞凋亡(P<0.05);细胞内酸性自噬小体含量增加(P<0.05);实时定量RTPCR和Western blot结果显示,与对照组相比,Beclin-1、PTEN和LC3-ⅡmRNA和蛋白表达水平逐渐升高,LC3-I表达下降(P<0.05)。结论丹参酮ⅡA可诱导人胃癌SGC7901细胞自噬,其机制可能涉及上调自噬相关基因表达Beclin-1,促进自噬标志蛋白LC3-I向LC3-Ⅱ转化,促进自噬调控蛋白PTEN表达。

AIM To investigate the effect of tanshinone II A （ Tan II A） on the autophagy of human gastric cancer SGC7901 cells and possible mechanism. METHODS SGC7901 cells were cultured in vitro and incubated with different concentrations （0.5, 1, 2, 4 ptg/mL） Tan II A in different time （24, 48, 72 h）. MTT assay was developed to detect the cell proliferation. The cell apoptotic rate and the content of acidic vesicular organelles content were detected by flow cytometry in 48 h. The expressions of autophagy-associated protein Beclin 1, phosphatase and tensin homolog （PTEN）, LC3- I and LC3-II were detected by real-time RT-PCR and western blot. RESULTS Tan 11A （0. 5 p,g/mL） had no significant effect on proliferation of SGC7901 cells compared to DMSO group （P 〉 0. 05 ）. Tan II A showed a dose- and time- dependent growth inhibition at the concentration above 1 p,g/mL （P 〈 0.05）. Compared with DMSO group, the cell apoptotic rate and content acid vesicular organelles content increased with the treatment of Tan I1 A. The results of real-time RT-PCR and western blot showed that compared with DMSO group, the expressions of Beclin 1, PTEN and LC3- II were significantly up-regulated as well as the experssion of LC3-I was significantly down-regulated （P 〈 0. 05 ）. CONCLUSION Tan II A induces the auophagy of human gastric cancer SGC7901 cells in time- and dose-dependent manner in vitro, which may be related to the up-regulation of Beclin 1, PTEN and LC3- 11 , as well as down-regulation of LC3-I.