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目的:研究二氢青蒿素(DHA)对乳腺癌细胞系T47D的增殖、凋亡作用的影响,初步探讨其抗癌作用的分子机制。方法:以乳腺癌细胞T47D为研究对象,观测不同浓度和作用时间的DHA对T47D细胞的作用,MTT噻唑蓝比色法检测对细胞的增殖抑制作用,分别PI单染及Annexin-V双染法流式细胞术分析细胞周期分布和早期凋亡率的变化,RT-PCR和蛋白质印迹法检测细胞凋亡相关蛋白的表达。结果:DHA对T47D细胞有增殖抑制作用且呈剂量和时间依赖性,DHA作用24、48和72h的IC50值分别为60.03、33.86和17.18μmol/L;流式细胞仪检测细胞阻滞于G0/G1期,随药物剂量增大(20、40和60μmol/L),G0/G1期比例逐渐增高(42.76%、49.71%和63.01%),S期比例降低(48.45%、42.41%和30.98%);Annexin-V双染法示早期细胞凋亡率呈明显剂量依赖性升高〔(20.81±0.68)%、(30.90±0.73)%、(45.32±2.17)%(n=3)〕;RT-PCR检测显示,DHA作用后细胞凋亡诱导因子BimmR-NA的表达量升高;蛋白质印迹法检测DHA作用48h凋亡相关蛋白tbid和Caspase-8表达增强,且呈一定剂量依赖关系。结论:DHA能显著抑制人乳腺癌细胞T47D生长,影响细胞周期,诱导细胞凋亡,其分子机制可能与促使Bim、tbid和Caspase-8表达增强的内源性凋亡途径有关。
Objective: To study the effect of dihydroartemisinin (DHA) on the proliferation and apoptosis of breast cancer cell line T47D and to explore the molecular mechanism of its anti-cancer effect. Methods: T47D breast cancer cells were used to observe the effect of DHA on T47D cells at different concentrations and time. MTT assay was used to detect the inhibitory effect on proliferation of T47D cells. PI staining and Annexin-V double staining Flow cytometry analysis of cell cycle distribution and early apoptosis rate changes, RT-PCR and Western blotting detection of apoptosis-related protein expression. RESULTS: DHA had a dose-and time-dependent inhibitory effect on the proliferation of T47D cells. The IC50 values of DHA for 24 h, 48 h and 72 h were 60.03, 33.86 and 17.18 μmol / L, respectively. The cell cycle arrest in G0 / G1 phase, the proportion of G0 / G1 phase increased gradually (42.76%, 49.71% and 63.01%) and the proportion of S phase decreased (48.45%, 42.41% and 30.98%) with the increase of drug dose (20, 40 and 60μmol / (20.81 ± 0.68)%, (30.90 ± 0.73)%, (45.32 ± 2.17)% (n = 3)], respectively; Annexin-V double staining showed that the rate of early apoptosis was significantly increased in a dose- The results of PCR showed that the expression of BimmR-NA was up-regulated after DHA treatment. The expression of tbid and Caspase-8 increased at 48h after DHA treatment in a dose-dependent manner. Conclusion: DHA can significantly inhibit the growth of human breast cancer cell T47D, affect the cell cycle and induce apoptosis. The molecular mechanism may be related to the endogenous apoptotic pathway that promotes the expression of Bim, tbid and Caspase-8.