百里醌对人胶质瘤细胞凋亡的作用及其机制研究

目的分析百里醌对胶质瘤U87细胞生长抑制和凋亡诱导的功能。方法体外胶质瘤细胞株U87以及人星形胶质细胞株NHA中添加不同浓度百里醌后,CCK-8法检测细胞活力;克隆形成实验观察U87细胞形成细胞克隆的能力;流式细胞术检测细胞周期和ROS含量;Hoechst染色法和流式细胞术测定细胞凋亡;流式细胞术(JC-1荧光染色)观察细胞线粒体膜电位变化;Western blot试验检测U87细胞中细胞周期相关蛋白(Cyclin B1、Cdc2、Cdc25c、p53和p73)、细胞凋亡相关蛋白(Fas-L、Fas、Bax、tBid、PARP、caspase 9和caspase 3)和DNA损伤相关蛋白(p-ATM、p-ATR、γ-H2AX、p-Chk1、p-Chk2和p-p53)表达。结果百里醌可显著抑制U87细胞活力和增殖,呈一定的剂量依赖关系,同时上调p-ATM、p-ATR、γ-H2AX、p-Chk1、p-Chk2和p-p53的表达;百里醌使U87细胞周期停滞在G2/M期,下调Cyclin B1、Cdc2和Cdc25c的表达,同时上调p53和p73的表达;百里醌可诱导U87细胞发生凋亡,上调U87细胞线粒体膜电位,同时促进细胞中Fas-L、Fas、Bax、tBid、cleaved-PARP、cleaved-caspase 9和cleaved-caspase 3的表达;caspase广谱抑制剂Z-VAD-FMK可削弱百里醌的凋亡诱导作用;U87细胞内的ROS表达水平经百里醌作用得以上调,呈一定的剂量依赖关系,百里醌对U87细胞的促进凋亡功能在一定程度上被ROS清除剂N-乙酰-L-半胱氨酸(NAC)逆转。结论百里醌可抑制体外胶质瘤细胞生长,并诱导细胞凋亡,其作用路径可能通过ROS通路实现。

Apoptosis induction and underlying mechanisms of thymoquinone on human glioma cells

Objective To explore the apoptosis induction and underlying molecular mechanisms of thymoquinone on human glioma cells.Methods Human glioma cell line U87 and normal human astrocytes NHA were treated with thymoquinone in vitro.CCK-8 and cell clone formation assays were employed to detect the cell viability and proliferation activity in vitro.The cell cycle and production of ROS were analyzed by flow cytometry.Hoechst 33342 staining and Annexin V/PI apoptosis assays were utilized to explore the apoptosis of U87 cells.The mitochondrial membrane potential of U87 cell was observed by JC-1 staining.The cell cycle regulating proteins(Cyclin B1,Cdc2,Cdc25 c,p53 and p73),cell apoptosis related factors(Fas-L,Fas,Bax,tBid,PARP,caspase 9 and caspase 3) as well as DNA damage signaling factors(p-ATM,p-ATR,γ-H2 AX,p-Chk1,p-Chk2 and p-p53) were determined by western blot.Results Thymoquinone treatment suppressed the viability and proliferation of U87 cells in a concentration-dependent manner,and the levels of p-ATM,p-ATR,γ-H2 AX,p-Chk1,p-Chk2 and p-p53 were all increased by the treatment of thymoquinone.Thymoquinone also increased cell arrest in the G2/M phase by decreasing Cyclin B1,Cdc2,and Cdc25 c,and up-regulating p53 and p73.Thymoquinone induced U87 cell apoptosis through the up-regulation of Fas-L,Fas,and Bax as well as the proteolysis of PARP,caspase 9 and caspase 3.Thymoquinone also contributed to the loss of mitochondrial membrane potential.The apoptotic cell death induced by thymoquinone was inhibited by pretreatment with Z-VAD-FMK,a pan-caspase inhibitor.Moreover,the apoptotic effect of thymoquinone was reactive oxygen species(ROS)-dependent,as evidenced by the inhibition of thymoquinone-induced PARP cleavage and ROS generation via N-acetylcysteine(NAC)-induced ROS scavenging.Conclusion Thymoquinone exerts a promotive effect on the apoptosis of human glioma cells through the ROS pathway.