穿心莲内酯对人肾细胞癌786-0细胞增殖、迁移和凋亡的影响

目的：研究穿心莲内酯（Andro）对肾细胞癌（RCC）786-0细胞的增殖、迁移和克隆形成能力的抑制作用及诱导其程序性凋亡的作用，并探讨其相关作用机制。方法：取对数生长期786-0细胞，加入不同浓度（5、10、20、40和80 μmol·L^-1） Andro作为实验组，以0 μmol·L^-1 Andro组作为空白对照组，MTS法检测各组细胞增殖率。取对数生长期786-0细胞，加入不同浓度（0.50、1.25和2.50 μmol·L^-1） Andro作为实验组，以0 μmol·L^-1 Andro组作为空白对照组，采用克隆形成实验检测各组细胞克隆形成能力。取对数生长期786-0细胞，加入不同浓度（10、20和40 μmol·L^-1） Andro作为实验组，以0 μmol·L^-1 Andro组作为空白对照组，采用划痕实验检测各组细胞迁移能力，流式细胞术检测各组细胞的凋亡率，Western blotting法检测各组细胞中凋亡相关蛋白表达水平。结果：与空白对照组比较，10、20、40和80 μmol·L^-1 Andro组细胞增殖率明显降低（P<0.05或P<0.01）。与空白对照组比较，0.50和1.25 μmol·L^-1 Andro组细胞克隆形成率明显降低（P<0.05或P<0.01）。与空白对照组比较，10、20和40 μmol·L^-1 Andro组细胞划痕愈合率均明显降低（P<0.01），20和40 μmol·L^-1 Andro组细胞凋亡率均明显升高（P<0.01）。与空白对照组比较，40 μmol·L^-1 Andro组细胞中DNA损伤蛋白γ-H2AX表达水平明显升高（P<0.01），Caspase-8表达水平明显降低（P<0.05），Caspase-8剪切体表达水平明显升高（P<0.01）。结论：Andro能够有效地抑制RCC细胞的增殖、迁移和克隆形成能力，并诱导其凋亡，其诱导凋亡作用的机制与诱导DNA损伤及JNK/H2AX和Caspase-8介导的细胞程序性凋亡途径有关。

Effects of andrographolide in proliferation,migration and apoptosis of renal cell carcinoma 786-0 cells

Objective:To study the inhibitory effects of andrographolide (Andro) on the proliferation,migration and clone formation ability of renal cell carcinoma (RCC) cells and the induction on the apoptosis,and to clarify their related mechanisms.Methods:The RCC cells were treated with different concentrations (5,10,20,40 and 80 μmol · L-1) of Andro as experimental groups,and 0μmol · L 1 Andro group was used as blank control group,MTS assay was used to detect the proliferation rates of RCC cells in various groups.The RCC cells were treated with different concentrations (0.50,1.25 and 2.50 μmol · L-1) of Andro as experimental groups,and 0 μmol · L 1Andro group was used as blank control group.Clonogenic assays was used to detect the colony formation ability of RCC cells in various groups.The RCC cells were treated with different concentrations (10,20and 40 μmol · L-1) of Andro as experimental groups,and 0 μmol · L-1 Andro group was used as blank control group.Wound healing assay was used to detect the migration ability of RCC cells in various groups.Flow cytometry was used to detect the apoptotic rates of RCC cells in various groups.Western blotting was performed to detect the expression levels of apoptosis related proteins in RCC cells in various groups.Results:Compared with blank control group,the proliferation rates of RCC cells in 10,20,40 and 80 μmol · L-1 Andro groups were markedly decreased (P＜0.05 or P＜0.01).Compared with blank control group,the colony formation rates of RCC cells in 0.50 and 1.25μmol · L-1 Andro groups were markedly decreased (P＜0.05 or P＜0.01).Compared with blank control group,the scratch healing rates of RCC cells in 10,20 and 40 μmol · L-1 Andro groups were markedly decreased (P＜0.01),and the apoptotic rates of RCC cells in 20 and 40 μmol · L-1 Andro groups were markedly increased (P＜0.01).Compared with blank control group,the expression level of γ-H2AX protein in 40μmol · L-1 Andro group was markedly increased (P＜0.01),the expression level of Caspase-8 protein was decreased (P＜0.05),and the expression level of cleaved Caspase-8 protein was markedly increased (P＜0.01).Conclusion:Andro can effectively suppress the proliferation,migration and colony formation ability of RCC cells and induce the apoptosis of RCC cells.The mechanism of apoptosis might be related to inducing the DNA damage and the apoptotic pathways induced by JNK / H2AX and Caspase-8.