抗体芯片筛选黄芩苷诱导胃癌SGC-7901细胞凋亡的作用靶点

Target Screening for Baicalin Induced Apoptosis in Human Gastric Cancer SGC-7901 Cells Based on Apoptosis Antibody Array

目的采用凋亡抗体芯片筛选黄芩苷诱导胃癌SGC-7901细胞凋亡的作用通路和靶点,探讨黄芩苷诱导胃癌细胞凋亡的相关机制。方法采用MTT法检测黄芩苷对SGC-7901细胞的增殖抑制作用;采用流式细胞术检测细胞凋亡情况;采用人凋亡抗体芯片双抗体夹心检测法筛选出黄芩苷(120μmo L·L^(-1))组与对照组的差异表达蛋白;采用Western Blot法对筛选出的差异蛋白Caspase-3、Caspase-8、Fas L、XIAP进行验证,增加对Fas、Caspase-9、TRAIL蛋白的检测。结果与对照组比较,20~320μmo L·L^(-1)的黄芩苷在24~96 h不同时间点可抑制胃癌SGC-7901细胞的增殖(P<0.05,P<0.01,P<0.001),且呈浓度和时间依赖性;80,120,160μmo L·L^(-1)黄芩苷均可诱导胃癌SGC-7901细胞凋亡(P<0.01),且呈浓度依赖性;凋亡抗体芯片检测结果表明,120μmo L·L^(-1)黄芩苷组的bad、Caspase-3、Caspase-8、Fas L、HSP60、TRAIL R4、IGF-I、IGF-II、p21、p27、p53和SMAC等蛋白表达明显上调,而XIAP蛋白表达显著下调,差异均有统计学意义(P<0.05,P<0.01)。Western Blot验证实验结果表明,160μmo L·L^(-1)浓度组的Fas L蛋白相对表达量显著上升(P<0.001);120,160μmo L·L^(-1)浓度组的Caspase-3、Caspase-8、Caspase-9、Fas、TRAIL蛋白相对表达量显著上升(P<0.01,P<0.001),XIAP蛋白相对表达量显著下调(P<0.001)。结论黄芩苷诱导胃癌细胞凋亡的抗肿瘤作用机制与Fas L、TRAIL介导的死亡受体有关;抗体芯片是有效的靶点筛选手段,可用于药物作用靶点和药理机制研究。

Objective To screen apoptosis relevant pathways and targets using antibody array on apoptosis of gastric cancer SGC-7901 cells induced by baicalin.Methods MTT assay was used to detect the growth of gastric cancer SGC-7901 cells treated with baicalin.Cell apoptosis was evaluated by flow cytometry;and subsequently,antibody array with the method of double antibody sandwich was used to screen aberrantly expressed protein targets of gastric cancer SGC-7901 cells treated with 120μmoL·L^-1 baicalin;Western Blot technique was used to validate the aberrantly expressed apoptotic proteins,including Caspase3,Caspase8,FasL and XIAP.In addition,Fas,Caspase9 and TRAIL were also detected to explore the apoptotic mechanism.Results Compared with control group,20-320μmoL·L^-1 baicalin could inhibit the proliferation of gastric cancer SGC-7901 cells with a time and concentration dependent manner（P〈0.05,P〈0.01,or P〈0.001）.Baicalin at 80,120 and 160μmoL·L^-1 could induce apoptosis of gastric cancer SGC-7901 cells with a concentration dependent manner（P〈0.01）.The antibody microarray screening found that 120μmoL·L^-1 baicalin could upregulate the expression of BCL2 associated agonist of cell death bad）,Caspase3,Caspase8,FasL,Hsp60,TRAIL R4,IGF-I,IGF-II,p21,p27,p53 and SMAC,and downregulate the expression of XIAP protein（P〈0.05,P〈0.01）.Western Blot confirmed that 160 μmoL·L^-1 baicalin could upregulate the expression of FasL（P〈0.001）;120,and 160μmoL·L^-1 baicalin could upregulate the expression of Caspase3,Caspase8,Caspase9,Fas,TRAIL（P0.01,P0.01）,and downregulate the expression of XIAP（P0.01）,which was consistent with the antibody array.Conclusion Baicalin could induce apoptosis in gastric cancer cell,and the mechanism of the anti-tumor effect is related to death receptor pathway mediated by FasL and TRAIL.In addition,antibody array is an effective method to study the pharmacological mechanisms of drug targets.aberrantly expressed apoptotic proteins, including Caspase3, Caspase8, FasL and XIAP. In addition, Fas, Caspase9 and TRAIL were also detected to explore the apoptotic mechanism. Results Compared with control group, 20-320 μmoL- L-1 baicalin could inhibit the proliferation of gastric cancer SGC-7901 ceils with a time and concentration dependent manner （P 〈 0.05, P〈 0.01, or P〈 0.001）. Baicalin at 80, 120 and 160 μmoL·L-1 could induce apoptosis of gastric cancer SGC-7901 cells with a concentration dependent manner （P 〈 0.01）. The antibody microarray screening found that 120 IxmoL-L-1 baicalin could upregulate the expression of BCL2 associated agonist of cell death （bad）, Caspase3, Caspase8, FasL, Hsp60, TRAIL R4, IGF-I, IGF-II, p21, p27, p53 and SMAC, and downregulate the expression of XIAP protein （P 〈 0.05, P〈 0.01） . Western Blot confirmed that 160 μmoL·L-1 baicalin could upregulate the expression of FasL （P〈0.O01） ; 120, and 160 μmoL＇L-1 baicalin could upregulate the expression of Caspase3, Caspase8, Caspase9, Fas, TRAIL（P 〈 0.01, P 〈 0.01）, and downregulate the expression of XIAP （P〈0.01） , which was consistent with the antibody array. Conclusion Baicalin could induce apoptosis in gastric cancer cell, and the mechanism of the anti-tumor effect is related to death receptor pathway mediated by FasL and TRAIL. In addition, antibody array is an effective method to study the pharmacological mechanisms of drug targets.