Wnt/β-catenin信号通路在白藜芦醇抗急性T淋巴细胞白血病中的作用机制研究

The effect mechanism of resveratrol against T-cell acute lymphoblastic leukemia by Wnt/β-catenin signaling pathway

目的:通过细胞水平和动物模型探讨Wnt/β-catenin信号通路在白藜芦醇体内外抗急性T淋巴细胞白血病(T-cell acute lymphoblastic leukemia,T-ALL)中的作用及其可能机制。方法:细胞实验(人淋巴细胞白血病Molt-4和Jurkat细胞)分为空白对照组(Control组)、二甲基亚砜组(DMSO组)和白藜芦醇处理组(Res组),动物实验分为正常对照组(Control组)、T-ALL模型组(T-ALL组)和白藜芦醇处理组(Res组)。采用CCK-8法检测白藜芦醇对Molt-4和Jurkat细胞增殖能力的影响,选取适合的给药浓度进行后续实验;尾静脉注射ICN1-GFP^(+)T-ALL细胞建立T-ALL小鼠模型;采用流式细胞术检测小鼠外周血中ICN1-GFP^(+)T-ALL细胞百分比,监测T-ALL小鼠造模后的一般情况;应用实时荧光定量PCR(RT-PCR)分别检测细胞和小鼠脾脏组织中c-Myc和Cyclin D1 mRNA的表达水平;应用蛋白免疫印迹法(Western Blot法)分别检测细胞和小鼠脾脏组织中β-catenin、TCF-1、LEF-1、c-Myc和Cyclin D1的蛋白表达水平。结果:白藜芦醇明显抑制Molt-4和Jurkat细胞增殖能力(P<0.01),抑制作用随白藜芦醇浓度增加逐渐增强(P<0.01);经白藜芦醇处理后,Molt-4和Jurkat细胞中c-Myc和Cyclin D1 mRNA表达水平明显降低(P<0.01),β-catenin、TCF-1、LEF-1及其靶蛋白c-Myc和Cyclin D1表达水平均显著降低(P<0.05);T-ALL小鼠脾脏组织中c-Myc和Cyclin D1 mRNA表达水平显著增高(P<0.01),β-catenin、TCF-1、LEF-1及靶蛋白c-Myc和Cyclin D1表达水平显著升高(P<0.01);经白藜芦醇处理后,小鼠外周血中GFP^(+)白血病细胞比例明显下降(P<0.01),c-Myc和Cyclin D1 mRNA表达水平明显降低(P<0.01),各蛋白表达水平明显下调(P<0.01)。结论:在T-ALL细胞株和动物模型中,白藜芦醇可能通过下调Wnt/β-catenin信号通路中β-catenin、TCF-1和LEF-1的表达进而抑制靶蛋白c-Myc和Cyclin D1水平,发挥抗急性T淋巴细胞白血病作用。

Objective:To explore the roles and possible mechanisms of Wnt/β-catenin signaling pathway in resveratrol against T-cell acute lymphoblastic leukemia(T-ALL)in vitro and in vivo by cellular level and animal model.Methods:Human T-ALL cell lines(Molt-4 cells and Jurkat cells)were divided into control group,DMSO group and resveratrol treated group.C57 BL/6 mice were randomly divided into three groups,including control group,T-ALL group and T-ALL mice with resveratrol treated group(resveratrol group).CCK-8 assay was performed to detect cell activity in Molt-4 cells and Jurkat cells.Mice injected with ICN1-GFP^(+)T-ALL cells by tail vein injection were used to establish the T-ALL model.The percentage of ICN1-GFP^(+)T-ALL cells in peripheral blood of mice was detected by flow cytometry.Real-time quantitative PCR(RT-PCR)was used to detect the expressions of c-Myc and Cyclin D1 in T-ALL cells and mice spleen tissues.The expressions ofβ-catenin,TCF-1,LEF-1,c-Myc and Cyclin D1 in T-ALL cell lines and mice spleen were detected by Western Blot.Results:The cell activity of Molt-4 cells and Jurkat cells was significantly suppressed after treating with resveratrol(P<0.01),and the inhibition effect was strengthened with the increasing dose of resveratrol(P<0.01).After treating with resveratrol,the expression of c-Myc and Cyclin D1 mRNA were significantly reduced(P<0.01).The protein expressions ofβ-catenin,TCF-1,LEF-1,c-Myc and Cyclin D1 in resveratrol group were reduced significantly(P<0.05).In vivo experiments,compared with the control group,the protein expressions ofβ-catenin,TCF-1,LEF-1,c-Myc,Cyclin D1,and the mRNA expression of c-Myc and Cyclin D1 were increased significantly in the mice spleen of T-ALL group(P<0.01).After resveratrol administration,the percentage of ICN1-GFP^(+)T-ALL cells in the peripheral blood of mice decreased notably(P<0.01)and the protein expression ofβ-catenin,TCF-1,LEF-1,c-Myc,Cyclin D1,and the mRNA expression of c-Myc,Cyclin D1 were significantly reduced in resveratrol group(P<0.01).Conclusion:In T-ALL cell lines and animal models,resveratrol may play an anti-acute T-lymphoblastic leukemia role by down-regulating the expression ofβ-catenin,TCF-1 and LEF-1 in the Wnt/β-catenin signaling pathway and then inhibiting the revels of traget proteins c-Myc and Cyclin D1.