虫草素通过调控Dickkopf相关蛋白/β-catenin信号通路诱导自噬和凋亡抑制弥漫大B细胞淋巴瘤

Cordycepin induces autophagy and apoptosis through regulating Dickkopf-related protein 1/β-catenin signal pathway to inhibit diffuse large B cell lymphoma

目的:探究虫草素是否通过Dickkopf相关蛋白1(Dkk1)/β-catenin信号诱导自噬和凋亡抑制弥漫大B细胞淋巴瘤(DLBCL)。方法:选择DLBCL SU-DHL-4细胞进行研究。MTT法检测细胞活性,用不同浓度虫草素处理DLBCL SU-DHL-4细胞,分为对照组和虫草素20、40、80μg/mL组。si-DDK1转染SU-DHL-4细胞,分为对照组、虫草素80μg/mL组和虫草素80μg/mL+si-DDK1组。流式细胞术检测细胞凋亡率,免疫荧光细胞化学检测微管相关蛋白轻链3(LC3)阳性细胞量,免疫印迹检测cleaved caspase-8、cleaved caspase-3、cleaved caspase-9、cleaved多腺苷二磷酸核糖聚合酶-1(PARP1)、becline-1、自噬相关蛋白7(Atg7)、LC3Ⅱ,DDK-1和β-catenin蛋白相对表达量。结果:与对照组相比,经虫草素40、80μg/mL干预后,SU-DHL-4细胞凋亡率升高,LC3+阳性表达量显著增加,cleaved caspase-8、cleaved caspase-3、cleaved caspase-9、cleaved PARP1、becline-1、ATG7、LC3Ⅱ/LC3Ⅰ和DDK-1蛋白相对表达量均上调,而β-catenin蛋白相对表达量下调。与虫草素80μg/mL组相比,在虫草素80μg/mL+si-DDK1组中DDK-1蛋白相对表达量下调,而β-catenin蛋白相对表达量上调;同时SU-DHL-4细胞凋亡率降低,LC3+阳性表达量显著降低,cleaved caspase-8、cleaved caspase-3、cleaved caspase-9、cleaved PARP1、becline-1、ATG7和LC3Ⅱ/LC3Ⅰ蛋白相对表达量均下调。结论:虫草素可通过调控Dkk1/β-catenin信号诱导自噬和凋亡来抑制DLBCL。

Objective:To investigate whether cordycepin could induce autophagy and apoptosis through regulating Dickkopf-related protein 1(Dkk1)/β-catenin signal to inhibit diffuse large B cell lymphoma(DLBCL).Methods:DLBCL SU-DHL-4 cells were selected for research.Cell viability was detected by MTT assay.DLBCL SU-DHL-4 cells were treated with different concentrations of cordycepin and divided into control group,cordycepin 20μg/mL group,cordycepin 40μg/mL group,and cordycepin 80μg/mL group.SU-DHL-4 cells were transfected with si-DDK1 and divided into control group,cordycepin 80μg/mL group,and cordycepin 80μg/mL+si-DDK1 group.Cell apoptosis rate was detected by the flow cytometry.The number of microtubule-associated protein light chain 3(LC3)positive cells was detected by fluorescence immunocytochemistry.The protein relative expression levels of cleaved caspase-8,cleaved caspase-3,cleaved caspase-9,cleaved poly-ADP-ribose polymerase(PARP1),becline-1,autophagy-related protein 7(Atg7),LC3Ⅱ,DDK-1 andβ-catenin were detected by Western blotting.Results:Compared with the control group,after intervention with 40 and 80μg/mL cordycepin,the apoptosis rate of SU-DHL-4 cells was increased,and the positive expression of LC3+was risen significantly,and the relative protein expression levels of cleaved caspase-8,cleaved caspase-3,cleaved caspase-9,cleaved PARP1,becline-1,ATG7,LC3Ⅱ/LC3Ⅰ,and DDK-1 were all up-regulated while the relative protein expression ofβ-catenin was down-regulated.Compared with the cordycepin 80μg/mL group,the relative protein expression of DDK-1 was down-regulated in the cordycepin 80μg/mL+si-DDK1 group,while the relative protein expression ofβ-catenin was up-regulated.At the same time,SU-DHL-4 cell apoptosis rate was reduced,and the positive expression of LC3+was significantly reduced,and the relative protein expression levels of cleaved caspase-8,cleaved caspase-3,cleaved caspase-9,cleaved PARP1,becline-1,ATG7,and LC3Ⅱ/LC3Ⅰwere all downregulated.Conclusion:Cordycepin can induce autophagy and apoptosis through regulating Dkk1/β-catenin signal to inhibit DLBCL.