毛壳素作为TrxR1抑制剂诱导卵巢癌细胞凋亡的机制研究

Mechanism study of Chaetocin as a TrxR1 inhibitor inducing apoptosis in ovarian cancer cells

目的探究硫氧还蛋白还原酶1(TrxR1)在毛壳素(Chaetocin)诱导卵巢癌细胞凋亡中的作用及相关分子通路。方法体外培养卵巢癌细胞系OVCAR-3,CCK-8法和试剂盒检测Chaetocin对OVCAR-3细胞增殖和细胞中TrxR1活性的影响。利用分子模拟对接和分子动力学分析Chaetocin与TrxR1的作用方式,将过表达TrxR1的慢病毒载体(TrxR1-OE)及空载体(Vec)转染入OVCAR-3细胞中,并将其分为:转染Vec的对照组(Vec组)、过表达TrxR1的TrxR1-OE组、Chaetocin处理转染过表达TrxR1或Vec的OVCAR-3细胞组(Chaetocin+TrxR1-OE组和Chaetocin+Vec组)。DCFH-DA法检测各组OVCAR-3细胞中ROS表达,Annexin V-FITC/PI法检测各组细胞的凋亡率,Western blot检测细胞中凋亡相关蛋白Cle-PARP、Bax、Cle-caspase-3及JNK/c-JUN信号通路中p-JNK/JNK和p-c-JUN/c-JUN比值。结果Chaetocin可显著抑制OVCAR-3细胞的增殖活力,并可与TrxR1直接结合来抑制OVCAR-3细胞中TrxR1活性。TrxR1-OE组中ROS含量,细胞凋亡率,细胞中Cle-PARP、Bax、Cle-caspase-3表达和p-JNK/JNK、p-c-JUN/c-JUN比值与Vec组相比,差异均无统计学意义(P>0.05),Vec+Chaetocin组、TrxR1-OE+Chaetocin组与Vec组相比均显著升高(P<0.05)。与Vec+Chaetocin组相比,TrxR1-OE+Chaetocin组中ROS含量,细胞凋亡率,细胞中Cle-PARP、Bax、Cle-caspase-3表达和p-JNK/JNK、p-c-JUN/c-JUN比值均显著降低,差异有统计学意义(P<0.05)。结论Chaetocin可在体外通过激活JNK/c-JUN通路来抑制TrxR1活性促进卵巢癌细胞中ROS的积聚,从而诱导细胞发生caspase途径的凋亡。

Objective Investigate the role of Thioredoxin Reductase 1(TrxR1)and the associated molecular pathways in Chaetocin-induced apoptosis in ovarian cancer cells in vitro.MethodssOvarian cancer cell line OVCAR-3 was cultured in vitro.The CCK-8 assay was used to evaluate the effects of Chaetocin on cell proliferation and TrxR1 activity in OVCAR-3 cells.Molecular docking and molecular dynamics analysis were performed to investigate the interaction between Chaetocin and TrxRl.Transfected the lentiviral vector(TrxR1-OE)overexpressing TrxR1 and the empty vector(Vec)into OVCAR-3 cells,and divided them into:control group transfected with Vec(Vec group),TrxR1-OE group overexpressing TrxR1,and OVCAR-3 cell group transfected with TrxR1 or Vec treated with Chaetocin(Chaetocin+TrxR1-OE group and Chaetocin+Vec group).DCFH-DA assay was used to measure the expression of reactive oxygen species(ROS)in OVCAR-3 cells of each group.Annexin V-FITC/PI assay was performed to determine the apoptosis rate of cells in each group.Western blot analysis was conducted to detect the expression of apoptosis-related proteins Cleaved-PARP,Bax,Cleaved-caspase-3,as well as the ratio of p-JNK/JNK and p-c-JUN/c-JUN in the JNK/c-Jun signaling pathway.ResultsChaetocin significantly inhibited the proliferation of OVCAR-3 cells and directly interacted with TrxRI to inhibit its activity.Compared to the Vec group,there were no significant changes in ROS levels,apoptosis rate,expression of Cleaved-PARP,Bax,Cleavedcaspase-3,and the ratio of p-JNK/JNK and p-c-JUN/c-JUN in the TrxR1-OE group(P>0.05).Compared with the Vec group,the Vec+Chaetocin group and TrxR1-OE+Chaetocin group showed significant increases(P<0.05).Compared with the Vec+Chaetocin group,the TrxR1-OE+Chaetocin group showed a significant decrease in ROS content,cell apoptosis rate,expression of Cle-PARP,Bax,Clecaspase-3 in cells,and p-JNK/JNK,p-c-JUN/c-JUN ratios,with statistical significance(P<0.05).Conclusion Chaetocin can induce the accumulation of reactive oxygen species(ROS)and promote caspase-dependent apoptosis in ovarian cancer cells by activating the JNK/c-JUN pathway and inhibiting TrxRl activity in vitro.