金诺芬对卵巢癌细胞活性的影响及其分子机制  

Effects of auranofin on the activity of ovarian cancer cells and its molecular mechanisms

作　　者：陈艳雅 黄丽珊 招锦兰 袁佩欣 叶玉锦 李仲均[1] Chen Yanya;Huang Lishan;Zhao Jinlan;Yuan Peixin;Ye Yujin;Li Zhongjun(Dept of Obstetrics and Gynecology,Dongguan Hospital Affiliated to Southern Medical University,Dongguan 523000;Dept of Rehabilitation Medicine,Dongguan Hospital Affiliated to Southern Medical University,Dongguan 523000)

机构地区：[1]南方医科大学附属东莞医院妇产科,东莞523000 [2]南方医科大学附属东莞医院康复医学科,东莞523000

出　　处：《安徽医科大学学报》2023年第4期541-546,共6页Acta Universitatis Medicinalis Anhui

基　　金：广东省医学科学技术研究基金(编号:A2021085);东莞市社会科技发展重点项目(编号:202050715001215)。

摘　　要：目的 探讨金诺芬对卵巢癌细胞活性的影响及其可能的分子机制。方法 细胞计数试剂盒-8(CCK-8)法测定金诺芬处理卵巢癌细胞系SKOV3、Caov3和SW626细胞和永生化的正常人胚肾HEK-293T细胞的剂量反应存活率曲线和半抑制剂量(IC_(50))。流式细胞术测定细胞周期。酶标仪测定细胞内总谷胱甘肽(GSH)、还原型GSH和氧化型谷胱甘肽(GSSG)的水平、硫氧还蛋白还原酶(TrxR)和活性氧自由基(ROS)的水平,并计算还原型GSH/GSSG的比值。Western blot测定细胞内细胞周期蛋白依赖性激酶(CDK)4、CDK6、细胞周期蛋白D1(Cyclin D1)和P53、p-P53、啮齿类双分蛋白2(MDM2)的表达情况。结果 与HEK-293T细胞相比,卵巢癌SKOV3、Caov3和SW626细胞的剂量反应存活率曲线和IC_(50)值显示卵巢癌细胞对金诺芬的敏感性更高(P<0.05)。与未处理组相比,经各自IC_(50)浓度金诺芬处理的SKOV3、Caov3细胞内总GSH、还原型GSH/GSSG的比值和TrxR的活性均降低(t=25.11、31.18,14.72、19.92,43.30、10.74,P<0.05),ROS水平均升高(t=23.82、27.71,P<0.05);G_(0)/G_(1)期细胞数量增多,S期和G2期细胞数量减少(P<0.05);且CDK4、CDK6、Cyclin D1及MDM2的表达水平均下调(t=7.51、15.59,17.32、11.26,20.78、20.78,24.25、17.32,P<0.05),而P53和p-P53的表达水平均上调(t=17.32、24.25,12.12、10.39,P<0.05)。结论 金诺芬通过抑制TrxR的活性引起卵巢癌细胞内氧化应激,并通过部分降解MDM2以稳定并激活P53,将癌细胞阻滞于G_(0)/G_(1)期,发挥抗卵巢癌活性。Objective To explore the activity of auranofin against ovarian cancer cells and its possible molecular mechanism.Methods The dose-response survival curve and IC_(50) of auranofin on ovarian cancer cell lines,SKOV3,Caov3 and SW626 cells and immortalized normal human embryonic kidney HEK-293T cells were determined by CCK-8 method.Cell cycle was determined by flow cytometry.The levels of total glutathione(GSH),reduced GSH and glutathione disulfide(GSSG),thioredoxin reductase(TrxR)and reactive oxygen species(ROS)in cells were determined by microplate reader,and the reduced GSH/GSSG ratio was calculated.Western blot was used to determine the expression of cyclin dependent kinases(CDK)4,CDK6,Cyclin D1,P53,p-P53 and MDM2 in SKOV3 and Caov3 ovarian cancer cells.Results Compared with HEK-293T cells,the dose-response survival curves and IC_(50) values of SKOV3,Caov3 and SW626 cells showed that ovarian cancer cells were more sensitive to auranofin(P<0.05).After SKOV3 and Caov3 cells were treated with the dose of respective IC_(50) concentrations of auranofin,compared with the untreated cells group,the Auranofin IC_(50) group cells′intracellular levels of GSH,the ratio of reduced GSH/GSSG and the activity of TrxR decreased(t=25.11/31.18,14.72/19.92,43.30/10.74,all P<0.05),and the levels of ROS increased(t=23.82/27.71,P<0.05);cells number at G_(0)/G_(1) phases increased,with cells number at S and G 2 phases decreased(P<0.05);and the expression levels of cell cycle-related proteins CDK4,CDK6,Cyclin D1 and the P53-specific E3 ubiquitin ligase MDM2 were down-regulated(t=7.51/15.59,17.32/11.26,20.78/20.78,24.25/17.32,all P<0.05),while the expression levels of P53 and p-P53 were up-regulated(t=17.32/24.25,12.12/10.39,all P<0.05).Conclusion Auranofin causes oxidative stress in ovarian cancer cells by inhibiting TrxR activity,and by partially degrading MDM2 to stabilize and activate P53,so as to block the cancer cells in G_(0)/G_(1) phase,and exert anti-ovarian cancer activities.

关 键 词：卵巢癌 金诺芬 硫氧还蛋白还原酶 细胞周期阻滞 

分 类 号：R737.31[医药卫生—肿瘤] R96[医药卫生—临床医学]