华蟾素调控PI3K/AKT通路逆转卵巢癌A2780/DDP细胞顺铂耐药的作用机制  

Mechanism of cinobufagin regulating PI3K/AKT signaling pathway to reverse cisplatin resistance in ovarian cancer A2780/DDP cells

作　　者：舒美玲 吴悦[1,2] 叶映泉 张爽爽 张梅 Shu Meiling;Wu Yue;Ye Yingquan;Zhang Shuangshuang;Zhang Mei(Dept of Integrated Traditional Chinese and Western Medicine,Anhui Medical University,Hefei 230032;Dept of Integrated Chinese and Western Medicine Oncology,The First Affiliated Hospital of Anhui Medical University,Hefei 230022)

机构地区：[1]安徽医科大学中西医结合临床医学系,合肥230032 [2]安徽医科大学第一附属医院中西医结合肿瘤科,合肥230022

出　　处：《安徽医科大学学报》2024年第4期671-677,741,共8页Acta Universitatis Medicinalis Anhui

基　　金：安徽省2021年重大疑难疾病(卵巢癌)中西医协同攻关项目(编号:皖中医药发展秘〔2022〕70号);安徽省第十三批“115”产业创新团队。

摘　　要：目的研究华蟾素(CBG)对人卵巢癌细胞顺铂耐药的逆转作用和机制。方法A2780细胞株及其顺铂耐药细胞株A2780/DDP是临床常见的卵巢癌细胞,故选择这两种细胞株作为研究对象。通过CCK-8法检测细胞活力,平板克隆和5-乙炔基-2′脱氧尿嘧啶核苷(EdU)实验检测细胞的增殖能力,赫斯特染色(Hoechst)法观察细胞凋亡情况,细胞划痕实验和Transwell实验评估细胞的迁移和侵袭能力,Western blot和定量逆转录PCR(RT-qPCR)法检测磷脂酰肌醇3-激酶/蛋白激酶(PI3K/AKT)信号通路和上皮-间质转化(EMT)的相关蛋白和mRNA的表达差异。结果与A2780细胞相比,A2780/DDP细胞的耐药指数分别约为5.636、5.864、5.695,采用CBG(2、4、6 mg/ml)处理A2780/DDP耐药细胞后,逆转耐药指数分别为1.617、2.570、3.461。CBG呈浓度依赖性地上调细胞凋亡水平、抑制细胞的增殖、迁移和侵袭能力(P<0.05)。Western blot结果显示:与A2780细胞相比,对照组(A2780/DDP)细胞中P-PI3K/PI3K和P-AKT/AKT的蛋白水平相对比值以及N钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、蜗牛蛋白(Snail)的蛋白表达更高,E钙黏蛋白(E-cadherin)蛋白表达更低(t_(P-PI3K/PI3K)=8.115,t_(P-AKT/AKT)=17.62、t_(N-cadherin)=6.126、t_(Vimentin)=4.001、t_(Snail)=17.333、t_(E-cadherin)=4.620,P<0.01);随着CBG剂量升高,耐药细胞中的P-PI3K、P-AKT、N-cadherin、Vimentin、Snail的蛋白表达水平降低,而E-cadherin的蛋白表达量增加(F_(P-PI3K)=268.5、F P-AKT=190.5、F_(N-cadherin)=24.02、F_(Vimentin)=57.65、F_(Snail)=87.24、F_(E-cadherin)=135.8,P<0.05)。RT-qPCR结果显示:随着CBG浓度增加,PI3K、AKT、N-cadherin、Vimentin、Snail的mRNA表达水平随之降低,相反E-cadherin的mRNA表达水平逐渐升高(F PI3K=101.1、F_(AKT)=558.3、F_(N-cadherin)=86.97、F_(Vimentin)=105.9、F_(Snail)=85.71、F_(E-cadherin)=80.96,P<0.01)。结论CBG具有逆转卵巢癌A2780/DDP细胞株顺铂耐药的作用,其机制可能与CBG调控PI3K/AKT信号通路和Objective To investigate the reversal effect and mechanism of cinobufagin(CBG)on cisplatin resistance in human ovarian cancer cells.Methods A2780 cell line and its cisplatin-resistant cell line A2780/DDP are common ovarian cancer cells in clinic,so these two cell lines were selected as the research objects.The cell viability was detected by cell Counting Kit-8(CCK-8)assay,and the cell proliferation ability was detected by plate cloning and 5-ethynyl-2′-deoxyuridine(EdU)assay.Hoechst staining was used to observe cell apoptosis.Cell scratch test and Transwell test were used to evaluate cell migration and invasion ability.Western blot and quantitative reverse transcription PCR(RT-qPCR)were used to detect the protein and mRNA expressions of phosphatidylinositol 3-kinase/protein kinase(PI3K/AKT)signaling pathway and epithelial-mesenchymal transition(EMT).Results Compared with A2780 cells,the drug resistance indexes of A2780/DDP cells were 5.636,5.864,5.695,respectively.After treatment of A2780/DDP cells with CBG(2,4,6 mg/ml),the reversal resistance indexes were 1.617,2.570,3.461,respectively.CBG treatment significantly increased the level of apoptosis and inhibited the proliferation,migration and invasion of the cells in a concentration-dependent manner(P<0.05).Western blot results showed that compared with A2780 cells,the relative ratio of P-PI3K/PI3K and P-AKT/AKT protein levels,as well as the protein expression of N-cadherin,Vimentin,and Snail were higher in the control group(A2780/DDP)cells,while the protein expression of E-cadherin was lower(t_(P-PI3K/PI3K)=8.115,t_(P-AKT/AKT)=17.62,t_(N-cadherin)=6.126,t_(Vimentin)=4.001,t_(Snail)=17.333,t_(E-cadherin)=4.620,P<0.01);As the dose of CBG increased,the protein expression levels of P-PI3K,P-AKT,N-cadherin,Vimentin,and Snail in drug-resistant cells decreased,while the protein expression level of E-cadherin increased(F_(P-PI3K)=268.5,F P-AKT=190.5,F_(N-cadherin)=24.02,F_(Vimentin)=57.65,F_(Snail)=87.24,F_(E-cadherin)=135.8,P<0.05).qRT-PCR results showed that with the

关 键 词：华蟾素 卵巢癌 顺铂耐药 逆转耐药 PI3K/AKT 上皮间质转化 作用机制 

分 类 号：R737.3[医药卫生—肿瘤]