依他尼酸联合顺铂化疗促肺癌A549细胞凋亡的作用及机制研究  

Ethacrynic acid promotes apoptosis in lung cancer A549 cells when combined with cisplatin chemotherapy

作　　者：黄玲萍[1,2] 谢丽霞[1] 邱钰超 胡萍[1] 叶小群[1] 

机构地区：[1]南昌大学第二附属医院呼吸内科,南昌330006 [2]江西省井冈山大学临床医学院内科教研室,江西吉安343000 [3]江西省新钢中心医院呼吸内科,江西新余338001

出　　处：《第三军医大学学报》2017年第17期1720-1727,共8页Journal of Third Military Medical University

基　　金：江西省科学技术厅社会发展攻关项目(20141BBG70035);江西省重大科技资助项目(20143ACB20011)~~

摘　　要：目的探讨依他尼酸(ethacrynic acid,EA)杀伤肺癌A549细胞球的作用及机制研究。方法无血清培养基中培养A549细胞球,应用Western blot检测CD133、SOX2、Ep CAM和ABCG2的蛋白表达水平。应用1、2、5、10、20 mg/m L的浓度顺铂(cisplatin,DDP)分别处理A549及细胞球48 h,用MTT检测48 h内细胞的存活率。应用比色法检测10、50、100、200μmol/L EA对A549细胞球中谷胱甘肽S-转移酶(glutathione S-transferase,GST)活性的抑制作用。应用流式细胞术、Western blot、Real-timePCR、相差显微镜观察检测200μmol/L EA处理A549细胞球前后ROS水平、细胞成球能力、β-catenin、Sox2和ABCG2 mRNA和蛋白以及β-catenin启动子活性的变化情况。应用β-catenin腺病毒感染A549细胞球后,再用200μmol/L EA的处理A549细胞球,利用Real-time PCR和Western blot检测β-catenin S、Sox2和ABCG2 mRNA和蛋白的变化,并用MTT检测A549细胞球增殖的抑制作用。结果成功培养出悬浮的A549细胞球,其高表达肿瘤干细胞标志物CD133、SOX2、Ep CAM以及耐药相关蛋白ABCG2,并可耐受不同浓度DDP的杀伤作用。200μmol/L EA处理A549细胞球后ROS水平明显升高,而GST活性、β-catenin、Sox2和ABCG2 mRNA和蛋白的表达、β-catenin的启动子活性、A549细胞球的成球能力显著下降,并且200μmol/L EA联合5 mg/m L DDP可增强对抑制A549细胞球增殖的作用和增加细胞凋亡的水平(P<0.05)。过表达β-catenin后,200μmol/L EA对β-catenin、Sox2和ABCG2 mRNA和蛋白的抑制作用较空载体组显著减弱。此外,过表达β-catenin可显著缓解200μmol/L EA联合5 mg/m L DDP对A549细胞球的增殖抑制作用。结论 EA通过抑制GST活性和β-catenin水平,发挥抑制A549细胞球增殖和干性,促进其凋亡的作用。EA可望成为治疗肺癌及肺癌干细胞的药物。Objective To investigate the killing effect of ethacrynic acid （EA） on lung cancer A549 cells derived spheres and explore the underlying mechanism. Methods A549 spheres were cultured in serum-free medium, and the protein expression of CD133, SOX2, EpCAM and ABCG2 was detected by Western blotting. MTT assay was used to evaluate the cell viability of A549 spheres and A549 cells after treated by 1, 2, 5, 10 and 20 mg/mL cisplatin （DDP） for 48 h. The activity of glutathione S-transferase （GST） was measured by colorimetric method after A549 spheres were treated with 10, 50, 100 and 200 μmol/L EA, respectively. Flow cytometry, Western blotting, real-time PCR and luciferase assay were used to analyze the levels of cellular reactive oxygen species （ROS） , formation of A549 spheres, mRNA and protein expression levels ofβ-catenin, Sox2 and ABCG2, and promoter activity of β-catenin upon 200 μmol/L EA treated cells for 48 h. A549 sphere was infected with β-catenin adenovirus for 24 h, followed by 200 μmol/L EA treatment （ in presence or absence of 5 mg/mL DDP） for 24 h. The expression of β-catenin, Sox2 and ABCG2 at mRNA and protein levels was detected by real-time PCR and Western blotting, and cell growth of A549 spheres was evaluated by MTT assay. Results The A549 spheres, with high expression of tumor stem cells markers CD133, SOX2, EpCAM and drug resistance related molecule ABCG2, and resistance to DDP at different doses, were successfully derived. After 200μmol/L EA had treated A549 sphere for 48 h, the levels of ROS were significantly increased （P 〈 0. 05 ） , and the mRNA and protein levels of β-catenin, Sox2 and ABCG2, and promoter activity of β-catenin were notably decreased （P 〈 0. 05 ）. The treatment of 200 μmol/L EA enhanced the inhibitory effect on proliferation and the promoting effect on apoptosis in A549 spheres induced by 5 mg/mL DDP （ P 〈 0. 05 ）. Up-regulation of β-catenin by adenoviral infection partly reversed the effects of 200 μmol/L EA on suppressing

关 键 词：肺癌干细胞 依他尼酸 GST Β-CATENIN 

分 类 号：R73-361[医药卫生—肿瘤] R734.2[医药卫生—临床医学]