儿茶素通过促进胃癌细胞铁死亡增强化疗药物5-FU的杀伤作用  

Catechin enhanced the killing effects of 5 - FU on gastric cancer cells throughpromoting ferroptosis of gastric cancer cells

作　　者：徐涛 井文君 刘静 XU Tao;JING Wenjun;LIU Jing(Instrumental Analysis Center of QAU,Shandong Qingdao 266109,China;Department of Oncology,Qingdao Central Hospital,Qingdao University,Shandong Qingdao 266042,China)

机构地区：[1]青岛农业大学分析测试中心,山东青岛266109 [2]青岛大学附属青岛市中心医院肿瘤综合二科,山东青岛266042

出　　处：《现代肿瘤医学》2024年第16期2981-2989,共9页Journal of Modern Oncology

基　　金：国家自然科学基金资助项目(编号:82000290);青岛农业大学高层次人才启动基金资助项目(编号:663/1120108,663/1121022)。

摘　　要：目的:探讨儿茶素对胃癌化疗效果的影响及机制。方法:使用5-FU(100μg/mL)处理胃癌细胞AGS和GC7901,分别处理0 h、3 h、6 h、9 h、12 h、16 h、24 h,通过PI染色及流式细胞术检测胃癌细胞AGS及GC7901的死亡情况;Western Blot实验检测正常胃黏膜上皮细胞RGM-1、胃癌细胞AGS和GC7901铁死亡相关特征蛋白谷胱甘肽过氧化酶4(glutathione peroxidase 4,GPX4)、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)及重肽铁蛋白(ferritin heavy polypeptide,FTH)的表达,Image J软件测量灰度值;将胃癌细胞分别设置为对照组、5-FU处理组、5-FU联合Catechin处理组和5-FU联合DMSO处理组,分别通过PI染色及流式细胞术检测细胞死亡,通过C11-BODIPY581/591染色及流式细胞术检测脂质过氧化物积累,通过Western Blot实验检测铁死亡相关特征蛋白的表达,通过吸光光度法检测细胞裂解物中游离Fe^(2+)的含量;分离线粒体和细胞质组分,检测各个分组中线粒体内游离Fe^(2+)含量和细胞质内游离Fe^(2+)含量,并通过Mito-Ferro Green染色进一步验证增量Fe^(2+)的细胞内定位;Western Blot方法检测血红素加氧酶1蛋白(heme oxygenase 1,Hmox1)在各分组中的表达并通过Image J软件测量灰度值;将胃癌细胞分别设置为对照组、5-FU处理组、5-FU组联合Catechin处理组、5-FU联合Catechin和siRNA-NC处理组、5-FU联合Catechin和siRNA-Hmox1处理组,检测线粒体游离Fe^(2+)含量、脂质过氧化物积累及细胞死亡。结果:胃癌细胞在5-FU处理0~12 h内,随处理时间增长死亡率逐渐升高,然而在处理12~24 h内死亡率变化不明显表现出耐药性;胃癌细胞中铁死亡相关特征蛋白GPX4、SLC7A11和FTH的表达量显著高于正常胃黏膜上皮细胞(P<0.05);与对照组相比,处理24 h后,5-FU联合Catechin处理组细胞死亡数量、脂质过氧化物积累都显著升高(P<0.05),同时铁死亡相关特征蛋白GPX4、SLC7A11、FTH的表达量显著降低(P<0.05);5-FU�Objective:To investigate the effects and mechanisms of catechin on the chemotherapy of gastric cancer.Methods:Gastric cancer cells(AGS and GC7901)were treated with 5-FU(100μg/mL)for 0 h,3 h,6 h,9 h,12 h,16 h,24 h separately.PI staining and flow cytometry were used to detect cell deaths of gastric cancer cells.Western Blot assays were performed to detect expression levels of ferroptosis-associated signature proteins glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),and ferritin heavy polypeptide(FTH)in gastric epithelial cells(RGM-1)and gastric cancer cells(AGS and GC7901).The gray-scale values were evaluated by Image J.Gastric cancer cells were divided into 4 groups:Control group,5-FU treatment group,5-FU treatment combined with catechin treatment group,and 5-FU treatment combined with DMSO treatment.Cell deaths were detected by PI staining and flow cytometry.Lipid peroxides accumulation was detected by C11-BODIPY581/591 staining and flow cytometry.Ferroptosis-associated signature proteins were detected by Western Blot assays.The free Fe^(2+)levels were detected by absorption spectrophotometry.The free Fe^(2+)levels were detected in the isolated mitochondria and cytoplasm respectively and the intracellular localization of the increased Fe^(2+)were detected by Mito-Ferro Green staining and fluorescence microscopy.The expression levels of Hmox1 were detected in each group by Western Blot assays and the gray-scale values were evaluated by Image J.Gastric cancer cells were divided into 5 groups:Control group,5-FU treatment group,5-FU treatment combined with catechin treatment group,5-FU treatment combined with catechin and siRNA-NC group,5-FU treatment combined with catechin and siRNA-Hmox1 group.Fe^(2+)levels in mitochondria,lipid peroxides accumulation and cell deaths were detected respectively in each group.Results:Cell deaths of gastric cancer cells increased when underwent 5-FU treatment within 12 h.However,the percentage of cell deaths did not increase obviously within 5-FU treatment fr

关 键 词：儿茶素 5-氟尿嘧啶 细胞铁死亡 二价铁离子 血红素加氧酶1 

分 类 号：R735.2[医药卫生—肿瘤]