摘要
目的:研究DL-丁胱亚磺酰亚胺(DL-BSO)导致结肠癌细胞发生铁死亡的作用机制。方法:(1)采用梯度浓度的DL-BSO干预人结肠癌HCT-116和SW480细胞,CCK-8法检测细胞活力的改变。(2)采用实验浓度的DL-BSO干预2种细胞,分为对照组和实验组,Transwell和划痕实验检测细胞迁移能力,Western blot法检测目的蛋白表达水平。(3)用necrostatin-1(Nec-1)和ferrostatin-1(Fer-1)预处理2种细胞,再用实验浓度的DL-BSO进行干预,分为对照组、DL-BSO组、抑制剂组和DL-BSO联合抑制剂组,CCK-8法检测细胞活力,以铁离子比色法试剂盒检测铁离子浓度,以谷胱甘肽(GSH)试剂盒检测GSH浓度,以Western blot法检测谷胱甘肽过氧化物酶4(GPX4)和胱氨酸/谷氨酸反向转运体(xCT)表达水平,以脂质过氧化传感器C11-BODIPY-591荧光显影和流式细胞术检测活性氧(ROS)水平。结果:DL-BSO干预后,HCT-116和SW480细胞活力均显著减弱,DL-BSO的最适浓度和处理时间分别为300μmol/L和36 h。DL-BSO处理使HCT-116和SW480细胞横向及纵向迁移能力均减弱,铁死亡关键蛋白GPX4和xCT表达下调,ROS水平和铁离子浓度升高,GSH浓度降低。DL-BSO的作用不受Nec-1的回调,但受到Fer-1的回调。结论:DL-BSO能通过铁死亡途径导致结肠癌细胞发生死亡,并减弱细胞的侵袭和迁移能力。
AIM:To investigate the mechanism of DL-buthionine-(S,R)-sulfoximine(DL-BSO)leading to ferroptosis of colon cancer cells.METHODS:(1)Human colon cancer HCT-116 and SW480 cells were treated with different concentrations of DL-BSO,and the cell viability was detected by CCK-8 assay.(2)The cells were treated with DLBSO at experimental concentration,and divided into control group and DL-BSO group.The cell migration ability was detected by Transwell and scratch assays,and the expression levels of the target proteins were detected by Western blot.(3)The cells were pretreated with necrostatin-1(Nec-1)or ferrostatin-1(Fer-1),intervened with DL-BSO at experimental concentration,and divided into control group,DL-BSO group,inhibitor group and DL-BSO combined inhibitor group.The cell viability was detected by CCK-8 assay,and the iron ion concentration was detected by colorimetry.The gluthione(GSH)level was determined by commercially available kits,and the glutathione peroxidase 4(GPX4)and cystine/glutamate antiporter(xCT)expression levels were detected by Western blot.The level of reactive oxygen species(ROS)was detected by lipid peroxidation sensor C11-BODIPY-591 fluorescence visualization and flow cytometry.RESULTS:After DL-BSO intervention,cell viability was significantly reduced,and the optimal concentration and treatment time of DLBSO were measured at 300μmol/L and 36 h,respectively.Cell lateral and longitudinal migration ability was also reduced.The expression of GPX4 and xCT,key proteins of ferroptosis,was down-regulated.The ROS level and iron ion concentration were increased,and GSH concentration was decreased.The effects of DL-BSO were counteracted by Fer-1 rather than Nec-1.CONCLUSION:Treatment with DL-BSO causes death of colon cancer cells through the ferroptosis pathway and diminish their invasion and migration ability.
作者
郑拥
孙玥
辛宝山
罗斌
刘刚
史良会
ZHENG Yong;SUN Yue;XIN Baoshan;LUO Bin;LIU Gang;SHI Lianghui(Department of Gastrointestinal Surgery,the First Affiliated Hospital of Wannan Medical College,Wuhu 241000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2023年第8期1399-1407,共9页
Chinese Journal of Pathophysiology
基金
安徽省教育厅自然科学研究计划项目(No.2022AH051241)
皖南医学院中青年科研基金(No.WK2021F07)。