二甲双胍诱导铁死亡抑制胶质瘤细胞增殖作用机制的研究  

Mechanism of metformin-induced ferroptosis in inhibition of glioma cell proliferation

作　　者：金鹏程 林杰 朱鹏磊 JIN Peng-cheng;LIN Jie;ZHU Peng-lei(Department of Neurosurgery,Wenzhou People’s Hospital,Wenzhou 325000,China)

机构地区：[1]浙江省温州市人民医院神经外科,温州325000

出　　处：《浙江中西医结合杂志》2023年第2期112-116,122,共6页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省温州市基础性医疗卫生科技项目(No.Y2020983)。

摘　　要：目的 探讨二甲双胍通过诱导铁死亡抑制胶质瘤细胞增殖的作用和机制。方法 常规培养正常人神经胶质细胞和胶质瘤细胞,酶联免疫吸附试验(ELISA)检测细胞脂质过氧化产物[5-、12-和15-羟二十碳四烯酸(HETE)]的变化,Western blot检测细胞铁死亡标志蛋白谷胱甘肽过氧化物酶(GPX4)和链脂肪酸-CoA连接酶(ACSL-4)表达;二甲双胍处理胶质瘤细胞U87,细胞计数试剂盒(CCK-8)检测细胞活力改变,克隆形成实验检测细胞克隆形成能力,乳酸脱氢酶(LDH)试剂盒检测细胞LDH释放量,5-乙炔基-2’-脱氧尿苷(EdU)染色检测细胞增殖,丙二醛(MDA)试剂盒检测细胞MDA水平,流式细胞术检测细胞活性氧(ROS)水平,Western blot检测细胞铁死亡标志蛋白GPX4和ACSL-4蛋白表达;二甲双胍联合铁死亡抑制剂(ferrostatin-1)处理U87细胞,进一步验证胶质瘤细胞铁死亡机制。结果 与正常胶质细胞比较,胶质瘤细胞脂质过氧化产物5-、12-和15-HETE水平降低,铁死亡标志蛋白GPX4表达增多,ACSL-4蛋白表达减少;二甲双胍处理U87细胞后,铁死亡蛋白GPX4表达减少,ACSL-4蛋白表达增多,MDA水平[(321±31)%比(100±12)%,P<0.01]和ROS水平增加,细胞活力[(57±11)%比(100±10)%,P<0.01]下降;ferrostatin-1联合二甲双胍处理U87细胞,GPX4表达增多,ACSL-4表达减少,MDA[(160±14)%比(295±21)%,P<0.001]和ROS水平降低,细胞活力[(81±5)%比(61±9)%,P<0.05]升高。结论 二甲双胍可能通过促进胶质瘤细胞U87铁死亡的发生从而抑制细胞增殖。Objective To evaluate the effects of metformin-induced ferroptosis in inhibition of glioma cell proliferation and the underlying molecular events.Methods Glioma and normal human glial cells were grown for ELISA or Western blot assessment of lipid peroxidation products(5-,12-and 15-HETE)and the ferroptosis markers(GPX4 and ACSL-4).Afterwards,glioma U87 cells were treated with or without metformin and metformin plus ferrostatin-1 for changes in cell viability using CCK-8 assay,clone formation using clone formation assay,cell proliferation using EdU staining,cellular ROS level using flow cytometry,MDA level using the MDA kit,LDH release using the LDH kit,and ferroptosis markers(GPX4 and ACSL-4 proteins) using Western blot.Results Compared with normal glial cells,levels of 5-,12-and 15-HETE and ACSL-4 protein were lower,whereas expression of ferroptosis marker GPX4 protein was high,indicating that glioma ferroptosis was decreased in different glioma cell lines.After metformin treatment,ferroptosis GPX4 protein was reduced,whereas ACSL-4 protein was increased compared to the control cells.Moreover,level of MDA(321 ±31 vs.100 ±12%;P <0.001)and ROS was induced;however,cell viability(57±11 vs.100±10%;P<0.001) was reduced after metformin treatment,indicating that metformin reduced cell proliferation but promoted ferroptosis in glioma cells.In addition,U87 cells were further treated with a ferroptosis inhibitor ferrostatin-1 to argue the effect of metformin in glioma cells and the data showed that addition of ferrostatin-1 increased GPX4 expression and reduced ACSL-4 expression and MDA level(160±14 vs.295±21%;P<0.001)and ROS production to induce glioma cell viability(81±5 vs.61±9%;P<0.05) vs.that of metformin-alone.Conclusion Metformin-reduced glioma cell proliferation could be through ferroptosis induction.

关 键 词：二甲双胍 胶质瘤 铁死亡 增殖 

分 类 号：R739.41[医药卫生—肿瘤]