摘要
目的:探讨二甲双胍是否能够逆转低氧诱导胰腺癌细胞对铁死亡诱导剂的抵抗,并进一步明确其作用机制。方法:人胰腺癌Patu8988细胞分别在常氧和低氧下培养不同时间(0、12、24、48 h),CCK-8法检测细胞增殖,免疫印迹法检测缺氧诱导因子-1α(hypoxia-inducible factor 1α,HIF-1α)蛋白表达;用不同浓度铁死亡诱导剂Erastin分别处理常氧和低氧培养的Patu8988细胞,CCK-8法检测细胞活性;低氧培养的Patu8988细胞分别给予以下处理:对照组、二甲双胍、Erastin、二甲双胍联合Erastin,用CCK-8法检测细胞活性,免疫印迹法检测谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白表达;用不同浓度二甲双胍处理低氧培养的Patu8988细胞,免疫印迹法分别检测腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK),雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR),HIF-1α等蛋白表达。结果:与常氧组相比,低氧抑制细胞增殖,且呈时间依赖性,在48 h差异最显著(P<0.05);Erastin浓度为10μmol/L时,低氧组细胞活性明显低于常氧组(P<0.05),为最佳实验浓度;低氧条件下,与单药处理组相比,二甲双胍联合Erastin可以显著抑制细胞活性,降低GPX4蛋白表达水平(P均<0.05);二甲双胍能够激活AMPK,抑制mTOR磷酸化以及HIF-1α蛋白水平(P均<0.05)。结论:二甲双胍能够通过AMPK-mTOR轴减少HIF-1α表达,从而逆转低氧诱导的胰腺癌细胞铁死亡抵抗。
Objective:To investigate whether metformin can reverse hypoxia-induced resistance to ferroptosis in pancreatic cancer cells and to further clarify its mechanism.Methods:Human pancreatic cancer cells Patu8988 were cultured under normoxia and hypoxia conditions for different time(0,12,24 or 48 h).CCK-8 assay was used to detect the cell viability and Western blotting was used to detect the protein expression of hypoxia-inducible factor 1α(HIF-1α);CCK-8 assay was used to detect the cell viability of Patu8988 cells treated with different concentrations of ferroptosis inducer Erastin in normoxia and hypoxia conditions;the hypoxia-cultured Patu8988 cells were divided into the following four treatment groups:control group,metformin group,Erastin group,metformin+Erastin group.CCK-8 assay was used to detect the cell viability,Western blotting was used to detect the protein expression level of glutathione peroxidase 4(GPX4).Patu8988 cells in hypoxia culture were treated with different concentrations of metformin,and Western blotting was used to detect the protein expression levels of AMP-activated protein kinase(AMPK),mammalian target of rapamycin(mTOR),HIF-1αand other proteins.Results:Compared with the normoxia group,hypoxia inhibited the cell viability in a time-dependence manner,and the difference between two groups was the most significant at 48 h(P<0.05).When the concentration of Erastin was 10μmol/L,the cell viability of the hypoxia group was significantly lower than that of the normoxia group(P<0.05),and 10μmol/L was the best experimental concentration.Compared with the group treated alone in hypoxia condition,the combined treatment could significantly inhibit the cell viability and reduce the protein expression level of GPX4(both P<0.05).By activating AMPK,inhibiting mTOR phosphorylation,metformin negatively regulated HIF-1αprotein expression level(both P<0.05).Conclusion:Metformin could reduce the accumulation of HIF-1αthrough the AMPK-mTOR axis,thus reversing hypoxia-induced resistance to ferroptosis in pancreatic cancer cells.
作者
蔡晓杰
王鸣
高洁
姜伟
宋廉
张礼荣
龚爱华
朱海涛
王冬青
CAI Xiao-jie;WANG Ming;GAO Jie;JIANG Wei;SONG Lian;ZHANG Li-rong;GONG Ai-hua;ZHU Hai-tao;WANG Dong-qing(School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013;Department of Radiology, Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001, China)
出处
《江苏大学学报（医学版）》
CAS
2020年第2期125-130,共6页
Journal of Jiangsu University：Medicine Edition
基金
江苏省研究生科研创新计划立项(KYCX18_2287)。
