该文旨在探讨金丝桃素诱导乳腺癌细胞MCF-7、MDA-MB-231铁死亡的作用机制.采用CCK-8法检测不同浓度金丝桃素对MCF-7、MDA-MB-231及MCF-10A细胞活力的影响,并检测Fer-1(ferrostatin-1)对金丝桃素所影响的细胞活力的恢复作用;克隆形成实验检测金丝桃素对MCF-7、MDA-MB-231细胞克隆形成的抑制作用;DCFH-DA探针对细胞内活性氧(reactive oxygen species,ROS)的积累进行检测;乳酸脱氢酶(lactate dehydrogenase,LDH)释放检测实验检测LDH的变化情况;组织铁测定试剂盒对细胞内Fe2+的积累进行检测;还原型谷胱甘肽(glutathione,GSH)测定试剂盒检测GSH水平;免疫印迹法检测金丝桃素处理后乳腺癌细胞内溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11/xCT)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白的表达情况.结果显示,金丝桃素对乳腺癌细胞MCF-7的半抑制浓度(IC50)为10μmol/L,MDA-MB-231的IC50为15 μmol/L,且对正常细胞MCF-10A无明显影响.金丝桃素能够导致MCF-7和MDA-MB-231细胞内ROS的积累、LDH的释放.此外,铁死亡抑制剂Fer-1能够逆转被金丝桃素所影响的MCF-7和MDA-MB-231细胞活力.同时金丝桃素还能够升高MCF-7和MDA-MB-231细胞内Fe2+含量,降低细胞内GSH水平,下调xCT以及GPX4的表达.结果表明,金丝桃素可能通过诱导乳腺癌细胞发生铁死亡从而抑制乳腺癌细胞增殖.
Mechanism of Hypericin Inducing Ferroptosis in Breast Cancer Cells
This study was aimed to investigate the mechanism of hypericin inducing ferroptosis in breast cancer cells MCF-7 and MDA-MB-231.CCK-8 assay was used to detect the effects of various concentrations of hy-pericin on the viability of MCF-7,MDA-MB-231 and MCF-10A cells,test the recovery effect of Fer-1(ferrostatin-1)on the cell viability affected by hypericin.Clonogenic assay was employed to test the inhibitory effect of hypericin on the clonal formation of MCF-7 and MDA-MB-231 cells.DCFH-DA probe was used to detect the accumulation of intracellular ROS(reactive oxygen species).LDH(lactate dehydrogenase)release assay was conducted to mea-sure changes in LDH.Tissue iron assay kit was used to detect the accumulation of intracellular Fe2+.Reduced GSH(glutathione)assay kit was employed to measure GSH levels.And immunoblotting was performed to assess the expression of SLC7A11/xCT(solute carrier family 7 member 11)and GPX4(glutathione peroxidase 4)proteins in breast cancer cells after hypericin treatment.The results showed that hypericin had a semi-inhibitory concen-tration(IC50)of 10 μmol/L for breast cancer cells MCF-7 and 15 μmol/L for MDA-MB-231 cells,and had no significant effect on normal cells MCF-10A.Hypericin could cause the accumulation of ROS and LDH release in MCF-7 and MDA-MB-231 cells.Additionally,the ferroptosis inhibitor Fer-1 could reverse the impact of hy-pericin on the viability of MCF-7 and MDA-MB-231 cells.At the same time,hypericin could also increase the intracellular Fe2+content in MCF-7 and MDA-MB-231 cells,decrease the levels of GSH,and downregulate the expression of xCT and GPX4.The results indicated that hypericin might inhibit the proliferation of breast cancer cells by inducing ferroptosis.
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