基于p53/SLC7A11/GPX4信号轴探讨七叶内酯诱导小鼠乳腺癌4T1细胞铁死亡的作用机制

Mechanism of esculetin promoting ferroptosis of mouse breast cancer 4T1 cells based on p53/SLC7A11/GPX4 axis

目的:探讨七叶内酯对小鼠乳腺癌4T1细胞铁死亡的影响及其作用机制。方法:对七叶内酯与p53、溶质载体家族7成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)进行分子对接,并绘制Kaplan-Meier和time ROC曲线。将4T1细胞分为对照组、1/2 IC_(50)组、IC_(50)组、2 IC_(50)组、erastin组和卡培他滨组。采用CCK-8法观察4T1细胞活力情况,筛选药物干预浓度;电镜观察4T1细胞线粒体形态,评估线粒体损伤情况;试剂盒检测4T1细胞活性氧(ROS)、Fe2+、丙二醛(MDA)、谷胱甘肽(GSH)和GPX4的水平;采用划痕和Transwell小室实验检测4T1细胞侵袭和迁移能力;Western blot检测p53、SLC7A11、GPX4和酰基辅酶A合成酶长链家族成员4(ACSL4)的表达水平。结果:与对照组相比,七叶内酯1/2 IC_(50)、IC_(50)和2 IC_(50)组细胞活力,GSH和GPX4表达水平,侵袭和迁移能力,以及SLC7A11和GPX4蛋白表达水平均降低,细胞线粒体变小,膜密度增高,嵴减少(P<0.05),Fe2+、ROS和MDA水平及p53和ACSL4蛋白表达水平升高(P<0.05)。结论:七叶内酯可促进4T1细胞铁死亡,其作用机制可能与p53/SLC7A11/GPX4信号通路相关。

AIM:To investigate the mechanism through which esculetin induces ferroptosis of mouse breast cancer 4T1 cells.METHODS:Molecular docking of esculetin with p53,solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)proteins was performed,and Kaplan-Meier curves and time-dependent receiver operating characteristic curves were drawn.The 4T1 cells were divided into 6 groups,namely the control(CON),1/2 IC_(50),IC_(50),2 IC_(50),erastin,and capecitabine groups.The appropriate drug concentration for treating 4T1 cells was screened by CCK-8 assay.The invasion and migration abilities of 4T1 cells following esculetin treatment at the selected drug concentration were analyzed by scratch test and Transwell assay.Mitochondrial morphological change were examined by electron microscopy.The levels of ferroptotic cell death were then quantified by Fe2+,reactive oxygen species(ROS),malondialdehyde(MDA),glutathione(GSH),and GPX4 assays.Western blot was performed to evaluate the protein levels of p53,SLC7A11,GPX4 and acyl-CoA synthetase long-chain family member 4(ACSL4).RESULTS:Compared with CON group,the esculetin 1/2 IC_(50),IC_(50)and 2 IC_(50)groups showed smaller size,increased membrane density,and decreased ridge of mitochodria,as well as decreased levels of GSH and GPX4,reduced cell invasion and migration abilities,and de-creased levels of SLC7A11 and GPX4 proteins(P<0.05).Furthermore,these groups exhibited increased levels of Fe2+,ROS,and MDA,as well as elevated p53 and ACSL4 protein abundance(P<0.05).CONCLUSION:Esculetin pro-motes ferroptosis of 4T1 cells through a mechanism involving the p53/SLC7A11/GPX4 regulatory axis.