重楼总皂苷诱导乳腺癌MCF-7细胞铁死亡抗肿瘤机制研究

Anti-tumor mechanism of total saponins of Paridis Rhizoma on inducing ferroptosis of breast cancer MCF-7 cells

探讨重楼总皂苷诱导乳腺癌MCF-7细胞的铁死亡作用及机制,为重楼总皂苷临床治疗乳腺癌提供理论依据。通过噻唑蓝(methyl thiazolyl tetrazolium,MTT)实验检测不同浓度重楼总皂苷对MCF-7细胞增殖能力的影响;倒置显微镜观察MCF-7细胞形态改变;平板克隆实验检测MCF-7细胞克隆形成能力;乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验检测MCF-7细胞膜的完整性;划痕实验检测MCF-7细胞迁移能力;利用荧光倒置显微镜检测MCF-7细胞中活性氧(reactive oxygen species,ROS)的水平,利用试剂盒检测MCF-7细胞中Fe2+的含量;透射电镜观察MCF-7细胞线粒体超微结构;采用Western blot检测重楼总皂苷对MCF-7细胞中p53、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long-chain family member 4,ACSL4)、转铁蛋白受体1(transferrin receptor protein 1,TFR1)表达的影响。结果显示,1.5、3、4.5、6、7.5、9μg·mL^(-1)重楼总皂苷均能显著抑制MCF-7细胞增殖,IC50为4.12μg·mL^(-1),对细胞形态有明显的损伤,导致细胞形成空泡,逐渐皱缩并脱落;抑制MCF-7细胞的克隆形成能力,破坏细胞膜促使细胞内LDH释放,缩短MCF-7细胞的迁移距离抑制迁移能力;显著提高MCF-7细胞中ROS的含量,诱导细胞氧化损伤,导致细胞内Fe^(2+)堆积,线粒体结构也发生明显的改变,线粒体膜密度增加,嵴减少甚至消失;促进p53蛋白表达,下调SLC7A11和GPX4表达并增强ACSL4和TFR1表达。因此,重楼总皂苷可显著抑制乳腺癌MCF-7细胞的增殖和迁移能力、破坏细胞结构,其机制可能与诱导细胞铁死亡有关。

This study aims to investigate the mechanism of total saponins of Paridis Rhizoma in inducing the ferroptosis of MCF-7 cells and provide a theoretical basis for the clinical treatment of breast cancer with total saponins of Paridis Rhizoma.The methyl thia-zolyl tetrazolium(MTT)assay was employed to examine the effects of different concentrations of total saponins of Paridis Rhizoma on the proliferation of MCF-7 cells.A phase contrast inverted microscope was used to observe the morphological changes of MCF-7 cells.The colony formation assay was employed to test the colony formation of MCF-7 cells.The lactate dehydrogenase(LDH)release test was conducted to determine the cell membrane integrity of MCF-7 cells.The cell scratch assay was employed to examine the migration of MCF-7 cells.After that,the level of reactive oxygen species(ROS)in MCF-7 cells was observed by an inverted fluorescence micro scope,and the content of Fe2*in MCF-7 cells was detected by the corresponding kit.Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of MCF-7 cells.Western blot was employed to determine the expression of ferroptosis-related proteins,such as p53,solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),acyl-CoA synthetase longchain family member 4(ACSL4),and transferrin receptor protein 1(TFR1)in MCF-7 cells.The results showed that 1.5,3,4.5,6,7.5,and 9μg·mL^(-1)total saponins of Paridis Rhizoma significantly inhibited the proliferation of MCF-7 cells,with the ICso of 4.12μg·mL^(-1).Total saponins of Paridis Rhizoma significantly damaged the morphology of MCF-7 cells,leading to the formation of vacuoles and the gradual shrinkage and detachment of cells.Meanwhile,total saponins of Paridis Rhizoma inhibited the colony formation of MCF-7 cells,destroyed the cell membrane(leading to the release of LDH),and shortened the migration distance of MCF-7 cells.Total saponins of Paridis Rhizoma treatment significantly increased the content of ROS,induced oxidative damage,and led to the accumulation of Fe^(2+) in MCF-7 cells.Furthermore,total saponins of Paridis Rhizoma changed the mitochondrial structure,increased the mitochondrial membrane density,led to the decrease or even disappear of ridges,promoted the expression of p53 protein,down-regulated the expression of SLC7A11 and GPX4,and up-regulated the expression of ACSL4 and TFR1.In summary,total saponins of Paridis Rhizoma can significantly inhibit the proliferation and migration of MCF-7 cells and destroy the cell structure by inducing ferroptosis.