消瘤藤总香豆素诱导结直肠癌细胞铁死亡的作用研究

Study on the Effect of Total Coumarins from Pileostegia tomentella Inducing Ferroptosis of Colorectal Cancer Cells

为探究广西传统瑶药消瘤藤总香豆素(Total Coumarins from Pileostegia tomentella,TCPT)诱导人结直肠癌(Colorectal Cancer,CRC)HT-29细胞铁死亡的作用及其机制,本研究采用不同浓度TCPT处理人结直肠癌HT-29细胞,利用细胞增殖活性检测试剂盒(Cell Counting Kit-8,CCK-8)结合流式细胞术(Flow Cytometry,FCM)检测细胞活性的变化情况,采用透射电子显微镜观察细胞线粒体的形态变化,利用二氯荧光素(DCFH-DA)染料探针法检测细胞内铁依赖性活性氧(Reactive Oxygen Species,ROS)含量的变化,利用特异性荧光探针BODIPY 581/589 C11检测细胞内脂质过氧化物含量,利用蛋白质印记(Western blotting)检测铁死亡相关蛋白谷胱甘肽过氧化物酶4(Glutathione Peroxidase 4,GPX4)、胱氨酸-谷氨酸反向转运体亚基(Cysteine-glutamate antiporter subunit,xCT)、核受体共激活因子4(Nuclear receptor Coactivator 4,NCOA4)和二价金属离子转运体(Divalent Metal Transporter 1,DMT1)的表达水平。结果表明:人结直肠癌HT-29细胞经TCPT处理后,细胞活力呈浓度依赖性下降,细胞线粒体出现细胞质骤减、嵴塌陷、“同心圆”等典型的铁死亡形态特征,细胞内的ROS和脂质过氧化物水平呈剂量依赖性显著升高。HT-29细胞铁死亡标志蛋白GPX4、xCT的表达水平下调,而NCOA4、DMT1的表达水平上调。综上所述,消瘤藤总香豆素可使细胞内ROS和脂质过氧化物水平明显升高,从而诱导结直肠癌HT-29细胞铁死亡,这可能是其抗结直肠癌的作用机制之一。

Total Coumarins from Pileostegia tomentella(TCPT)was a traditional Yao medicine in Guangxi,to explore the effect and mechanism of TCPT in inducing ferroptosis of colorectal cancer HT-29 cells,human Colorectal Cancer(CRC)HT-29 cells were treated with different concentrations of TCPT in this study,and the changes of cell viability were detected by Cell Counting Kit-8(CCK-8)combined with Flow Cytometry(FCM).The morphological changes of mitochondria were observed by transmission electron microscopy,and the changes of intracellular iron-dependent Reactive Oxygen Species(ROS)content were detected by dichlorofluorescein(DCFH-DA)dye probe method.The specific fluorescent probe BODIPY 581/589 C11 was used to detect intracellular lipid peroxide content.The expression levels of ferroptosis-related proteins Glutathione Peroxidase 4(GPX4),Nuclear receptor Coactivator 4(NCOA4),Cysteine-glutamate antiporter subunit(xCT)and Divalent Metal Transporter 1(DMT1)proteins in HT-29 cells were detected by Western blotting.The results showed that after TCPT treatment,the cell viability of human colorectal cancer HT-29 cells decreased in a concentration-dependent manner.Cell mitochondria showed typical ferroptosis morphological characteristics such as cytoplasmic sudden reduction,ridge collapse,and‘concentric circles’.The levels of intracellular ROS and lipid peroxides in the cells were significantly increased in a dose-dependent manner.The expression levels of ferroptosis marker proteins GPX4 and xCT in HT-29 cells were down-regulated,while the expression levels of NCOA4 and DMT1 were up-regulated.In summary,the total coumarin from Pileostegia tomentella can significantly increase the levels of intracellular ROS and lipid peroxidation,thus inducing ferroptosis in colorectal cancer HT-29 cells,which may be one of its anti-colorectal cancer mechanisms.