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

关键词:  瑶药  消瘤藤  总香豆素  铁死亡  结直肠癌

Key words:  Yao medicine  Pileostegia tomentella  total coumarins  ferroptosis  Colorectal Cancer (CRC)

摘要:

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。

Abstract:

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.