土贝母苷甲通过促进GPX4泛素化降解诱导结直肠癌细胞铁死亡  

Tubeimoside I induced ferroptosis in colorectal cancer cells by promoting ubiquitinated degradation of GPX4

作　　者：王萍[1] 王长福 韩士林 匡海学[1] 王秋红[1,2] WANG Ping;WANG Chang-fu;HAN Shi-lin;KUANG Hai-xue;WANG Qiu-hong(Key Laboratory of Basic and Application Research of Beiyao Ministry of Education,Heilongjiang University of Chinese Medicine,Harbin 150040,China;Guangdong Standardized Processing Engineering Technology Research Center of Traditional Chinese Medicine,Gaungdong Pharmaceutical Univerisity,Guangzhou 510006,China)

机构地区：[1]黑龙江中医药大学教育部北药基础与应用研究重点实验室,哈尔滨150001 [2]广东药科大学广东省中药饮片规范化炮制工程技术研究中心,广州510006

出　　处：《天然产物研究与开发》2025年第3期403-410,共8页Natural Product Research and Development

基　　金：国家重点研发计划中医药现代化研究重点专项(2018YFC1707100);2022年全国名老中医药专家传承工作室建设项目(2022-75);黑龙江省教育厅“头雁”团队支持项目(2019-5)。

摘　　要：本研究旨在探究土贝母苷甲(tubeimoside I,TBMS 1)诱导的结肠癌细胞HCT116细胞铁死亡及分子机制。体外培养结直肠癌HCT116细胞,MTT法检测不同浓度TBMS 1作用HCT116细胞的存活率,并根据此结果进行分组;克隆形成实验检测TBMS 1对HCT116细胞增殖能力的影响;Western blot法检测细胞中溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)和谷胱甘肽过氧化物酶(glutathione peroxidase 4,GPX4)的表达;流式细胞仪及荧光显微镜检测细胞中脂质活性氧(lipid reactive oxygen species,Lipid ROS)水平的变化;丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)、亚铁离子(Fe^(2+))试剂盒分别检测细胞中MDA,GSH以及Fe^(2+)的相对水平;免疫共沉淀法检测GPX4蛋白的泛素化水平。研究结果表明随TBMS 1药物浓度的增加,HCT116细胞的存活率逐渐降低,克隆形成能力逐渐降低;细胞中加入TBMS 1后,GSH水平降低,而Fe^(2+)和MDA水平增加;HCT116细胞中加入TBMS 1作用后,细胞中氧化态的Lipid ROS绿色荧光明显增加且Lipid ROS水平明显增加;在细胞中同时加入TBMS 1和铁死亡抑制剂ferrostatin-1(Fer-1)能够逆转TBMS 1导致的细胞死亡,且细胞中加入不同浓度的TBMS 1能够抑制GPX4蛋白的表达,而对SLC7A11蛋白的表达几乎没有影响;与对照组相比,单独加入TBMS 1对GPX4蛋白表达的抑制能够被Fer-1所逆转,且蛋白免疫共沉淀结果显示,TBMS 1促进了GPX4蛋白的泛素化和降解。本研究结果发现TBMS 1可能通过促进GPX4蛋白的泛素化和降解诱导结直肠癌HCT116细胞铁死亡并抑制其恶性增殖。This study aims to investigate tubeimoside I(TBMS 1)-induced ferroptosis and molecular mechanism in colorectal cancer HCT116 cells.HCT116 cells were cultured in vitro,and MTT assay was used to detect the survival rate of cells with different concentrations of TBMS 1,and the cells were grouped according to this result.Colone formation assay was used to detect the effect of TBMS 1 on the proliferative ability of HCT116 cells;Western blot assay was used to detect the expression of SLC7A11 and GPX4 in the cells;flow cytometry and fluorescence microscopy were used to detect the changes of lipid reactive oxygen species(Lipid ROS)in the cells;malondialdehyde(MDA),glutathione(GSH),and Fe^(2+)iron kit were used to detect the relative level of MDA,GSH,and Fe^(2+),respectively;and the immunoprecipitation assay was used to detect the ubiquitylation level of the GPX4 protein.Compared with 0μmol/L TBMS 1,the viability of HCT116 cells gradually decreased with the increase of TBMS 1 concentration,and the clone formation ability gradually decreased,and the GSH level decreased,while the Fe^(2+)and MDA levels increased after treatment by TBMS 1.The green fluorescence of Lipid ROS in the oxidative state and the level of Lipid ROS in the cells were significantly increase after the addition of TBMS 1 to HCT116 cells compared to the effect of 0μmol/L TBMS 1.Simultaneous addition of TBMS 1 and ferroptosis inhibitor ferrostatin-1(Fer-1)to the cells was able to reverse the cell death caused by TBMS 1,and the addition of different concentrations of TBMS 1 to the cells was able to inhibit the expression of GPX4 protein,while it had almost no effect on the expression of SLC7A11 protein.The inhibition of GPX4 protein expression by the addition of TBMS 1 alone was able to be reversed by Fer-1 compared to the control group,and protein immunoprecipitation showed that TBMS 1 promoted ubiquitination and degradation of GPX4 protein.TBMS 1 may induce ferroptosis and inhibit malignant proliferation of colorectal cancer HCT116 cells by promoting ubi

关 键 词：土贝母苷甲 结直肠癌 增殖 谷胱甘肽过氧化物酶4 铁死亡 泛素化 

分 类 号：R93[医药卫生—生药学]