Abstract:

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 Fe²⁺ iron kit were used to detect the relative level of MDA,GSH,and Fe²⁺,respectively;and the immunoprecipitation assay was used to detect the ubiquitylation level of the GPX4 protein.Compared with 0 μmol/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 Fe²⁺ 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 0 μmol/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 ubiquitination and degradation of GPX4.

Key words: tubeimoside I, colorectal cancer, proliferation, glutathione peroxidase 4, ferroptosis, ubiquitin