Objective To investigate the effects of STIP1 homology and U-box containing protein 1(STUB1)on the ubiquitination of glutathione peroxidase 4(GPX4)and ferroptosis in colon cancer cells HCT116,as well as the impact on CD8+T cell-mediated killing of HCT116 cells.Methods HCT116 cells were divided into control group,empty plasmid transfection(pcDNA3.1-vector)group,STUB1 overexpression plasmid transfection(pcDNA3.1-STUB1)group,and co-transfection(pcDNA3.1-STUB1+pcDNA3.1-GPX4)group.Cell proliferation ability was assessed by CCK-8 assay.Clonogenic ability was determined by clone formation assay.Malondialdehyde(MDA)levels in cells were measured using an MDA kit.Intracellular ferrous ion(Fe2+)levels were detected with an Fe2+probe.Changes in mitochondrial membrane potential were detected using JC-1 dye.Protein expression levels of STUB1,solute carrier family 7 member 11(SLC7A11),and GPX4 were determined by western blot.The binding between STUB1 and GPX4 was assessed by co-immunoprecipitation.The effect of STUB1 on GPX4 protein ubiquitination was detected using a ubiquitin antibody.HCT116 cells transfected with different plasmids were co-cultured with human peripheral blood CD8+T cells,and the killing ability of CD8+T cells against HCT116 cells was measured using a lactate dehydrogenase(LDH)kit.Perforin,granzyme,and interferon-γ levels in the co-culture supernatant were determined by ELISA.Results Compared with the control group,the pcDNA3.1-STUB1 group showed decreased cell proliferation ability,mitochondrial membrane potential,and protein expression levels of SLC7A11 and GPX4,along with increased STUB1 protein expression,MDA,Fe2+levels,and GPX4 ubiquitination in HCT116 cells.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited increased cell proliferation ability,mitochondrial membrane potential,and expression levels of SLC7A11 and GPX4,along with decreased MDA and Fe2+levels in HCT116 cells.After co-culture of HCT116 cells with CD8+T cells,the pcDNA3.1-STUB1 group showed significantly increased killing rate of CD8+T cells against HCT116 cells,as well as elevated levels of perforin,granzyme,and interferon-γ in the co-culture supernatant compared with the control group.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited decreased killing rate of CD8+T cells against HCT116 cells and reduced levels of perforin,granzyme,and interferon-γ in the co-culture supernatant.Conclusion Overexpression of STUB1 promotes GPX4 ubiquitination in colon cancer cells HCT116,induces ferroptosis,and enhances the killing effect of CD8+T cells on HCT116 cells.

Objective To investigate the effects of STIP1 homology and U-box containing protein 1(STUB1)on the ubiquitination of glutathione peroxidase 4(GPX4)and ferroptosis in colon cancer cells HCT116,as well as the impact on CD8+T cell-mediated killing of HCT116 cells.Methods HCT116 cells were divided into control group,empty plasmid transfection(pcDNA3.1-vector)group,STUB1 overexpression plasmid transfection(pcDNA3.1-STUB1)group,and co-transfection(pcDNA3.1-STUB1+pcDNA3.1-GPX4)group.Cell proliferation ability was assessed by CCK-8 assay.Clonogenic ability was determined by clone formation assay.Malondialdehyde(MDA)levels in cells were measured using an MDA kit.Intracellular ferrous ion(Fe2+)levels were detected with an Fe2+probe.Changes in mitochondrial membrane potential were detected using JC-1 dye.Protein expression levels of STUB1,solute carrier family 7 member 11(SLC7A11),and GPX4 were determined by western blot.The binding between STUB1 and GPX4 was assessed by co-immunoprecipitation.The effect of STUB1 on GPX4 protein ubiquitination was detected using a ubiquitin antibody.HCT116 cells transfected with different plasmids were co-cultured with human peripheral blood CD8+T cells,and the killing ability of CD8+T cells against HCT116 cells was measured using a lactate dehydrogenase(LDH)kit.Perforin,granzyme,and interferon-γ levels in the co-culture supernatant were determined by ELISA.Results Compared with the control group,the pcDNA3.1-STUB1 group showed decreased cell proliferation ability,mitochondrial membrane potential,and protein expression levels of SLC7A11 and GPX4,along with increased STUB1 protein expression,MDA,Fe2+levels,and GPX4 ubiquitination in HCT116 cells.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited increased cell proliferation ability,mitochondrial membrane potential,and expression levels of SLC7A11 and GPX4,along with decreased MDA and Fe2+levels in HCT116 cells.After co-culture of HCT116 cells with CD8+T cells,the pcDNA3.1-STUB1 group showed significantly increased killing rate of CD8+T cells against HCT116 cells,as well as elevated levels of perforin,granzyme,and interferon-γ in the co-culture supernatant compared with the control group.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited decreased killing rate of CD8+T cells against HCT116 cells and reduced levels of perforin,granzyme,and interferon-γ in the co-culture supernatant.Conclusion Overexpression of STUB1 promotes GPX4 ubiquitination in colon cancer cells HCT116,induces ferroptosis,and enhances the killing effect of CD8+T cells on HCT116 cells.