The Role And Mechanism Of HO-1 In Erastin Induced Ferroptosis And Autophagy In Colorectal Cancer

Background:Colorectal cancer,one of the most common cancers of the digestive system,is the leading cause of cancer death worldwide.At present,colorectal cancer is mainly treated by traditional therapies,such as surgical resection,chemotherapy,radiotherapy,and so on.Ferroptosis is a newly discovered form of cell death in recent years,mainly characterized by intracellular mitochondrial shrinkage,lipid peroxide accumulation,reduced glutathione content,increased reactive oxygen species,and reduced glutathione peroxidase 4 expressions.As a ferroptosis inducer,Erastin has been shown to induce cell death in a wide range of tumor cells,but the specific mechanism still needs further investigation.In addition,we speculate that Erastin may also induce other cell death modes.Recent studies have reported that ferroptosis is closely related to autophagy,and the occurrence of ferroptosis is often accompanied by the occurrence of autophagy.Heme oxygenase 1(HO-1)is a rate-limiting enzyme in the catabolism of heme.It has been reported that HO-1 can inhibit the proliferation of tumor cells,but its specific mechanism remains unclear.In this study,we detected the expression of HO-1 in colorectal cancer cells after Erastin induction and used Hemin exogenous supplement of HO-1 to further explore the specific mechanism of HO-1 in Erastin-induced ferroptosis and autophagy.Objective:To determine whether Erastin can induce ferroptosis and autophagy in colorectal cancer cells;To explore the role and mechanism of HO-1 in Erastin-induced ferroptosis and autophagy in colorectal cancer cells.To explore the relationship between Erastin induced ferroptosis and autophagy in colorectal cancer cells.To determine whether Hemin can induce ferroptosis and autophagy in colorectal cancer cells,providing a new idea for the treatment of colorectal cancer.Methods:In this study,colorectal cancer cell lines HT29 and HCT116 were selected for related experiments.1.The morphological changes of mitochondria after Erastin treatment were observed by transmission electron microscopy;After Erastin and Erastin combined with ferroptosis inhibitor Ferrostatin 1(Fer-1)treatment,CCK8 assay was used to detect the cell viability;Western Blot was used to detect the expression of GPX4;MDA and GSH kits were used to detect intracellular MDA and GSH content;flow cytometry was used to detect intracellular ROS changes.2.After Erastin and Erastin combined with Fer-1 treatment,the mRNA and protein expression of HO-1 were detected by q RT-PCR and Western Blot.After Erastin and Erastin combined with Hemin treatment,CCK8 assay was used to detect the cell viability;q RT-PCR was used to detect HO-1 mRNA expression;Western Blot was used to detect HO-1 and GPX4 protein expression;MDA and GSH kits were used to detect intracellular MDA and GSH content.3.The autophagosomes were observed by transmission electron microscopy after Erastin treatment.After Erastin and Erastin combined with the autophagy inhibitor3-methyladenine(3-MA)treatment,CCK8 assay was used to detect the cell viability;the mRNA and protein expression of ATG5 and LC3B were detected by q RT-PCR and Western Blot.4.After Erastin and Erastin combined with 3-MA treatment,the mRNA and protein expression of HO-1 were detected by q RT-PCR and Western Blot.After Erastin and Erastin combined with Hemin treatment,the mRNA and protein expression of HO-1,ATG5 and LC3B were detected by q RT-PCR and Western Blot.5.After Erastin,Erastin combined with Fer-1 and Erastin combined with 3-MA treatment,SLC7A11 protein expression was detected by Western Blot.After Erastin and Erastin combined with Hemin treatment,SLC7A11 protein expression was detected by Western Blot.6.After Erastin and Erastin combined with Hemin treatment,GSH kits were used to detect intracellular GSH content.After Erastin combined with GSH and Erastin combined with Hemin and GSH treatment,SLC7A11 protein expression was detected by Western Blot;GSH kits were used to detect intracellular GSH content.7.After Erastin combined with GSH and Erastin combined with Hemin and GSH treatment,CCK8 assay was used to detect the cell viability;GPX4,ATG5 and LC3B protein expression was detected by Western Blot.8.After Erastin and Erastin combined with Fer-1 treatment,the mRNA and protein expressions of ATG5 and LC3B were detected by q RT-PCR and Western Blot.After Erastin and Erastin combined with 3-MA treatment,Western Blot was used to detect the expression of GPX4;MDA and GSH kits were used to detect intracellular MDA and GSH content.9.After Hemin and Hemin combined with Fer-1 treatment,CCK8 assay was used to detect the cell viability;Western Blot was used to detect the expression of GPX4;MDA and GSH kits were used to detect intracellular MDA and GSH content.After Hemin and Hemin combined with 3-MA treatment,CCK8 assay was used to detect the cell viability;Western Blot was used to detect the expression of ATG5 and LC3B.Results:1.After Erastin treatment,mitochondrial shrinkage and mitochondrial crest reduction were observed by transmission electron microscopy.After Erastin treatment,the cell viability decreased,the expression of GPX4 protein decreased,the content of MDA increased,the content of GSH decreased and the level of ROS increased.After Erastin and Fer-1 treatment,cell viability was increased,GPX4 protein expression was increased,MDA content was decreased,GSH content was increased and ROS level was decreased compared with Erastin treatment group.2.The mRNA and protein expression of HO-1 increased after Erastin treatment compared with the untreated group.The mRNA and protein expression of HO-1 were decreased after Erastin and Fer-1 treatment compared with Erastin treatment group.After Erastin and Hemin treatment,cell viability decreased,HO-1 mRNA and protein expression increased,MDA content increased and GSH content decreased compared with Erastin treatment group.3.Autophagosomes were observed by transmission electron microscopy after Erastin treatment.After Erastin treatment,the cell viability decreased and the mRNA and protein expression of ATG5 and LC3B increased compared with the untreated group.After Erastin and 3-MA treatment,the cell viability was increased,and the mRNA and protein expression of ATG5 and LC3B were decreased compared with Erastin treatment group.4.The mRNA and protein expression of HO-1 increased after Erastin treatment compared with the untreated group.The mRNA and protein expression of HO-1 were decreased after Erastin and 3-MA treatment compared with Erastin treatment group.The mRNA and protein expressions of HO-1,ATG5 and LC3B were increased after Erastin and Hemin treatment compared with the Erastin treatment group.5.The protein expression of SLC7A11 was increased after Erastin treatment compared with the untreated group.The protein expression of SLC7A11 decreased after Erastin combined with Fer-1 or 3-MA treatment,respectively compared with the Erastin treatment group.The protein expression of SLC7A11 increased after Erastin and Hemin treatment compared with Erastin treatment group.6.The GSH content of Erastin and Hemin treatment group was reduced compared with the Erastin treatment group.After Erastin and GSH treatment,SLC7A11 protein expression decreased and GSH content increased compared with Erastin treatment group.After Erastin,Hemin and GSH treatment,SLC7A11 protein expression decreased and GSH content increased compared with Erastin and Hemin treatment group.7.After Erastin and GSH treatment,the cell viability increased,the protein expression of GPX4 increased and the protein expression of ATG5 and LC3B decreased compared with Erastin treatment group.After Erastin,Hemin and GSH treatment,the cell viability increased,the protein expression of GPX4 increased and the protein expression of ATG5 and LC3B decreased compared with Erastin and GSH treatment group.8.The mRNA and protein expression of ATG5 and LC3B were decreased after Erastin and Fer-1 treatment compared with Erastin treatment group.After Erastin and3-MA treatment,GPX4 protein expression was increased,MDA content was decreased and GSH content was increased compared with Erastin treatment group.9.Compared with the untreated group,the cell viability decreased,the protein expression of GPX4 decreased,the protein expression of ATG5 and LC3B increased,the content of MDA increased,and the content of GSH decreased compared with Hemin treatment group.After Hemin and Fer-1 treatment,cell viability was increased,GPX4protein expression was increased,MDA content was decreased,GSH content was increased compared with Erastin treatment group.After Erastin and 3-MA treatment,the cell viability was increased,and the protein expression of ATG5 and LC3B were decreased compared with Erastin treatment group.Conclusions:1.Erastin induced ferroptosis in colorectal cancer cells.2.HO-1 promotes Erastin-induced ferroptosis in colorectal cancer cells.3.Erastin can induce autophagy in colorectal cancer cells.4.HO-1 promotes Erastin-induced autophagy in colorectal cancer cells.5.HO-1 promoted Erastin-induced ferroptosis and autophagy in colorectal cancer cells by inhibiting the function of system XC~-,and Erastin-induced ferroptosis and autophagy were mutually promoted.6.Hemin can induce ferroptosis and autophagy in colorectal cancer cells.