Mechanism Of Necroptosis Of Colorectal Cancer Cells Induced By Proso Millet Peroxidase

Cell necrosis is a kind of cell death caused by chemical,physical or biological factors.It is always considered to be a passive form of death caused by injury or pathological tissue without gene regulation.In recent years,it has been found that some cell necrosis is also strictly regulated by internal molecules,such as receptor interacting protein kinase 1(RIPK1)and receptor interacting protein kinase 3(RIPK3)regulated cell death,it has the phenotype of common cell necrosis named Necroptosis.The molecular mechanism of necroptosis is significantly different from the apoptosis.When the activity of caspase,the key enzyme of apoptosis,is inhibited,the necroptosis induced by necroptosis inducer can be significantly enhanced.Induction of necroptosis in tumor cells,especially those resistant to common apoptotic drugs,is also considered as a potential therapy for cancer.In this paper,a novel protein was obtained by buffer extraction,ammonium sulfate precipitation,cation exchange chromatography and molecular exclusion chromatography.It was found that the protein is a secretory cationic peroxidase containing heme cogroups by MALDI-TOF/TOF-MS and UV scanning and named PmPOD(proso millet peroxidase,PmPOD).In order to further clarify the mechanism of tumor cell death induced by PmPOD,HT29 and HCT116 cells were used as cell models in this study.The mechanism of death induced by PmPOD and its influencing factors were studied.The cell viability was detected by MTT and ATP assay and combined with the apoptosis inhibitor z-VAD-fmk and necroptosis specific inhibitor Nec-1,we preliminarily determined the mode of PmPOD induced cell death in colorectal cancer cell line.Hoechst 33342 and PI double staining and lactate dehydrogenase release assay determine the damage effect of PmPOD on cell membrane.The intracellular localization of RIPK3-GFP andMLKL-DsRed2 after transfection of pCMV-RIPK3-GFP and pCMV-MLKL-DsRed2 plasmids was observed by laser confocal microscopy.The secretion of TNF-?,a key signal factor induced by PmPOD,and the expression of necroptosis key molecules were analyzed by ELISA and qRT-PCR techniques.Methylation specific PCR and Western Blot were used to detect the effect of PmPOD on the methylation of RIPK3 promoter region and the effect of RIPK3 expression on PmPOD induced necroptosis.We detected the changes of ROS and related antioxidant enzymes in PmPOD induced HT29 and HCT116 necroptosis.At the same time,we detected the effect of RIPK1 and RIPK3 on the production of ROS and necroptosis.The results showed that the mechanism of HCT116 and HT29 death induced by PmPOD was not apoptosis but necroptosis.The apoptosis inhibitor z-VAD could significantly enhance the cell death induced by PmPOD,while the necroptosis inhibitor Nec-1 could significantly inhibit the cell death induced by PmPOD.This mode of death is regulated by RIPK1 and RIPK3,and performed by MLKL.RIPK3-GFP and MLKL-DsRed were transferred from the cytoplasm to plasma membrane after PmPOD treatment,leading to the rupture of the cell membrane.The results of Western Blot showed that the phosphorylation level of RIPK3 and MLKL was increased significantly after the treatment of PmPOD.It was also found that PmPOD could induce the production of TNF-? through up-regulation of transcription gene.In HCT116 cells,PmPOD can restore the expression of RIPK3 in HCT116 cells by demethylation of the RIPK3 promoter region.The same,demethylation reagent 5-AD and the overexpressed RIPK3 can both enhance RIPK3 expression.The expression of RIPK3 can synergize with PmPOD to induce the necroptosis effect of HCT116 cells.In general,there are at least two mechanisms of necroptosis induced by PmPOD,autocrine production of TNF-? and restore RIPK3 expression by demethylation.At the same time,it was found that PmPOD induced the increase of reactive oxygen species(ROS),and the antioxidant NAC and BHA could attenuate cell necroptosis,indicating that ROS might be a regulator of PmPOD induced necroptosis.After PmPOD,the activity of malondialdehyde(MDA)increased,the content of superoxide dismutase(SOD)and glutathione(GSH)decreased,and these changes can be reversed by necroptosis inhibitors,indicating that PmPOD can cause oxidative damage to cells,which may be a stress response caused by the increase of ROS.These results provide an important basis and experimental basis for the in-depth study of the mechanism of PmPOD induced necroptosis of tumor cells.