Mechanism Of Ionizing Radiation-mediated GPX4 Acetylation Modification Inducing Ferroptosis In Thyroid Cancer Cells

Thyroid cancer is the most common malignant tumor of endocrine system,its incidence increases year by year,and advanced thyroid cancer treatment effect is poor,high recurrence rate.Objective to find a new method to improve the curative effect of thyroid cancer,especially for clinically drug-resistant thyroid cancer,due to limited surgical treatment.Radiation therapy is one of the most effective cancer treatments that can eradicate cancer cells by targeting the delivery of ionizing radiation(IR).However,most cancer cells will show radioresistance phenomenon,so it is an urgent scientific problem to improve the sensitivity of cancer cells to radiotherapy combined with other treatments.Ferroptosis is an atypical form of cell death,which is characterized by large accumulation of lipid peroxides and dysregulation of the antioxidant mechanism dependent on glutathione in cells,and is closely related to the occurrence and development of tumors.Glutathione peroxidase 4(GPX4)is an antioxidant enzyme capable of hydrolyzing lipid peroxides accumulated in cells,scavenging oxygen free radicals and negatively regulating iron death.In theory,down-regulation of GPX4 expression levels or enzyme activity in tumor cells can promote cell ferroptosis,so the combination of ionizing radiation and ferroptosis pathways may be useful in the treatment of radio-resistant cancers,including thyroid cancer.Acetylation is a widespread posttranslational modification(PTM)in cells.It is one of the important epigenetic regulatory mechanisms involved in various biological processes and signal transduction in cells.In recent years,the role of PTM in iron death has attracted much attention.Some studies have found that deubiquitination enzyme inhibitors can promote the degradation of GPX4 and trigger the process of iron death.However,how the acetylation modification of GPX4 affects the pathway of iron death has not been reported.In our preliminary study,we found that IR treatment increased GPX4 acetylation levels in thyroid cancer cells.Therefore,in this paper,it is intended to determine whether IR can induce ferroptosis of thyroid cancer cells by mediating GPX4 acetylation,so as to provide a new therapeutic strategy for thyroid cancer.Therefore,in this study,TPC1,K1 thyroid cancer cell lines and HEK293 T were used as cell models.After the cells were treated with IR irradiation and iron death regulator,the correlation between IR and ferroptosis was determined by detecting relevant indicators of iron death.Then,by detecting the effect of GPX4’s acetylation level on its protein stability,we can determine whether IR mediates GPX4’s acetylation to induce ferroptosis,and further clarify the interaction relationship and molecular mechanism between IR,iron death and GPX4’s acetylation.The results of this study will provide a new theoretical basis for the tumor targeting intervention strategy of IR combined with key factors related to the ferroptosis pathway.The work of this paper is summarized as follows:First,the death mode of thyroid cancer cells induced by IR irradiation was identified: different doses of IR were used in combination with different cell death inhibitors,and through the detection of cell viability,the death mode of thyroid cancer cells induced mainly by ferroptosis accompanied by a certain degree of apoptosis was determined.Second,biological experiments confirmed that IR induced ferroptosis and down-regulated GPX4 protein levels: By detecting ROS/MDA/GSH/CCK-8 and protein expression levels,it was confirmed that IR could induce ferroptosis in TPC1 and K1 of thyroid cancer cells,inhibit cell proliferation,down-regulate GPX4 and XCT and up-regulate the expression level of ACSL4 protein.Thirdly,the identification of iron-death inducers targeting GPX4 to enhance the sensitivity of thyroid cancer cells to radiotherapy: after treating thyroid cancer cells with Erastin or RSL3,the targeting of GPX4 was determined by Western Blot and CCK-8 detection.Fourth,GPX4 was identified as the key factor of IR induced cell death: The GPX4 plasmid was overexpressed in thyroid cancer cells,and the key factor of GPX4’s involvement in IR induced death of thyroid cancer cells was determined by detecting the expression levels of MDA,GSH,CCK-8 and protein.Fifth,GPX4 acetylation was down-regulated by IR and ferroptosis inducers:293T cells were transfected with Flag-GPX4,and GPX4 could be acetylated by Flag-IP and Acetyl-IP experiments.Subsequently,the cells were treated with IR and ferroptosis inducer,and immunoprecipitation combined with Western Blot analysis showed that IR and Erastin could down-regulate GPX4 acetylation level.Sixth,it was confirmed that acetylation of GPX4K148 was related to the death pathway of IR and ferroptosis: Using Phosphositeplus to predict the potential acetylation sites of GPX4(K47R,K148 R,K162R),construct a point mutant plasmid,verify the successful construction of the plasmid,and conduct Acetyl-IP and Flag-IP.The results indicate that K148 is the main acetylation modification site of GPX4.In conclusion,we speculate that IR can induce ferroptosis in thyroid cancer cells by mediating acetylation of GPX4 at K148.The experimental results obtained in this paper will provide a new perspective for further research on potential targets in the treatment of diseases related to ferroptosis.