The Role And Mechanism Of PTGS2/COX-2 In Ferroptosis Of Non-Small Cell Lung Carcinoma

Objectives Lung cancer is the most lethal malignant tμMor on the earth and seriously threatens hμMan lives.There is no effective treatment due to advanced stage when they were diagnosed.Ferroptosis is a new way of progress cell death that triggered with iron-dependent lipid peroxidation,tightly corelate with oncogenesis and therapeutic susceptibility.It was reported that prostaglandin-endoperoxide synthase 2（PTGS2）,encoding cyclooxygenase 2（COX-2）,took part in regulation of lipid metabolism,was overexpressed in non-small cell lung carcinoma（NSCLC）,was responsible for poor prognosis of lung cancer patients,whose inhibitor induced apoptosis by blocking the activity of PI3K/AKT signaling pathway.On the other hand,inhibited phosphatidylinositol 3-kinase/protein kinase B（PI3K/AKT）signaling pathway promoted prostate cancer cell ferroptosis.However,upregulated PTGS2 was a marker on the downstream of ferroptosis induced by（1S,3R）-RAS-selective lethal 3（RSL3）,targeting glutathione peroxidase 4（GPX4）and inducing ferroptosis,in leukemia cell BJe LR.The sensitivity of certain cancer to ferroptosis depends on their molecular genetic background.It is common to find mutant phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha（PIK3CA）and activated PI3K/AKT signaling pathway in NSCLC cells,we want to learn about the susceptibility of NSCLC cells to RSL3,and figure out the relationship among PTGS2,ferroptosis regulated gene and PI3K/AKT signaling pathway.The study may offer new mechanism of ferroptosis in NSCLC cells,and guides a new way in individual therapy.Methods First of all,established NSCLC cell ferroptosis model with various cells by classic ferroptosis inducer RSL3.Secondly,characterize morphological and biochemical change with CCK-8,Iron Assay kit,C11-BODIPY dye and transmission electron microscope.Thirdly,measure ferroptosis regulated genes expression level by RT-q PCR and western blot.Lastly,genetic（lentivirus transfection）and pharmacological（selective inhibitors）interventions were applied to alter PTGS2/COX-2 expression level in NSCLC,followed with testing of ferroptosis key related genes whose relationships were analyzed.Results（1）Successfully established NSCLC cell ferroptosis model which exhibited mitochondrial abnormalities,such as condensation or swelling,increased membrane density,reduced or absent crista,as well as rupture of the outer membrane,but no margination and chromatin;increased intracellular lipid peroxidation detected by C11-BODIPY（enhanced green fluorescent）;cell death induced by RSL3 only could be rescued with ferroptosis inhibitor Ferrostatin-1/DFO,but not apoptosis inhibitor Z-VAD-FMK,necroptosis inhibitor Necrosulfonamine,or autophagy inhibitor Chloroquine;increased concentration of intracellular Fe²⁺indexed by Iron Assay kit;expression level of GPX4,ferroptosis suppressor protein 1（FSP1）and solute carrier family 7 member 11（SLC7A11）were sharply decreased,while PTGS2,iron responsive element binding protein 2（IREB2）,transferrin receptor（TFRC）and（ACSL4）increased.The PIK3CA m RNA level was significantly changed under the treatment of RSL3.（2）Sensitivity to RSL3 and PTGS2/COX-2 expression level of NSCLC cells was devised with histologic subtypes,metastatic status and genetic variation.COX-2inhibitor Celecoxib enhanced the suppressed effect of RSL3 in NSCLC,which could be blocked with apoptosis inhibitor and ferroptosis inhibitor.（3）Celecoxib reduced COX-2 protein level in NSCLC,inhibited the expression level of phosphorylated-PI3K,phosphorylated-AKT and mechanistic target of rapamycin kinase（m TOR）,while reducing expression of FSP1 and increasing ACSL4 and IREB2 level.On the contrast,when NCI-H1299 was stably overexpressed PTGS2/COX-2,PI3K/AKT/m TOR pathway was activated,so as to FSP1,ACSL4and IREB2 were suppressed.Conclusions Both sensitivity to RSL3 and PTGS2 level was correlated with histologic subtypes,metastatic status and genetic variation.The PIK3CA m RNA level was remarkably affected by RSL3.COX-2 inhibitor induced apoptosis and ferroptosis,and enhanced the antitμMor function of RSL3.Suppressed-COX-2triggered NSCLC cells ferroptotic death by blocking the PI3K/AKT/m TOR signaling axis.