PERK/ATF3-Reduced ER Stress On High Potassium Environment In The Suppression Of Tumor Ferroptosis

Objective: The objective of this study is to explore the effect of tumor high-potassium microenvironment on ferroptosis and elucidate the possible molecular mechanism.Providong potential targets for tumor microenvironment therapy.Methods: A high-potassium medium was constructed first,and an isotonic medium with the same concentration was configured to exclude the effect of mere addition of potassium chloride on the change of osmotic pressure.Different concentrations of high potassium medium combined with ferroptosis inducers(Erastin;RSL3)was the experimental group,normal potassium concentration medium combined with iron death inducer was the control group;CCK-8 assay,cell cloning assay and Edu proliferation assay were used to detect the effect of high concentration potassium on ferroptosis of tumor cells.RNA sequencing was used to screen the potential pathway of iron death resistance caused by high potassium;The relative contents of GSH and MDA in cells treated with high potassium and GSK2606414 were detected by full-wavelength microplate.The relative levels of ROS and calcium ions in tumor cells were detected by flow cytometry.The protein expressions of ATF3,PERK,IRE1 and ATF6α were analyzed by WB.The ATF3 knockout plasmid was constructed to detect the effect of ATF3 on ferroptosis.Cells pretreated with high potassium for one week were implanted subcutaneously in mice to verify the effect of high potassium on ferroptosis in vivo.The effect of high concentration potassium on the expression of ATF3 and ferroptosis index COX2 was verified by immunohistochemistry.Results:1.CCK-8 results showed that high potassium significantly reversed ferroptosis caused by Erastin,the effect was greatest when potassium concentration reached 35 m M(P<0.001);but high potassium cannot rescue ferroptosis by RSL3.2.The results of cell cloning and Edu proliferation experiments showed that the cells in the high potassium treatment group were still proliferative after Erastin induction.3.RNA sequencing revealed that the endoplasmic reticulum related genes were significantly up-regulated after Erastin treatment,while the endoplasmic reticulum was not significantly up-regulated in the high potassium combined with Erastin treatment group,suggesting that high potassium may affect ferroptosis through the endoplasmic reticulum.4.Using different ER inhibitors combined with Erastin treatment,it was found that inhibition of ER PERK related pathways could significantly reverse Erastin induced ferroptosis.5.RNASequence results analyzed that high potassium may affect ferroptosis through the endoplasmic reticulum associated protein ATF3.Western blot results showed that intracellular ATF3 levels were significantly increased with Erastin and significantly decreased with high potassium concentration.6.Knockout of ATF3 significantly improved cell sensitivity to ferroptosis(P<0.001),and overexpression of ATF3 promoted the ferroptosis in tumor cells.7.In vivo results showed that the tumors were larger in the high potassium pretreatment combined with Erastin group than in the Erastin group(P<0.001).Immunohistochemical results showed that the expression of COX2 and ATF3 in Erastin group was lower than that in the control group.Conclusions: High potassium concentration inhibited ferroptosis in tumor cells by inhibiting ATF3 expression...