Molecular Mechanism Of Lactate Regulating Ferroptosis Through GPX4 To Promote Etoposide Resistance In NSCLC Cells

Objective: Etoposide is sensitive to different kinds of lung cancer,that is,it is sensitive to small cell lung cancer,but insensitive to non-small cell lung cancer.Iron death,characterized by lipid peroxidation,is a new type of programmed death discovered in recent years.Etoposide promotes the metabolic reprogramming of non-small cell lung cancer(NSCLC)cells to produce lactic acid,which is involved in a variety of biological functions of cancer.The relationship between lactic acid and ferroptosis and whether etoposide regulates ferroptosis through lactic acid is still unclear.Therefore,this paper mainly explores the relationship between the metabolism remodeling of lactic acid in NSCLC cells and ferroptosis,as well as the role and molecular mechanism of etoposide resistance.Materials and Methods: Human lung cancer cells H1299,A549,H460 and H1688 were selected as subjects.(1)MTT assay was used to detect the sensitivity of different lung cancer cell lines to etoposide.(2)MTT assay was used to investigate whether ferroptosis inhibitors could inhibit the lethality of etoposide in different lung cancer cell lines;(3)The intracellular iron concentration and lipid reactive oxygen level of different lung cancer cells treated with etoposide were detected by iron ion kit and lipid reactive oxygen probe.(4)The effect of etoposide on the expression of GPX4 protein was investigated by WB.(5)MTT assay was used to detect whether manipulation of GPX4 expression affected the sensitivity of etoposide;(6)Whether etoposide regulates the expression of GPX4 at the transcriptional level was investigated by q PCR and dual luciferase reporter gene technology;(7)IP test and overexpression combined knockdown test were used to detect the role of NEDD4 L in regulating GPX4 expression;(8)WB test and knock down test were used to detect whether lactic acid was involved in mediating the expression of GPX4;(9)To investigate whether lactic acid regulates GPX4 at the transcriptional level by q PCR and dual luciferase reporter gene technology;(10)WB technique was used to explore the pathway through which lactic acid regulates GPX4 expression.Results:(1)MTT results showed that the sensitivity of etoposide to SCLC was higher than that to NSCLC;(2)MTT results showed that ferroptosis inhibitors could alleviate etoposide-induced cell death in SCLC cells.(3)Iron ion detection and ROS probe detection showed that etoposide up-regulated iron ion content and lipid ROS level in SCLC cells;Downregulation of iron ion content in NSCLC cells had no effect on the level of lipid reactive oxygen species.(4)WB experiment proved that etoposide could up-regulate the expression of GPX4 protein in NSCLC cells and down-regulate the expression of GPX4 protein in SCLC cells;(5)MTT assay showed that GPX4 expression could affect the sensitivity of etoposide;(6)q PCR and dual luciferase reporter gene techniques showed that etoposide had no effect on GPX4 transcription level;(7)IP and WB test results showed that etoposide regulates GPX4 expression through SGK1-NEDD4 L pathway;(8)WB experiment showed that etoposide promoted the metabolic reprogramming of NSCLC cells to produce lactic acid,which was involved in the regulation of GPX4;(9)q PCR and dual luciferase reporter gene techniques proved that lactic acid had no effect on the transcription level of GPX4;(10)WB technique proved that lactic acid regulates the expression of GPX4 through the p38-SGK1-NEDD4 L pathway.Conclusion:(1)Etoposide can not only promote the apoptosis of small cell lung cancer cells,but also induce the ferroptosis,but only induce the apoptosis of non-small cell lung cancer cells.(2)Lactic acid induced ferroptosis resistance of NSCLC cells by upregulating GPX4 and made them resistant to etoposide.(3)Etoposide regulates the p38-SGK1-NEDD4 L pathway through lactic acid to up-regulate the expression of GPX4.