Study On The Mechanism Of Vitamin C Sensitizing Erastin-induced Ferroptosis In Pancreatic Cancer Cells

Background:Ferroptosis is an emerging form of regulated cell death,and studies have shown that the ferroptosis activator erastin can selectively trigger cell death in cancer cells containing mutant RAS.This has raised new hope for the treatment of drug-resistant tumours.However,some pancreatic cancer cells are less sensitive to erastin-induced iron death,while normal pancreatic cells are also susceptible to this ferroptosis.Therefore,there is an urgent need to find drugs that enhance the sensitivity of erastin-insensitive cells to erastin,while limiting its side effects on normal cells.In recent years,attention has been drawn to the role of vitamin C in the regulation of iron death,and there are many intersections between its role as an antioxidant in vivo and erastin-induced ferroptosis.Therefore,in this paper,we will investigate the relationship between vitamin C,erastin,ferroptosis and pancreatic cancer,with the aim of providing a new reference for the treatment of pancreatic cancer.Aims:We used molecular bioassays（in vivo,in vitro）and bioinformatics to analyse the effects of vitamin C and erastin alone or in combination on pancreatic cancer cells and normal cells.And to investigate the potential molecular biological mechanism of vitamin C in erastin-induced iron death in pancreatic cancer cells through relevant experiments,with a view to providing new ideas for reference in the treatment of pancreatic cancerMethods:1.The effect of vitamin C on the proliferation of pancreatic cancer cells as well as normal cells of the pancreas was analysed by the CCK8 method.The types of vitamin C-mediated cell death in pancreatic cancer cells were explored by adding different cell death inhibitors.Cell death related indicators were determined by GSH and ROS assay kits.2.To investigate the expression and survival curve of GLUT-1 in pancreatic cancer by bioinformatics methods and ROC curve to verify the efficiency.Interfering plasmid sh-GLUT1was transfected into pancreatic cancer cell lines and cell viability of vitamin C treated pancreatic cancer cells was measured by CCK-8.Cell viability of vitamin C-treated pancreatic cancer cells was assayed by CCK-8 using GLUT1 inhibitor（STF31）.3.Cell viability of different types of pancreatic cancer cells after treatment with different concentrations of erastin was assessed by CCK-8 assay.The effect of vitamin C or erastin alone,or in combination with both,on the proliferation of pancreatic cancer cells was measured by the CCK-8 assay.The effect of vitamin C or erastin alone,or in combination,on the proliferation of pancreatic cancer cells was measured using the relevant kits and flow cytometry,including GSH,MDA and lipid ROS levels.4.After treatment of pancreatic normal cells H6C7 and mouse embryonic fibroblasts MEF with different concentrations of erastin,cell viability was measured by CCK-8 assay.After treatment with vitamin C or erastin alone,or in combination with both,cell viability was measured by the CCK-8 method,and changes in cell morphology were observed under microscopy.The levels of ferroptosis in pancreatic normal and embryonic fibroblasts,including GSH,MDA and lipid ROS levels,were measured using the relevant kits and flow cytometry after administration of vitamin C or erastin alone or in combination with both.5.To investigate the effect of vitamin C or erastin alone,or in combination,on subcutaneous pancreatic tumours in mice by means of subcutaneous tumourigenesis experiments.H&E staining of the heart,liver,spleen,lungs and kidneys of mice was used to observe drug toxicity.6.The levels of ferrous ions in pancreatic cancer cell lines were determined using the Iron Content Assay Kit and the fluorescent imaging probe Bio Tracker 575 Red Fe²⁺Dye after the administration of vitamin C or erastin alone,or in combination with both.7.differential analysis of genetic differences and functional enrichment analysis of pancreatic cancer cells after combined treatment with erastin and vitamin C by RNA transcriptome sequencing.8.Validation of RNA sequencing results and putative associated genes and proteins by q PCR and Western blot.The entry and exit of target genes into and out of the nucleus was directly verified by nuclear plasma separation assay under electron microscopy.Results:1.Vitamin C induced tumour cell death in a dose-dependent manner in pancreatic cancer cells Pa Tu8988,Bx PC3 and PANC1 cells.The cytotoxicity to pancreatic cancer cells increased with increasing dose of vitamin C administration,but had little effect on normal H6C7 and MEF cells.2.Vitamin C-induced cell death was significantly rescued by the ferroptosis inhibitor DFO,but not by the apoptosis inhibitor ZVAD and the programmed cell necrosis inhibitor Nec-1,suggesting that the type of vitamin C-induced cell death in pancreatic cancer cells was iron death.3.Vitamin C significantly reduced GSH levels in pancreatic cancer cells Pa Tu8988 and Bx PC3cells;the addition of iron death inhibitor（DFO）rescued the vitamin C-induced decrease in GSH levels.Vitamin C increased the production of cytosolic lipid ROS in pancreatic cancer cells Pa Tu8988 and Bx PC3,but not in human normal ductal epithelial cell line H6C7 and mouse embryonic fibroblasts MEF.4.Bioinformatic analysis showed that GLUT1 was highly expressed in pancreatic cancer tumour specimens compared to normal tissue and that high GLUT1 expression was associated with a poorer prognosis of the tumour,with a 5-year AUC value of 0.735 for ROC diagnostic efficiency.5.STF31（GLUT1 inhibitor）significantly attenuated the decline in cell death induced by vitamin C in pancreatic cancer cells Pa Tu8988 and Bx PC3.In addition,analysis of CCK-8results showed that the killing effect of vitamin C on pancreatic cancer cells was reduced in GLUT1-interfered pancreatic cancer cells,i.e.vitamin C-induced cell death was somewhat inhibited.6.After different concentrations of erastin treated different types of pancreatic cancer cell lines,CCK-8 results showed that PANC1 was more sensitive to erastin,while Pa Tu8988 and Bx PC3were insensitive to erastin-induced ferroptosis7.In pancreatic cancer cells treated with vitamin C or erastin alone,or in combination with both,CCK-8 results showed that treatment with either erastin or vitamin C alone moderately inhibited the viability of pancreatic cancer cells,whereas the combination of erastin and vitamin C significantly reduced the proliferation of pancreatic cancer cells.8.In pancreatic cancer cells,the addition of erastin or vitamin C alone decreased GSH levels and increased MDA and lipid ROS levels;the combination of erastin and vitamin C resulted in a more pronounced decrease in GSH and a significant increase in MDA and lipid ROS levels than erastin and vitamin C alone.9.The CCK-8 results showed that erastin inhibited the growth of pancreatic normal cells H6C7and mouse embryonic fibroblasts MEF in a dose-dependent manner,suggesting that high doses of erastin may be cytotoxic to normal cells.10.CCK-8 results showed that vitamin C rescued the iron death induced by erastin in pancreatic normal cells H6C7 and mouse embryonic fibroblasts MEF.The combination of erastin and vitamin C resulted in higher GSH elevation and lower MDA and lipid ROS production when compared to the group treated with erastin alone.These results indirectly suggest that vitamin C protects H6C7 and MEF cells from erastin-induced ferroptosis.11.The results of the subcutaneous tumourigenesis assay in mice showed that the weight of subcutaneous tumours in the erastin and vitamin C combination treatment group was much smaller than that in the erastin or vitamin C alone treatment group.erastin and vitamin C combination significantly inhibited tumour growth and there was no significant difference in body weight between the groups.In addition,erastin and vitamin C treatment resulted in a decrease in GSH,as well as an increase in MDA and ferrous iron levels.Analysis of the results of H&E staining showed that erastin and vitamin C did not damage the heart,liver,spleen,lungs and kidneys at the given doses.12.Using both the Bio Tracker 575 Red Fe²⁺Dye method,a selective fluorescent imaging probe,and the Iron Content Assay Kit to detect ferrous iron levels,we found that compared to erastin or vitamin C treatment alone,the levels of variable iron pool（LIP）in Patu8988 and Bx PC3cells treated with the combination of erastin and vitamin C.13.RNA sequencing results showed that in pancreatic cancer Bx PC,288 genes were upregulated and 644 genes were downregulated in the vitamin C and erastin combination group,compared to the DMSO-treated control group.Among the differentially expressed genes enriched in the ferroptosis pathway mainly included HMOX1,CP and FTH1.14.m RNA levels of HMOX1 and FTH1 were significantly increased in Pa Tu8988 and Bx PC3cells after co-treatment with erastin and vitamin C by q PCR assays,whereas the expression of CP was decreased.Western blot experiments showed that co-treatment with erastin and vitamin C increased the expression of HMOX1,FTH1 NCOA4 and p-AMPK levels,but had no effect on CP.In addition we also examined the expression of iron death related proteins by Western blot experiments and showed that the protein of SLC7A11 and GPX4 was significantly reduced in the erastin and vitamin C combination group compared to the control group.15.Combining nuclear/cytoplasmic fractionation,immunofluorensece and Western blot experiments,we concluded that combination of erastin and vitamin C could activate AMPK/NRF2/HMOX1 signaling pathway in pancreatic cancer cells.Conclusions:Vitamin C sensitizes erastin-induced ferroptosis in pancreatic cancer cells and significantly reduces its cytotoxicity to normal cells.In particular,the combined treatment of erastin and vitamin C synergistically induced iron death,a process involving GSH depletion and the accumulation of divalent iron.In conclusion,the combination of erastin and vitamin C provides a new reference strategy for the treatment of pancreatic cancer.