Mechanisms Of Radiation-Induced Ferroptosis In Pancreatic Cancer Cells And Phenotypic Conversion Of Cancer-Associated Fibroblasts

Objective:Radiotherapy(RT)refers to the first-line treatment for tumors,however,radiotherapy resistance often exists during treatment.Previous studies have often focused on the tumor cells themselves,while neglecting the effect of the tumor microenvironment.Pancreatic cancer,that with highly interstitial fibrosis,has a tumor microenvironment that plays an important role in treatment resistance.The aim of this study was to investigate the role of radiotherapy on ferroptosis of tumor cells and the effect of radiotherapy on cancer-associated fibroblasts(CAFs),that will provide new directions for pancreatic cancers.Methods:(1)CCK8 and colony-forming assays were used to detect cell death in PANC-1 cells under radiotherapy conditions and a group with ferroptosis rescue agents ferrostatin-1(Fer-1)to observe cell salvage after radiotherapy.Western blot was used to measure the expression of ferroptosis protein GPX4 and SLC7A11.Glutathione,malondialdehyde(MDA)and Lipid ROS were detected on intracellular levels.(2)ELISA was used to measure glutamine and glutamate levels in PANC-1 cell supernatants after radiotherapy.Western blot was used to measure PANC-1intracellular expression of GLS1,a protein indicator of glutamine metabolism,after radiotherapy.(3)Western blot was used to measure HMGB1 expression both in PANC-1 cells and cell supernatants after radiotherapy.Immunofluorescence was used to observe localization of HMGB1 in PANC-1 cells after radiotherapy.Database was used to analyze expression and prognosis of HMGB1 in pancreatic cancer.(4)Western blot and flow cytometry was used to measure the expression of α-SMA and lipid droplet content in pancreatic stellate cells under radiotherapy and non-radiotherapy treatment conditions.qRT-PCR was used to measure the mRNA expression of markers of phenotype of cancer-associated fibroblasts after radiotherapy.Database was used to analyze the correlation between HMGB1 and markers of cancer-associated fibroblasts.Results:(1)Fer-1 can partially rescued the cell death induced by radiotherapy,and ferroptosis related proteins GPX4 and SLC7A11 decreased after radiotherapy,the intracellular levels of GSH decreased,and the levels of MDA and Lipid ROS increased,indicating that radiotherapy induced ferroptosis in tumor cells.(2)Levels of intracellular GLS1 protein in PANC-1 cells and levels of glutamine and glutamate in the cell supernatants were increased after radiotherapy,indicating that radiotherapy enhanced glutamine metabolism in tumor cells and increased cellular release of glutamine and glutamate.(3)The level of HMGB1 protein decreased in PANC-1 cells after radiotherapy,while the level of HMGB1 protein increased in cell supernatant.HMGB1 was translocated from the nucleus to the cytoplasm after radiotherapy,indicating the release of intracellular HMGB1 to extracellular cells after radiotherapy.(4)α-SMA in pancreatic stellate cells increased after co-culture with PANC-1 cells and the lipid droplet levels decreased after radiotherapy,indicating that radiotherapy promoted the activation of pancreatic stellate cells after co-culture with PANC-1 cells.(5)PANC-1 cells after radiotherapy promoted the transformation of CAFs to iCAFs,while intracellular HMGB1 protein levels were elevated.Exogenous HMGB1 promotes the transformation of CAFs to iCAFs for a short period of time.Conclusions:On the one hand,radiotherapy induce ferroptosis in tumor cells,on the other hand,radiotherapy promotes the release of HMGB1 from tumor cells,leading to the phenotypic conversion of CAFs in the tumor microenvironment and generating treatment resistance.