Metabolic Characterization Of Ferroptosis Induced By (1S,3R) RAS-selective Lethal 3 In HT-1080 Fibrosarcoma Cells

ObjectiveFerroptosis is a new type of iron-dependent cell death,which is related to ischemia-reperfusion injury,cancer,neurodegenerative diseases and other diseases.Targeting ferroptosis can treat iron overload-related diseases.In this study,ferroptosis was induced by(1S,3R)RAS-selective lethal 3(RSL3)in HT-1080 cells,and ferrostatin-1(Fer-1)was used to inhibite the cell death,to explore the effects of RSL3 and Fer-1 on HT-1080 cell viability and cell morphology.Using untargeted metabolomics based on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF MS)to explore the metabolic characteristics and metabolic markers related to ferroptosis,provide a metabolomics basis for research on RSL3-induced ferroptosis.MethodsTaking the human fibrosarcoma cells(HT-1080 cells)as the research object,use RSL3 and Fer-1 to incubte cells at different time gradients(3,6,12h).The experiment was divided into three groups: ferroptosis induction group(RSL3 group),ferroptosis suppression group(RSL3+Fer-1 group),solvent control group(DMSO group).Different concentrations(0,0.125,0.25,0.5,1,2 ?M)of RSL3 suspension were used to stimulate HT-1080 cells,and 0.5 and 1?M Fer-1 was used to inhibit the cell death.CCK-8 kit was used to detect the cells activity in each group.Reduced glutathione(GSH)kit was used to determine the level of GSH produced in each group of cells at different times.The optical microscope was used to observe the changes in cell morphology under the action of RSL3 and Fer-1 at different times.The intracellular fluid and extracellular fluid were collected for metabolomics analysis based on UPLC-QTOF MS.Metabolomics data was analyzed using Metaboanalyst 5.0 online software.Principal component analysis,t test,partial least square discriminant analysis,volcano map and heat map analysis were performed by Metaboanalyst 5.0 statistical analysis module;and according to the P value,the fold change,and the projection value of important variables,the differential metabolites between groups were selected.The functional analysis(MS peak to Pathways)module of Metabo Analyst5.0 was used for metabolic pathway analysis.Results1.The effect of RSL3 and Fer-1 on the activity of HT-1080 cells1u M RSL3 acts on HT-1080 cells,the cell survival rates at 3,6,12 h were89.64%,68.05%,30.39%,respectively.At the same time adding 0.5u M Fer-1,the cell survival rate was 100.37%,85.35%,71.08%,respectively.2.The effect of RSL3 and Fer-1 on the level of GSH in HT-1080 cellsIn the DMSO control group,RSL3 group,RSL3+Fer-1 group,the intracellular GSH levels at 3,6,12 h were not statistically significant(P>0.05).3.The effect of RSL3 and Fer-1 on HT-1080 cell morphologyUnder an inverted optical microscope,it can be observed that RSL3 incubation for 12 h can cause the visible cell death,addition of Fer-1 can significantly inhibit the cell death,but the changes in the internal structure of the cell cannot be observed.At3,6h,there were no obvious cell death,but significant changes have occurred in the intracellular and extracellular metabolites.4.UPLC-QTOF MS analysis resultsThe quality control samples in the principal component analysis graph were clustered together,indicating that the method was reproducible and the model was stable.The orthogonal partial least squares discriminant analysis model was well constructed in each model at different times,the cluster of RSL3 group vs DMSO group,RSL3+Fer-1 group vs RSL3 group were separated obviously,indicating that there were metabolic differences between the groups.The volcano map showed the overall change trend of metabolites,and the heat map showed the change in the content of different metabolites.Differential metabolites: In positive ion mode of the intracellular fluid,RSL3 group vs DMSO group,a total of 152 differential metabolites were identified,of which 135 metabolites were down-regulated and 17 metabolites were up-regulated;in RSL3+Fer-1 group vs RSL3 group,there were 64 different metabolites consistent with the results of RSL3 group vs DMSO group.In negative ion mode of the intracellular fluid,a total of 87 differential metabolites were identified in RSL3 group vs DMSO group,of which 70 metabolites were down-regulated and 17 metabolites were up-regulated;In RSL3+Fer-1 group vs RSL3 group,70 metabolites were also meaningful in the RSL3 group vs DMSO group.In the RSL3 group vs DMSO group,a total of 103 differential metabolites were identified in the positive ion mode of the extracellular fluid,of which 102 metabolites were down-regulated and 1 metabolite was up-regulated;in the RSL3+Fer-1 group vs RSL3 group,56 metabolites were also significant in RSL3 group vs DMSO group.The trends of differential metabolites in RSL3+Fer-1 group vs RSL3 group were opposite to RSL3 group vs DMSO group.The differential metabolites related to ferroptosis in the three modes were mainly phospholipid metabolites,including phosphatidylethanolamine compounds,such as PE-NMe(18:3(9Z,12 Z,15Z)/20:0),lysophospholipid Acylethanolamine compounds such as Lyso PE(0:0/16:1(9Z),lysophosphatidylcholine such as Lyso PC(20:1(11Z)),phosphatidylserine(PS),phosphatidylglycerol(PG),etc.Glutathione and S-Lactoylglutathione,NADH,NADPH and other metabolites were also significant.Differential metabolic pathways: In the intracellular fluid of RSL3 group vs DMSO group,the biosynthesis of squalene and cholesterol,fatty acid metabolism,de novo fatty acid biosynthesis,fatty acid activation,glycolipid metabolism,saturated fatty acid ?-oxidation,glycerophospholipid metabolism pathways were statistically significant(P<0.05).In the intracellular fluid of RSL3+Fer-1 group vs RSL3 group,porphyrin metabolism,aspartic acid and asparagine metabolism,saturated fatty acid?-oxidation,glutathione metabolism,pyrimidine metabolism,glycerophospholipid metabolism pathways were statistically significant(P<0.05).In the pathway analysis of extracellular fluid samples,only carnitine shuttle was statistically significant in RSL3 group vs DMSO group(P<0.05).Conclusion1.RSL3 caused significant metabolic changes in HT-1080 cells.There were 239 differential metabolites in the intracellular fluid in RSL3 group vs DMSO group,among them 205 metabolites were dowm-regulated and 34 metabolites were up-regulated.There were 103 differential metabolites in the extracellular fluid,all of which were down-regulated except for one up-regulated.Fer-1 can reverse the metabolic changes caused by RSL3.Phospholipids,glutathione and S-Lactoylglutathione,NADH,NADPH and other metabolites were of great significance in ferroptosis.2.Pathways such as squalene and cholesterol biosynthesis,glutathione metabolism,saturated fatty acid ?-oxidation and fatty acid metabolism may be the significant metabolic pathways in the process of ferroptosis.