Synergistic Effect Of Aerobic Glycolysis And Glutamate Metabolism In Glioma Cells

Aerobic glycolysis,as a unique metabolic mode of tumor cells,has become a focus of anti-tumor therapy research.However,many studies have demonstrated that tumor cells can survive when aerobic glycolysis is impaired.In addition to aerobic glycolysis,“glutamine addiction” has been considered to be the second major hallmarks of tumor cell metabolism.Glutamate produced by glutamine in tumor cells has multiple metabolic pathways,including being excreted by tumor cells in exchange for cystine,participating in the production of the antioxidant glutathione(GSH),and being converted to α-ketoglutarate(α-KG)enters the Tricarboxylic acid cycle(TCA)to provide energy.Metabolic flux analysis in proliferating glioblastoma cells demonstrate that the coordinated metabolism of glucose and glutamine enabled a metabolic platform supporting both bioenergetics and biosynthesis.Therefore,these observations suggest that a better understanding of the relationships between glucose and glutamine metabolism will lead to novel approaches to curb tumor growth.In this study,we found that inhibiting glycolysis or depriving glucose in glioma cells,abnormally increased glutamate efflux.We further discovered that inhibition of glycolysis or glucose starvation increases the expression levels of glutamate cystine antiporter(x CT)and γ-glutamate synthetase(γ-GCS)to produce more reduced glutathione(GSH)to regulate redox balance.Finally,our research demonstrated that combined glycolysis inhibitor and inhibitor of glutamate cystine antiporter(x CT)or glutamylcysteine synthase(γ-GCS)can effectively block prototype glutathione(GSH)production and caused up-regulation of reactive oxygen species(ROS),thereby better inhibiting the proliferation of glioma cells.In summary,our results indicated that glutamate metabolism promoted survival of glioma cells through redox regulation when glycosis is impaired,and that blocking glutamate’s redox pathway can improve the therapeutic effect of targeted glycolysis.The main results are as follows:1.Ophthalaldehyde(OPA)pre-column derivatization method was used to determine the optimal detection wavelength of glutamine and glutamate at335 nm by full-wavelength scanning of a UV spectrophotometer.Then,the glutamine and glutamate standards were detected by HPLC.It was found that the peak time of glutamine and glutamate was 7.3 min and 5.5 min,respectively,and the concentration was directly proportional to the peak area;2.Utilizing 2-DG or Oxa to inhibit glycolysis or glucose deprivation to treat glioma U251 and U87 cells.By detecting extracellular lactate secretion and glucose consumption,it was found that the 2-DG and Oxa semi-inhibitory concentration of glioma cells treated for 24 hours was 15 m M,The results demonstrated that inhibition of glycolysis or glucose deprivation significantly reduced glycolysis levels in U251 and U87 cells.High-performance liquid chromatography(HPLC)was used to detect the extracellular glutamate content,and the results demonstrated that under inhibiting glycolysis or depriving glucose,the glutamate efflux of U251 and U87 cells increased significantly;3.q PCR and Western blot techniques detected changes of key molecules in the glutamate metabolism pathway.The results discovered that under the conditions of glycolysis blocked,x CT and γ-GCS were highly expressed.The m RNA level of glutamate dehydrogenase that decomposed glutamate into the Tricarboxylic acid cycle did not change significantly and the activity was relatively reduced.4.By detecting the content of GSH and ROS in glioma cells,it was confirmed that when inhibiting glycolysis or depriving glucose,glutamate participated in intracellular GSH synthesis,thereby reducing ROS levels and ensuring cell survival.Glycolysis inhibitor combined with x CT inhibitor sulfasalazine(SSZ)at 450 μM or γ-GCS inhibitor Buthithine sulfoximine(BSO)at 1 m M,compared with each inhibitor alone,it significantly inhibited glioma cell viability.