Study Of The Anti-glioma Activities Of ZZZ-1 And Its Underlying Mechanisms Via ¹³C Isotope Tracing And LC-MS

Objectives:Glioma is an intractable CNS tumor with high morbidity and mortality.Generally,tumor tissues cannot be surgically resected due to its infiltrative growth.Decades of efforts have not yielded any definitive treatments to cure these tumors.Up to now,surgery followed by fractionated radiotherapy and adjuvant treatment with the alkylating agent TMZ is still the gold standard for GBM therapy.Recently,targeting the metabolic characteristic of cancer cells as a novel strategy of cancer therapy are getting more and more attention.However,the well-known metabolic regulator have failed to cure the glioma due to the significant heterogeneity and flexibility in energetic metabolism of GBM cells.Fortunately,ZZZ-1,a small molecule compound that is identified in our lab for first time showed a potent anti-GBM activity in vitro and in vivo.Based on our previous data,its anti-GBM efficiency are associated with regulating energetic metabolism of GBM cells.This study was devoted to explore the metabolic regulation effects of ZZZ-1 underlying the anti-glioma activities,especially on the mitochondrial OXPHOS,nucleotides biosynthesis and the following alteration of metabolic characteristics.Experiment design:(1)The anti-GBM activity were compared between ZZZ-1 and other metabolic regulating agents,such as 2-DG,DCA and Metformin in a panel of GBM cells including established GBM cells lines and patients-derived GBM cells,and then a representative cell line was used to determine the effect of ZZZ-1 on apoptosis induction.(2)To demonstrate the effects of ZZZ-1 on the glucose and glutamine utilization,the glucose and glutamine consumption,and also the lactate production were determined in a panel of GBM cells,and then the ATP level was compared between 2-DG and ZZZ-1,and were determined in glioma cells treated with ZZZ-1 alone and combined with OXPHOS inhibitor oligomycin.(3)To reveal the characteristic of glucose and glutamine metabolism in GBM cells,the U87MG cells were incubated in 13C-Glc or 13C-Gln,and then the abundance of 13C in intermediates involved in catabolism and anabolism were analyzed by LC-MS to trace the flux of glucose and glutamine metabolism.(4)To demonstrate the effect of ZZZ-1 on the glucose and glutamine catabolism,firstly,the U87MG cells were incubated in 13C-Glc and 13C-Gln and treated with ZZZ-1,and then the level of intermediates involved in glycolysis and TCA cycle were determined.Secondly,the level of TCA cycle intermediates,NADH,NAD+ and ATP were measured in a panel of GBM cells to test the increased activity of OXPHOS by ZZZ-1.(5)To demonstrate the effect of ZZZ-1 on the anabolism pathway that glucose and glutamine involved in,firstly,the mitochondrial and cytoplasmic level of TCA cycle intermediates,Gin and Glu were determined in U87MG cells to further verify the blockage of metabolic association between the mitochondria and the cytoplasm.Secondly,the U87MG cells were incubated in 13C-Glc and 13C-Gln and treated with ZZZ-1,and then the level of amino acids that derived from glucose or glutamine and its downstream nucleotides to reveal the alternation of de novo nucleotides synthesis pathway induced by ZZZ-1.(6)To explore the effect of ZZZ-1 on redox homeostasis,the level of intracellular NADPH,NADP+,GSH,GSSG and glutathione content in media were determined by LC-MS in a panel of cells treated with ZZZ-1,and then,the ROS level and MMP were determined in Tumor 3 cells by DCFH-DA and JC-1,respectively.Additionally,the GBM cells U87MG,C6 and Tumor 3 were treated with ZZZ-1,ROS scavenger GSH and NAC,OXPHOS inhibitor OM alone and combination,and then the ROS and cell viability were determined to explore the association between ROS elevation and cells viability decrease.Results:(1)Compared to other well-known metabolic regulators,such as 2-DG,DCA,Metformin,ZZZ-1 exerted a more potent anti-GBM activity,and significantly induced apoptosis.(2)The glucose were mainly flowed into the glycolysis,PPP pathway and purine base synthesis,and partly flowed into TCA cycle,while glutamine were flowed into TCA cycle,pyrimidine synthesis and glutathione synthesis,which means the predilection and cross-talk are exist in glucose and glutamine metabolism.(3)ZZZ-1 widely increased the consumption of glucose in early time points,which accompanied by decreased lactate production.ZZZ-1 also regulate the metabolism of glutamine in cellular contexts.Meanwhile,ZZZ-1 increased the glycolytic and TCA cycle intermediates derived from glucose and glutamine,ATP and NADH,which indicated that ZZZ-1 enforce the glucose and glutamine into TCA cycle for OXPHOS.(4)ZZZ-1 decreased the level of amino acids derived from glucose and glutamine and its downstream nucleotides,and increased the level of TCA cycle intermediates in mitochondria,which demonstrated that ZZZ-1 inhibit biosynthesis of nucleotides via blocking the metabolites in mitochondria.(5)ZZZ-1 wildly broken the balance of GSH/GSSG,NADPH/NADP+ and induced the inhibition of glutathione synthesis and the release of GSSG,which results in the disruption of redox homeostasis indicated by ROS accumulation and MMP decrease.Conclusions:ZZZ-1 regulate the metabolism of glucose and glutamine simultaneously,which fulfilled by TCA cycle and OXPHOS enhancement,biosynthesis inhibition and redox homeostasis disruption.In a word,ZZZ-1 enforce the GBM cells to use glucose and glutamine as the normal cells,which reverse the oncogenic metabolic characteristic of GBM cells,and overcome the metabolic heterogeneity and flexibility during GBM therapy.