Cancer Cells Coordinating The Homeostasis Of NADH And Energy By A Non-PPP Role Of G6PD

Bacground and ObjectiveUnrestricted proliferation,angiogenesis and genomic instability were the most typical characteristics of cancer cells,and cancer cells have made many metabolic changes to adapt to the accelerated proliferation rate and nutrients consumption.Because of the metabolic reprogramming,cancer cells were undergoing in an environment with high level of reactive oxygen species.In order to prevent cancer cells from dying due to the excessive accumulation of reactive oxygen species,cancer cells needed to reprogram metabolic process involved in maintaining the homeostasis of reductive equivalent,as to ensure producing enough reductive substances,such as reductive glutathione,which can clean up the overloaded reactive oxygen species in time.Pentose phosphate pathway was an important pathway of glucose metabolism in cancer cells,which linked the glucose and nucleotides metabolism,and also ensured the supplement of ribose for cell proliferation.In addition,the reductive equivalent-NADPH,which can be released through the oxidized pentose phosphate pathway,was widely involved in intracellular biomass synthesis and maintained the reductive state of glutathione and sulfhydrylase in the intracellular environment of cancer cells.Therefore,the increased metabolic flux of pentose phosphate pathway has been found in a variety of cancers.Glucose-6-phosphate dehydrogenase（Glucose-6-phosphate Dehydrogenase,G6PD）is the first rate-limiting enzyme of the oxidized of pentose phosphate pathway,which catalyzes the oxidation of glucose-6-phosphate to 6-phosphate glucuronolactone,and produces a molecule of NADPH and H⁺at the same time.According to the traditional view,G6PD is the main source of cytoplasmic NADPH,which not only provides the basic reductive equivalent to biosynthesize lipids in cancer cell,but also involves in maintaining the reductive status of glutathione in cell.Therefore,the enzyme activity of G6PD is considered to play an important role in promoting cancerigenesis and progression.However,recently,our team found the flux of glucose flow to pentose phosphate pathway decreased significantly when we cultured HeLa and other cancer cells with[3-²H]glucose under the stimulation of hypoxia or inhibitor of mitochondrial respiratory chain,while cancer cells often suffers mitochondrial abnormal function and ischemic and anoxic microenvironment,suggesting that NADPH,which produced by G6PD enzyme activity,may not be as important as the traditional view in the significance of resisting oxidative stress in cancer cells.As for cancer cells can also be substantively compensated the cytosolic NADPH pool by NADPH-generating enzymes,such as malate dehydrogenase（Malic Enzyme,ME）,isocitrate dehydrogenase（Isocitrate Dehydrogenase,IDH）and methylenetetrahydrofolatedehydrogenase（Methylenetetrahydrofolate Dehydrogenase,MTHFD）,which can also help cancer cells maintain the relative homeostasis of reductive glutathione,and it has been reported that those enzymes actually play an important role in maintaining intracellular reductive homeostasis in a variety of cancers.In addition,G6PD deficiency,a genetic disease with G6PD enzyme function deficiency,was characterized of reactive oxygen species and hemolytic crisis induced by eating broad bean or taking sulfonamides,which can be explained by that G6PD is the only source of cytosolic NADPH in red blood cells,and the erythrocyte membrane was destroyed by reactive oxygen species after stimulations of such drugs and hemolysis occurs.However,patients with G6PD-deficiency alleles are well tolerated,except that it could bring a risk of acute non-spherocytic hemolytic anemia（NSHA）triggered by exogenous oxidative stressors in red blood cells,while mice with G6PD knockout meted embryonic death,that is,the loss of G6PD activity may not that necessary to individual embryonic development,but G6PD itself was indispensable,and this also gives us a hint that G6PD proteins may have important functions besides its PPP activity,which are essential for embryonic development and individual survival.In order to clarify the specific physiological and pathological role of G6PD in cancer cells,and also to discover the potential functions of G6PD,we established several clonal cancer cell lines with G6PD knockout by CRISPR-Cas9,and then established the research model including wild type,G6PD-deficiency natural mutant and artificial site mutant G6PD.By using this research model,we want to observe and study a series of phenotypic changes of cancer cells with G6PD knockout,and to clarify the functional significance of G6PD enzyme activity and G6PD itself on cancer cells when against oxidative stress.In addition,both of transcriptology and metabonomics methods was used to reveal the most relevant metabolic changes by G6PD knockout,and also the role of G6PD in maintaining cancer cells’energy and reductive equivalent homeostasis.In general,we want to draw out the unknown side of G6PD,and then provide new understanding and idea in embryonic development and cancer transforming.Methods1.Established G6PD knockout monoclonal cell lines of HeLa,MDA-MB-231 and HCT116 by CRISPR-Cas9,and verified by Western Bloting and/or genome sequencing.2.Confirmed and established research models expressed wild type,G6PD-deficiency natural mutant and artificial site mutant G6PD in G6PD knockout monoclonal cell lines of HeLa,MDA-MB-231 and HCT116.3.Taken HeLa cells as the representative cell model,transcriptome RNAseq sequencing and non-targeted metabolic mass spectrometry were used to study the similarities and differences among KO G6PD,wtG6PD and enzyme activity deficient G6PD（G6PD-R257G）cells,and also to reveal the changes of signal and metabolic pathway most related to G6PD enzyme activity and G6PD protein itself.4.HeLa cells were used as the representative cell model to stimulate or change the culture environment.Cell lines of KO G6PD,wtG6PD and enzyme activity deficient G6PD（G6PD-R257G）were stimulated with hydrogen peroxide,hypoxia and inhibitors of mitochondrial respiratory chain,and the responses of the three cells to those stimulations and their corresponding metabolic changes,such as glycolysis,glutamine metabolism,NADH and NADPH homeostasis,were observed.5.Taken HeLa cells as the representative cell model,stable isotope labeling culture and LC-MS mass spectrometry were used to study the metabolic fluxes of PPP,glycolysis,citric acid cycle,fatty acid synthesis,NADH/NAD+and NADPH/NAD+in cell lines of KO G6PD,wtG6PD and enzyme activity deficient G6PD（G6PD-R257G）.6.By using HeLa cells as the representative cell model,LC-MS targeted lipid metabolism mass spectrometry was used to study the lipid metabolites in cell lines of KO G6PD,wtG6PD and enzyme activity deficient G6PD（G6PD-R257G）.7.HeLa cells were used as the representative cell model to study the specific mechanism why cancer cells been sensitive to various stimuli after knockout G6PD by using drugs or expressing tool enzymes.8.HeLa cells were used as the representative cell model to study the role of G6PD in affecting the AMPK signal pathway and coordinating energy and reductive equivalent homeostasis.Results1.HeLa,MDA-MB-231 and HCT116 G6PD knockout monoclonal cancer cell lines were successfully constructed,and wtG6PD and a series of enzyme activity defects G6PD were re-expressed.It was found that cancer cells with G6PD knockout were sensitive to stimulations such as hydrogen peroxide,hypoxia and mitochondrial respiratory chain blockage and cells died quickly,mostly like necrosis.2.G6PD（R257G） has shown loss of oxPPP activity,but could robustly rescued cells from hydrogen peroxide,hypoxia and mitochondrial respiratory chain blockage-induced cell death.And R257G mutant can restore most of the functions of WT G6PD,what is interesting,G6PD（R257G） also has shown many different metabolic characteristics from wtG6PD.3.After G6PD knockout,the ratios of NADPH/NADP+in cancer cells decreased significantly,while the ratios of NADH/NAD+increased significantly.Reducing the ratio of NADH/NAD+in KO G6PD cells by drugs or tool enzymes could increase the ratio of NADPH/NADP+in cancer cells and partly reverse the rapid death caused by hydrogen peroxide and mitochondrial respiratory chain blockage.4.After G6PD knockout,the flux of glycolysis in cancer cells was significantly increased,and the cancer cells showed many metabolic phenotypes of mitochondrial dysfunction.And the cancer cells with G6PD knockout could not effectively increase glycolysis when blocking the mitochondrial respiratory chain and then driving the cells to necrosis,while stimulating the glycolysis in KO G6PD cells could partly reverse the rapid death caused by hydrogen peroxide and mitochondrial respiratory chain blockage.5.After G6PD knockout,the reductive carboxylation pathway of glutamine in cancer cellswasobviouslyinhibited,whilethecompensatory glucose--pyruvate--malate--citrate--acetyl-coenzyme A metabolic pathway was increased.6.Knockout of IDH1 and IDH2 by CRISPR-Cas9 technique can increase the intracellular NADH/NAD+ratio.Both inhibiting of AMPK signal pathway and the glycolysis metabolism in cancer cells,which can cause rapid death of cancer cells similar to that of KO G6PD when using drugs to block the mitochondrial respiratory chain.7.After G6PD knockout,the response of cancer cells to activated AMPK became unregulated,and there is an important connection between the unregulated of AMPK activation and the rapid death of KOG6PD cells induced by mitochondrial respiratory chain blocking.8,Reducing the NADH/NAD+ratio with AKB,can obviously stimulate AMPK signal activation in HeLa^{KO G6PD}cell.Conclusions1.The cancer cells with G6PD knockout are very sensitive to hydrogen peroxide,hypoxia and AntiA stimulation,and can cause rapid necrosis by such stimulations.Although G6PD（R257G）shows very low enzyme activity of oxPPP,it can well reverse the rapid necrosis of cancer cells stimulated by hydrogen peroxide,hypoxia and AntiA.And R257G mutant can restore most of the functions of wtG6PD,what’s more,G6PD（R257G）also has shown many different metabolic characteristics from wtG6PD.Therefore,G6PD protein itself is also important for cancer cells to resist oxidative stress,and which is completely different from the mechanism by which G6PD or other key enzymes maintain reductive homeostasis by producing NADPH.2.Cancer cells show disturbance of mitochondrial respiratory chain after knockout of G6PD.And stimulation affecting the function of mitochondria will cause rapid necrosis of cells,but reducing the ratio of NADH/NAD+cells with drugs or tool enzymes can partly reverse the rapid necrosis of KO G6PD under such stimuli,and the function of G6PD protein to maintain NADH/NAD+homeostasis does not depend on its enzyme activity of NADPH synthesis.3.In general,when cancer cells are exposed to hypoxia or mitochondrial respiration blockage,cancer cells need to deal with the lack of energy and reductive power by greatly increasing glycolysis and glutamine reductive metablism,but if G6PD was deleted,the reductive carboxylation of glutamine cannot be activated.In addition,the early increase of glycolysis requires the activation of AMPK signal,while the activation of AMPK signal becomes unregulated after G6PD knockout.And what’more,the elevatory NADH/NAD+ratio during the process of glycolysis will feedback inhibit the conversion of glucose to pyruvate,which will eventually lead to rapid cell death due to lack of ATP synthesis.4.After G6PD knockout,cancer cells did not affect the metabolic conversion of glucose to fatty acids,but significantly inhibited the metabolic conversion of glutamine to fatty acids.When tracing back to the source of acetyl-Co A,it was found that cancer cells with G6PD knockout can compensate for the supplement of intracellular acetyl-CoA by glucose--pyruvate--malate--citrate--acetyl-Co A.However,the metabolic pathway of glutamine--a-ketoglutarate--isocitrate--citrate--acetyl coenzyme A was inhibited.5.Combined knockout of IDH1/2 could also significantly increase the ratio of intracellular NADH/NAD+.Although combined knockout of IDH1/2 was not sensitive to hypoxia or mitochondrial respiratory chain inhibitor,while combined with inhibition of AMPK signal activation,cancer cells knockout of IDH1/2 could cause the rapid cell death just like the G6PD knockout cells.It may suggest that the rapid cell death of the KO G6PD cells mentioned above is closely related to the unregulated of AMPK and the glutamine reductive metablism,and the key link among which may be the maintenance of NADH homeostasis and energy supply.6.The reductive metabolism of glutamine in cancer cells has an important relationship with the homeostasis of NADH/NAD+,and the reductive metabolism of glutamine affects the metabolism of energy generation by changing the NADH/NAD+ratio in cancer cell.