Reductive Carboxylation Pathway and Redox Potential in Cancer Cells With IDH2 Silencing

Abstract Anhydrous enol-oxaloacetate (AEO) has demonstrated the ability to enhance neuronal cell bioenergetics and activate brain mitochondrial biogenesis. Since oxaloacetate has demonstrated positive effects on brain bioenergetics in neurodegenerative diseases we have begun to investigate whether AEO may also have a positive effect on the altered cellular metabolism found in cancer cells, particularly Glioblastoma multiforme. The “Warburg effect” describes an abnormal metabolic state in cancer, distinct from normal tissue, in which energy is generated through enhanced conversion of pyruvate to lactate even in the presence of oxygen during glycolysis. Oxaloacetate (OAA) is a key anaplerotic substrate that is required to maintain TCA cycle flux. The role of oxaloacetate supplementation on the energy metabolism is not known in cancer cells. Goal of this study is to investigate the changes in metabolic fluxes in glucose metabolism with and without the presence of OAA in patient-derived GBM cells. We use GC-MS based 13C isotopomer analysis for this study. GBM cells are grown in 15mM glucose containing DMEM medium supplemented with 2mM oxaloacetate for 10 days. 6 hours prior to harvesting, [U-13C]glucose is introduced to the medium. 13C isotopomer analysis of GC-MS data showed that OAA supplementation for 10 days drastically decreased Warburg glycolysis by reducing 13C labeling (M+3) by 19.7% and 48.8% in pyruvate and lactate pools respectively in comparison with cells not treated with OAA. M+3 13C labeled pyruvate entered TCA cycle via acetyl-CoA, where we also observed reduced levels of M+2 13C labeled citrate (20.5%) and glutamate (23.9%) isotopomers. Pyruvate can also enter TCA cycle via pyruvate carboxylation pathway and this activity was also found to be slightly decreased in the OAA treated cells. All the differences were statistically significant. These results indicate that OAA can be used to alter bioenergetics of GBM cells, specifically glucose oxidation.

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Redox Potential Ultrasensitive Nanoparticle for the Targeted Delivery of Camptothecin to HER2-Positive Cancer Cells

Molecular Pharmaceutics ◽

10.1021/mp5000482 ◽

2014 ◽

Vol 11 (6) ◽

pp. 1897-1905 ◽

Cited By ~ 40

Author(s):

Remant Bahadur K.C. ◽

Varun Chandrashekaran ◽

Bei Cheng ◽

Hexin Chen ◽

Maria Marjorette O. Peña ◽

...

Keyword(s):

Redox Potential ◽

Cancer Cells ◽

Targeted Delivery ◽

Her2 Positive

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Abstract 308: Reductive Carboxylation Contributes to Cardiac Adaptation in Response to the Oncometabolite D2-hydroxyglutarate

Circulation Research ◽

10.1161/res.127.suppl_1.308 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Anja Karlstaedt ◽

Brandon Faubert ◽

Heidi M Vitrac ◽

Rebecca L Salazar ◽

Benjamin D Gould ◽

...

Keyword(s):

Cancer Cells ◽

Rat Heart ◽

Metabolic Flux ◽

Adult Mouse ◽

Epigenetic Modifications ◽

Oxidative Decarboxylation ◽

Flux Analysis ◽

Chip Sequencing ◽

Ventricular Cardiomyocytes ◽

Reductive Carboxylation

Cancer cells rewire metabolism to support tumor growth and proliferation. In isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. D2-HG causes inhibition of alpha-ketoglutarate dehydrogenase (AKGDH), which is associated with reduced cardiac contractile function. How tumor cells influence the metabolism of cardiomyocytes remains mostly unknown. Specific cancer cells use glutamine-dependent reductive carboxylation to circumvent defective mitochondrial metabolism by producing citrate and acetyl-CoA for lipid synthesis, which tumors require for growth. Here, we explore the hypothesis that inhibition of AKGDH by the oncometabolite D2-HG increases glutamine-dependent reductive carboxylation in the heart. We combined ex vivo rat heart perfusions with mass-spectrometry-based stable isotope tracer studies and in silico metabolic flux analysis. In response to D2-HG-mediated inhibition of AKGDH, we observed an increased reductive carboxylation of alpha-ketoglutarate to citrate rather than oxidative decarboxylation. This pathway increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. When we perfused rat hearts with 13C-labelled D2-HG, we observed a similarly increased formation of citrate. To identify which IDH isoform is responsible for redirecting carbon flux, we modulated IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes using siRNAs. Reduced expression of IDH1 impaired reductive formation of citrate. Importantly, we observed a significant correlation between reductive citrate formation and epigenetic modifications of histones, including increased histone 3 lysine 9 acetylation and di-methylation. To explore these observations, we conducted ChIP-sequencing and identified distinct transcriptional remodeling. Taken together, we demonstrate how oncometabolic stress in the heart causes redirection of central carbon metabolism via reductive carboxylation, and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

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Antagonism of Glycolysis and Reductive Carboxylation of Glutamine Potentiates Activity of Oncolytic Adenoviruses in Cancer Cells

Cancer Research ◽

10.1158/0008-5472.can-18-1326 ◽

2018 ◽

Vol 79 (2) ◽

pp. 331-345 ◽

Cited By ~ 8

Author(s):

Arthur Dyer ◽

Benjamin Schoeps ◽

Sally Frost ◽

Philip Jakeman ◽

Eleanor M. Scott ◽

...

Keyword(s):

Cancer Cells ◽

Oncolytic Adenoviruses ◽

Reductive Carboxylation

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Influence of reductive carboxylation on redox state of cancer cells

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2012.08.259 ◽

2012 ◽

Vol 53 ◽

pp. S123-S124

Author(s):

A. Dvorak⁎ ◽

J. Zelenka ◽

K. Smolkova ◽

L. Vitek ◽

P. Jezek

Keyword(s):

Cancer Cells ◽

Redox State ◽

Reductive Carboxylation

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The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

International Journal of Cell Biology ◽

10.1155/2012/273947 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 59

Author(s):

Katarína Smolková ◽

Petr Ježek

Keyword(s):

Cancer Cells ◽

Isocitrate Dehydrogenase ◽

Competitive Inhibition ◽

Malignant Tumors ◽

Krebs Cycle ◽

Protective Function ◽

Genome Wide ◽

Isocitrate Dehydrogenase 2 ◽

Reductive Carboxylation

Isocitrate dehydrogenase 2 (IDH2) is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP+-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD+accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction), which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall “anoxic” glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2—frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia—catalyze reductive carboxylation of 2-oxoglutarate and reduction toD-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations.D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factorα.

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TAMI-13. MODULATION OF REDUCTIVE CARBOXYLATION FLUX OF GLUTAMINE IN GLIOBLASTOMA CELLS BY USING OSCILLATING MAGNETIC FIELD

Neuro-Oncology ◽

10.1093/neuonc/noab196.797 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi200-vi200

Author(s):

Kumar Pichumani ◽

Omkar Ijare ◽

Shashank Hambarde ◽

Martyn Sharpe ◽

Blessy John ◽

...

Keyword(s):

Magnetic Field ◽

Test Group ◽

Treated Group ◽

Glutamine Metabolism ◽

Hypoxic Conditions ◽

Oscillating Magnetic Field ◽

Carboxylation Pathway ◽

Humidified Air ◽

13C Isotopomer Analysis ◽

Reductive Carboxylation

Abstract Increased cell proliferation in glioblastoma (GBM) leads to hypoxia in the tumor microenvironment. This is a major concern in GBM patients as it promotes tumor invasion. Glutaminolysis is a hallmark of cancer cells and under hypoxic conditions glutamine metabolism proceeds through reductive carboxylation pathway. Recently, we have shown that oscillating magnetic field (OMF) produces oncolytic effects which can influence cellular metabolism. Here, we have explored the effect of OMF on glutamine metabolism in GBM cells. Patient-derived GBM cells were grown in high glucose (25 mM) DMEM supplemented with 20% fetal bovine serum (FBS), 2.0 mM glutamine and 1.0 mM pyruvate at 37 °C under humidified air and 5% CO2. Cells were divided into 2 groups (Test and Sham; n = 4 each group). When reached confluency (~2.0×106 cells/mL), cells in both groups were treated with 4.0 mM of [U-13C]glutamine in DMEM (supplemented with 20% FBS, and 1.0 mM pyruvate). The “Test” group was subjected to OMF for 3 hours, and the “Sham” group was treated similar to the “Test” group but with non-magnetic rods of the same dimensions as the magnets in the Test group. After 3 h, cells were harvested in 50% methanol analyzed by GC-MS. The 13C-isotopomer analysis showed that glutamine metabolism in GBM cells proceeds through reduction carboxylation, confirmed by the higher levels of M+5 citrate (15.42 ± 1.28 % ) than M+4 citrate (2.05 ± 0.28 %). When GBM cells were treated with OMF, a statistically significant decrease in the citrate M+5 was observed, compared to the “Sham” treated group (15.42 ± 1.28 % vs. 8.89 ± 1.30 %; p = 0.0003). This decrease in M+5 citrate upon OMF treatment clearly indicates that the OMF decreases the reductive carboxylation flux of glutamine in GBM cells which would have therapeutic value in treating GBM patients.

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The oxoglutarate reductive carboxylation pathway: A review

Life Sciences ◽

10.1016/0024-3205(79)90073-0 ◽

1979 ◽

Vol 24 (22) ◽

pp. 2011-2022 ◽

Cited By ~ 3

Author(s):

Amedeo F. D'Adamo ◽

Katherine Dugan Tobin

Keyword(s):

Carboxylation Pathway ◽

Reductive Carboxylation

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Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited

10.1101/2020.06.08.134890 ◽

2020 ◽

Author(s):

Zhaoqi Li ◽

Brian W. Ji ◽

Purushottam D. Dixit ◽

Evan C. Lien ◽

Konstantine Tchourine ◽

...

Keyword(s):

Cell Proliferation ◽

Cancer Cells ◽

Cancer Cell ◽

Electron Acceptor ◽

De Novo ◽

Lipid Biosynthesis ◽

Cancer Cell Proliferation ◽

Lipid Uptake ◽

Expression Of Genes ◽

Carboxylation Pathway

AbstractIt is not well understood how physiological environmental conditions and nutrient availability influence cancer cell proliferation. Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can limit cancer cell proliferation1-5. However, it is currently unclear which specific metabolic processes are constrained by electron acceptor availability, and how they affect cell proliferation. Here, we use computational and experimental approaches to demonstrate that de novo lipid biosynthesis can impose an increased demand for NAD+ in proliferating cancer cells. While some cancer cells and tumors synthesize a substantial fraction of their lipids de novo6, we find that environmental lipids are crucial for proliferation in hypoxia or when the mitochondrial electron transport chain is inhibited. Surprisingly, we also find that even the reductive glutamine carboxylation pathway to produce fatty acids is impaired when cancer cells are limited for NAD+. Furthermore, gene expression analysis of 34 heterogeneous tumor types shows that lipid biosynthesis is strongly and consistently negatively correlated with hypoxia, whereas expression of genes involved in lipid uptake is positively correlated with hypoxia. These results demonstrate that electron acceptor availability and access to environmental lipids can play an important role in determining whether cancer cells engage in de novo lipogenesis to support proliferation.

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Cancer cells with defective oxidative phosphorylation require endoplasmic reticulum–to–mitochondria Ca2+ transfer for survival

Science Signaling ◽

10.1126/scisignal.aay1212 ◽

2020 ◽

Vol 13 (640) ◽

pp. eaay1212 ◽

Cited By ~ 4

Author(s):

Cesar Cardenas ◽

Alenka Lovy ◽

Eduardo Silva-Pavez ◽

Felix Urra ◽

Craig Mizzoni ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Oxidative Phosphorylation ◽

Cancer Cells ◽

Autophagic Flux ◽

Release Channel ◽

Atp Production ◽

Fusion Inhibition ◽

Intracellular Ca ◽

Reductive Carboxylation

Spontaneous Ca2+ signaling from the InsP3R intracellular Ca2+ release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca2+ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP3R activity or mitochondrial Ca2+ uptake increased α-ketoglutarate (αKG) abundance and the NAD+/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)–to–mitochondria Ca2+ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca2+ inhibited αKGDH activity and increased NAD+, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca2+ from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca2+ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca2+ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.

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