Smolkova 2015 Int J Biochem Cell Biol

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Smolková K, Dvořák A, Zelenka J, Vítek L, Ježek P (2015) Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells. Int J Biochem Cell Biol 65:125-33.

» PMID: 26007236

Smolkova K, Dvorak A, Zelenka J, Vitek L, Jezek P (2015) Int J Biochem Cell Biol

Abstract: Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite R-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO2-dependent reductive carboxylation glutaminolysis (RCG) and CO2-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring 13C incorporation from 1-13C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, 13C-citrate, 13C-malate, and 13C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5 mM glucose (2-, 2.5-, and 13-fold at 25 mM glucose), respectively, after 6 h. Such enrichment decreased by 6% with IDH1 silencing, but by 30-50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO2. At hypoxia (5% O2), IDH2-related and unrelated 13C-accumulation into citrate and malate increased 1.5-2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. 13C-2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal 13C-2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and 13C-2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.

Keywords: Reductive carboxylation, NADPH-dependent isocitratedehydrogenase IDH2, Hypoxia, Oncometabolite R-2-hydroxyglutarate, Breast carcinoma, HTB-126 cells, Breast adenocarcinoma, MDA-MB-231 cells

O2k-Network Lab: CZ Prague Jezek P

Labels: MiParea: Respiration, mtDNA;mt-genetics, Genetic knockout;overexpression  Pathology: Cancer  Stress:Oxidative stress;RONS  Organism: Human  Tissue;cell: Endothelial;epithelial;mesothelial cell, Other cell lines  Preparation: Intact cells 

Coupling state: LEAK, ROUTINE, ET  Pathway: ROX  HRR: Oxygraph-2k