Zelenka 2013 Abstract MiP2013
|Zelenka J, Smolkova K, Dvorak A, Vitek L, Jezek P (2013) Reverse carboxylation glutaminolysis in breast cancer cells. Mitochondr Physiol Network 18.08.|
The sustained high rate of tumor cell proliferation results in aglycemia, initiating the revival of OXPHOS in conjunction with the promotion of glutaminolysis , which proceeds either as OXPHOS-involved, encompassing forward Krebs cycle truncated after citrate synthase; or as reductive carboxylation ("anoxic") glutaminolysis (RCG), using the only two enzymes of Krebs cycle acting in the reversed mode – isocitrate dehydrogenase isoform-2 (IDH2) and reversed aconitase. In 2011, it became clear that RCG determines and accelerates tumorigenesis in grade 2 and 3 gliomas, secondary glioblastomas (less frequently in primary glioblastomas ) and acute myeloid leukemia. Reverse reaction of heterozygous mutants of IDH2 and cytosolic IDH1 produces from 2-oxoglutarate the oncometabolite D-2-hydroxyglutarate , which further promotes neoplasia, e.g. by competitive inhibition of histone demethylation, leading to genome-wide alternations in the methylation of histones and DNA.
We have provided evidence that RCG exists in breast carcinoma cells, compromising the Krebs cycle metabolite flux, using IDH2 silencing by miRNA/shRNA expression. Respiration of intact cells and maximal respiration of HTB-126 Glc cells with stably silenced IDH2 increased, indicating substrate (e.g. glutamine) utilization towards the Krebs cycle and NADH synthesis. Increased mitochondrial coupling and decreased uncoupling in miIDH2 cell lines implies an increased respiratory flux used for ATP synthesis, and decreased proton leak, respectively, both indicating higher effectivity of mitochondrial substrate utilization by inactivation of the reductive carboxylation pathway. ATP levels in cell lysates were concomitantly elevated upon IDH2 silencing, and were drastically decreased upon glutamine withdrawal demonstrating increased glutamine involvement in NADH synthesis and ATP production rather than its utilization in reductive carboxylation pathway when IDH2 was inactivated.
We have also confirmed reductive carboxylation flux by GC-MS studies, where 13C incorporation from 1-13C-glutamine into citrate and malate was decreased with IDH2 silencing compared to controls. Noteworthy, 13C icorporation into malate is only possible after ACL and MDH reactions in cytosol after preceding citrate export from mitochondria. We have found a substantial RCG increase under hypoxia. RCG was also elevated in the presence of OXPHOS blockers in SH-SY5Y neuroblastoma cells.
Labels: MiParea: Respiration, mt-Structure;fission;fusion, mtDNA;mt-genetics, Pharmacology;toxicology Pathology: Cancer
Organism: Human Tissue;cell: Other cell lines, HeLa Preparation: Intact cells
Coupling state: ROUTINE
1 - Institute of Physiology, Academy of Sciences, Prague; 2 - First Faculty of Medicine, Charles University, Prague, Czech Republic. - Email: [email protected]
Supported by grants no. P301/12/P381 from Czech Science Foundation and by GAUK 426411 from Grant Agency of Charles University.
- Smolková K, Plecitá–Hlavatá L, Bellance N, Benard G, Rossignol R, Ježek P (2011) Waves of gene regulation suppress and then restore oxidative phosphorylation in cancer cells. Intl J Biochem Cell Biol 43: 950–968.
- Mullen AR, Wheaton WW, Jin ES, Chen PH, Sullivan LB, Cheng T, Yang Y, Linehan WM, Chandel NS, Deberardinis RJ (2011) Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481: 385-388.
- Ward PS, Cross JR, Lu C, Weigert O, Abel-Wahab O, Levine RL, Weinstock DM, Sharp KA, Thompson CB (2012) Identification of additional IDH mutations associated with oncometabolite R(-)-2-hydroxyglutarate production. Oncogene 31: 2491-2498.