Dataset Information

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

ABSTRACT: High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a ¹³C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2-¹³C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non-oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3-¹³C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into ?-ketoglutarate and increased that for succinate, which is consistent with ROS-mediated conversion of ?-ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.

SUBMITTER: Carrigan JB

PROVIDER: S-EPMC4916734 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Publications

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

Carrigan John B JB Reed Michelle A C MA Ludwig Christian C Khanim Farhat L FL Bunce Christopher M CM Günther Ulrich L UL

ChemPlusChem 20160322 5

High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a <sup>13</sup>C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cel ...[more]

PMID: 27347458

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Project description:BackgroundMetabolic fluxes provide invaluable insight on the integrated response of a cell to environmental stimuli or genetic modifications. Current computational methods for estimating the metabolic fluxes from 13C isotopomer measurement data rely either on manual derivation of analytic equations constraining the fluxes or on the numerical solution of a highly nonlinear system of isotopomer balance equations. In the first approach, analytic equations have to be tediously derived for each organism, substrate or labelling pattern, while in the second approach, the global nature of an optimum solution is difficult to prove and comprehensive measurements of external fluxes to augment the 13C isotopomer data are typically needed.ResultsWe present a novel analytic framework for estimating metabolic flux ratios in the cell from 13C isotopomer measurement data. In the presented framework, equation systems constraining the fluxes are derived automatically from the model of the metabolism of an organism. The framework is designed to be applicable with all metabolic network topologies, 13C isotopomer measurement techniques, substrates and substrate labelling patterns. By analyzing nuclear magnetic resonance (NMR) and mass spectrometry (MS) measurement data obtained from the experiments on glucose with the model micro-organisms Bacillus subtilis and Saccharomyces cerevisiae we show that our framework is able to automatically produce the flux ratios discovered so far by the domain experts with tedious manual analysis. Furthermore, we show by in silico calculability analysis that our framework can rapidly produce flux ratio equations--as well as predict when the flux ratios are unobtainable by linear means--also for substrates not related to glucose.ConclusionThe core of 13C metabolic flux analysis framework introduced in this article constitutes of flow and independence analysis of metabolic fragments and techniques for manipulating isotopomer measurements with vector space techniques. These methods facilitate efficient, analytic computation of the ratios between the fluxes of pathways that converge to a common junction metabolite. The framework can been seen as a generalization and formalization of existing tradition for computing metabolic flux ratios where equations constraining flux ratios are manually derived, usually without explicitly showing the formal proofs of the validity of the equations.

Dataset Information

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

Publications

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

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