Figure 4.
Imatinib resistance results in the rewiring of glutamine/glutamate metabolism to proline, collagen and glutathione metabolism. (A) Extracellular metabolic fluxes of KU812 Parental and imatinib resistant (ImaR) cells under normoxia (Norm) and hypoxia (Hyp). Data are mean ± SD for n = 3 of n = 2 independent experiments. Glutamine consumption and glutamate production were obtained under normoxia. Data were normalized to cell number and incubation time. (B) Western blotting analysis of total protein fractions related to glutamine and proline metabolism of KU812 Parental and ImaR cells under normoxia or hypoxia. Data are mean ± SD for n = 3 of n = 2 independent experiments. Kidney-type glutaminase 1 (KGA-GLS1), glutaminase C (GAC-GLS1), glutaminase 2 (GLS2), and glutamine synthetase (GS) (C) Protein profile of the rewiring of glutamine metabolism in KU812 imatinib-resistant when compared to KU812 parental cells (mean ± SD for n = 2 replicates). Log2 fold change values were calculated and represented by green color = protein upregulation; and red = protein downregulation. (D) Representative experiment of the amino acids consumption and production rates (mean ± SD for n = 3 replicates) significantly different between KU812 Parental and ImaR cells under normoxia and hypoxia. (E) Intracellular concentrations of amino acids significantly different between in KU812 Parental and ImaR cells under normoxia. The data are mean ± SD for n = 3 replicates. (F) Fold difference value (mean ± SD for n = 3 replicates) in 13C Glutamine label incorporation in some metabolites of the KU812 ImaR cells compared to KU812 Parental cells in normoxia. Statistically significant differences in all the panels was determined by two-tailed independent sample Welch’s t-test when n < 3 and Student’s t-tests when n > 3: * p < 0.05, ** p < 0.01, *** p < 0.001, and p ≥ 0.05 (n.s. = non-significant differences). Abbreviations: ASNS, aspargine synthetase; ASS1, arginosuccinate synthase; GFPT1, Glutamine-Fructose-6-Phosphate Transaminase 1; GNPNAT1, Glucosamine-Phosphate N-Acetyltransferase 1; OAT, ornithine aminotransferase; P4H, prolyl 4-hydroxylase; P5CDH, delta-1-pyrroline-5-carboxylate dehydrogenase; P5CS, delta-1-pyrroline-5-carboxylate synthase; PGM, phosphoglucomutase; PYCR, pyrroline-5-carboxylate reductase; ME, malic enzyme; SLC, solute carrier; SNAT, system N amino acid transporter; and UAP1, UDP-N-Acetylglucosamine Pyrophosphorylase 1.