Abstract 62: Alteration of metabolic pathways of glucose consumption by CENU treatment in B16 melanoma tumors: A NMR spectroscopy-based [1,2-13C]glucose fluxomics analysis

Abstract

Abstract We have previously shown using proton NMR spectroscopy-based metabolomics that mice bearing B16 melanoma tumors treated by chloroethylnitrosourea (CENU) had a strong intratumoral decrease in glycolysis involving the accumulation of intracellular glucose and glucose-6-phosphate, together the accumulation of glutamate derivatives, despite no significant lactate content change (1). To get insights into the metabolic pathway followed by glucose carbons in CENU-treated tumor, we used double labelled glucose ([1,2-13C]glucose) and investigated the obtained isotopomers of lactate, the final product of glycolysis. Mice bearing B16 melanoma tumors were treated by CENU or saline solution (control) at days 11, 14 and 17 at dose 15µg/g weight. At day 23, mice were injected intraperitoneally with [1,2-13C]glucose at 25 mg in 150µl saline solution. Animals were sacrificed 30 minutes after glucose injection. Tumors were removed and kept at −80°C until use. The analysis of lactate isotopomers, a fluxomics approach, was done at 500 MHz using high resolution magic angle spinning (HRMAS) 1H-NMR spectroscopy by exploiting 1H-13C coupling signals. The two lactate signals, the methyl (C3) signal centered at 1.33 ppm and the methine (C2) signal centered at 4.12 ppm were differentially analyzed, and quantified from spectra witout and with broadband 13C-decoupling. In addition, the activity of pyruvate-kinase was measured using enzyme assay. In comparison with control tumors, CENU-treated tumors exhibited an overall decrease of labelled lactate both at C3 and C2 position, together with a significant increase in the relative proportion of C2 to C3 labelling (0.65±0.10 vs 0.91±0.13, P<0.01, n=3 vs n=3). Because lactate only labelled on C3 was a product of glucose metabolism through the pentose phosphate pathway, it was concluded that most of glucose in CENU-treated tumors was directly metabolized to lactate, thus dedicated to ATP production rather than macromolecular biosynthesis. Consistently, the activity of pyruvate kinase, the main source of glycolysis-derived ATP was increased under CENU treatment (+48%, P<0.01). Altogether these fluxomics data provide evidence of glycolysis adaptation that may be correlated to reduced proliferation, decreased aggressiveness, and redifferentiation, as previously shown in this model (2). In addition, this study suggests that metabolic targeting of glycolysis-related ATP production may improve CENU efficacy. 1- Morvan D, Demidem A. Cancer Res, 67; 2150-59, 2007 2- Demidem A et al. Cancer Res, 61; 2294-300, 2001 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 62.