Abstract 48: Directing glutamine and glucose metabolism with troglitazone and EGCG limits MCF-7 cell growth

Abstract

Abstract Tumor cell growth requires balanced glutaminolysis and glycolysis which in turn subserve mitochondrial membrane potential (Ψm) and cell growth (DNA synthesis). Troglitazone (TRO) reduces Ψm by 72% (p<0.001) within 15min. Both glutaminolysis (47% reduction in NH4+ p<0.05) and glycolysis (70% reduction in lactate formed p<0.001) are reduced. However, after 24h TRO exposure Ψm has returned to baseline levels associated with an adaptive increase in glutamine (2,200 ±166 to 2936±33nmol/mg) and glucose uptake (11,573±840 to 14,523±870nmol/mg). The Ψm restoration with TRO occurs with an increase in glycolysis (5,007±360 to 8,303±513nmol/mg) and ammoniagenesis (1,747±127 to 3,780±260 nmol/mg) both p<0.05. CCCP, a mitochondrial uncoupling reagent, reduced Ψm 84% acutely which is abrogated to 53% at 24h without an increase in glutamine uptake (1,763± 330nmol/mg). Partial restoration with CCCP occurs with only an increase in glycolysis (7,876±3,212nmol/mg) suggesting that a proton gradient is required for full Ψm restoration. The adaptive increase in glutamine uptake seen with TRO is coupled to accelerated flux via mitochondrial glutamate dehydrogenase (GDH) as evidenced by a 46% reduction in cellular glutamate (88±7 to 48±4nmol/mg, p<0.01) and an increase in NH4+ produced per glutamine utilized from 0.8 to 1.3. Alanine formation is decreased 75% (1,086±117 to 273±50nmol/mg, p<0.001) consistent with reduced flux via alanine aminotransferase (ALT). Inhibition of mitochondrial H+ ATPase with oligomycin in the presence of TRO prevented restoration of Ψm (99± 6 vs 48± 8 %, TRO vs TRO+Oligo, p<0.001) and decreased GDH flux (NH4+/Gln= 1.23 to 0.86, TRO vs TRO+Oligo p<0.01 with restoration of alanine formation (227± 122 to 1,017± 80nmol/mg, p<0.01) suggesting that the mitochondrial membrane proton gradient drives glutamate via GDH with ALT as a default pathway. Shifting glucose and glutamine metabolism to mitochondrial membrane restoration was associated with a small reduction in DNA synthesis suggesting that glutamate flux via GDH was supporting both and that blocking TRO-induced glutamate flux via GDH might limit either Ψm restoration or cellular proliferation, or, both. The green tea polyphenol, EGCG, added with TRO for 24h inhibited GDH flux as evidenced by 2-fold rise in glutamate content(48±4 to 87± 6nmol/mg, p<0.05) and increased alanine formed (672± 81 vs 1292±212nmol/mg, p<0.01). Associated with TRO+EGCG was a 10% reduction in Ψm and a >40% reduction in cellular proliferation consistent with a shift of glucose from glycolysis (5,261± 512 to 3,635±478 nmol/mg, p<0.05) supporting proliferation (lac/glc=0.60 to 0.46) to maintaining Ψm in place of glutamate. These results point to an interrelationship of glutamine and glucose metabolism in supporting Ψm in cancer cells and suggest that shifting Ψm dependency to GDH flux with TRO while blocking this flux with EGCG may limit tumor growth. 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 48.