Determination of the role of aspartate aminotransferase in the mechanism of action of oxamate.

A87 Tumor invasiveness and growth directly correlate with glycolytic flux to lactate suggesting that enhanced glucose metabolism may confer growth advantages to neoplastic cells. The glycolytic enzyme, lactate dehydrogenase (LDH) is believed to be a key regulator of glycolysis since it is induced by the oncogene c-myc and by Hypoxia Inducible Factor 1, and is highly expressed by neoplastic cells. LDH-A is the isozyme that favors the oxidation of NADH to NAD+ in order to reduce pyruvate to lactate. Lactate is exported via the monocarboxylate transporters and then converted back into glucose in the liver via the Cori cycle. We postulate that the rationale for this waste of anabolic carbons by rapidly dividing cells is the requirement of NAD+ for the oxidation of the glycolytic intermediate glyceraldehyde 3-phosphate to 3-phosphoglycerate. We exposed MDA-MB-231 breast adenocarcinoma cells to 13C-glucose and examined the effects of the LDH inhibitor oxamate (40mM) on the pooling of glycolytic products using 2D NMR spectroscopy. Surprisingly, we found that oxamate did not affect lactate secretion but rather markedly decreased the 13C enrichment of glutamine/glutamate and the pyrimidine bases, both products of the TCA cycle. In addition to inhibiting LDH, oxamate has been found to inhibit aspartate aminotransferase (AAT) which is required for the shuttling of cytosolic NADH into mitochondria for oxidative metabolism. Oxamate thus may suppress mitochondrial electron transport which is required for the activity of the TCA cycle. In support of this hypothesis, we observed a marked decreased in oxygen consumption (-35%) after exposure of MDA-MB-231 cells to oxamate (40mM). Suppression of de novo glutamate and pyrimidine synthesis by oxamate should limit cellular proliferation and we found that MDA-MB-231 breast adenocarcinoma tumor growth in Balb/C athymic mice is markedly suppressed by oxamate (i.p. injection 15 mg every 24 hours X 12 days, avg. decrease 34%±17%). Using a siRNA oligo directed towards AAT we transfected MDA-MB-231 cells and observed a marked decrease in proliferation of cultured cells as well as those grown in soft agar. Next we examined the effect of a pharmacological inhibitor of AAT, amino-oxyacetate (AOA) on these transformed cells. We found that AOA caused a similar decrease in cell proliferation as well as a co committed decrease in oxygen consumption. In addition we observed a decrease in the cellular NAD+/NADH ratio of those cells treated with AOA for 48 hours. We hypothesize that this change is ratio is due to the buildup of NADH through the inhibition of AAT in the malate-aspartate shuttle (the shuttle that is required for cells to transfer reducing equivalents from NADH across the mitochondrial membrane). Finally using 2D NMR spectroscopy we found that inhibition of AAT with AOA showed a similar decrease in 13C enrichment of glutamine/glutamate and the pyrimidine bases, as those cells treated with oxamate. We conclude that oxamate indeed acts to inhibit AAT and thus prevents the malate-aspartate shuttle from transferring reducing equivalents across the mitochondrial membrane, consequently causing a selective suppression of transformed cell growth. Understanding the role of aspartate aminotransferase in mechanism of action of oxamate has led us to a unique target for the development of anti-neoplastic agents.