Abstract 60: Altering tumor cell metabolism may contribute to the antitumor activity of ethyl pyruvate
Authors: Liang Xiaoyan , de Vivar Chavaz Antonio Romo , Schapiro Nicole E. , Thorne Stephen H. , Wang Tao , van Houten Ben , Lotze Michael T. , de Vera Michael 
Published: February 2011

Abstract
Pyruvate, a key metabolite in cellular energy metabolism, is the end-product of glycolysis and the starting substrate for the TCA cycle which generates NADH and ATP during oxidative phosphorylation. According to the Warburg hypothesis, increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors. We have previously shown that ethyl pyruvate (EP), the ethyl ester of pyruvate, significantly inhibits the growth of hepatic tumors. The mechanism of this anti-tumor effect is unclear, and here we hypothesized that EP alters tumor cell metabolism. We analyzed the bioenergetic phenotype of tumor cells by measuring cellular respiration and glycolysis rate using a novel real-time assessment of oxidative phosphorylation and glycolysis.
METHODS: C57BL/6 mice were injected with 2×105 MC38 colorectal and PANCO2 pancreatic tumor cells via the portal vein. In vitro, 4x104 MC38 and PANCO2 cells/well were seeded into XF24 V7 microplates. Cells were pretreated with/without EP (5-10mM) for 60 minutes before measurement. The XF24 Analyzer (Seahorse Bioscience) was used to measure extracellular flux changes of oxygen consumption rate (OCR, measure of oxidative phosphorylation) and the rate of proton excretion into the media (extracellular acidification rate, ECAR, measure of glycolytic rate).
RESULTS: Mice injected with MC38 and PANCO2 cells resulted in the growth of bulky tumors in the liver. EP (80 mg/kg ip) administered 30 minutes prior to infusion of tumor cells and continuing daily for 9 days significantly inhibited tumor growth in a dose-dependent fashion, with 80 mg/kg EP achieving &gt;70% reduction in the number of tumor nodules in comparison to untreated animals. TUNEL staining demonstrated a marked increase in tumor cell apoptosis in EP-treated mice. MC38 and PANCO2 tumor cells had substantially different bioenergetic phenotypes. MC38 cells had lower basal OCR (148 ± 13 vs 434 ± 32 pmoles/min, p&lt; 0.05) and higher basal ECAR than PANCO2 cells (56 ± 6 vs 8 ± 2 mpH/min, respectively, p&lt; 0.05). Use of 2-deoxyglucose (2-DG) to block glycolysis significantly enhanced OCR in PANCO2 from 437 ± 50 to 1486 ± 154 pmoles/min but not in MC38, suggesting that MC38 cells are more dependent on glycolysis. EP enhanced mitochondrial cellular respiration (OCR) in MC38 and decreased basal ECAR significantly without observable changes in PANCO2 cells, suggesting a reversal of the Warburg phenomenon. SUMMARY: Altering tumor cell metabolism by increasing mitochondrial oxidative phosphorylation and decreasing glycolysis may contribute to the antitumor activity of EP.
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 60.