Extracellular pH, lactate, and a hypothesis for NADH-associated proton dynamics in tumor cells

Abstract

Event Abstract Back to Event Extracellular pH, lactate, and a hypothesis for NADH-associated proton dynamics in tumor cells Angela M. Otto1* and Cornelia Janzon2 1 Technische Universitaet Muenchen, Institute of Medical Engineering (IMETUM), Germany 2 Klinikum Leverkusen, Abteilung Onkologie/Haematologie, Germany Background and aim. The microenvironment of tumor cells is characterized by a gradual deprivation of nutrients and oxygen, an accumulation of lactate, and increased acidification. The question is how variable combinations of these components change tumor cell growth and metabolic activity in quantitative terms. The aim here is to investigate how extracellular lactate and pH affect this metabolic activity under limiting nutrient conditions. Methods. The breast cancer cell line MCF-7 was cultivated in “low growth” medium (DMEM with reduced FCS, containing tumor-relevant concentrations of glucose, glutamine as well as lactate, and set at different pH (6.6 – 7.4) for up to 4 days. Growth was determined by cell count. The WST-assay was used as a measure of plasma membrane NADH-oxidase activity (1). The activities of pyruvate kinase and lactate dehydrogenase (LDH) in cell lysates were measured by the kinetics of NADH-based changes in absorption. Lactate production was determined in cell culture supernatants using LDH activity as a read out. Results. At pH 7.4 with combinations of variable glucose concentrations and 0.1 mM glutamine, tumor cell growth was low, but the activity of NADH oxidase activity markedly increased at 2.5. mM glucose. Pyruvate kinase activity was also increased, while LDH activity remained 2-3-fold lower than that of pyruvate kinase. The inhibition of mitochondrial respiration by rotenone transiently increased NADH oxidase activity further. Lactate (25 mM) further increased NADH oxidase activity, but enhanced growth only at limiting conditions. At an acidic pH (6.6), these tumor cells become virtually independent of glucose and glutamine levels for both growth and the metabolic parameters tested. The culture medium of such cells contained only 7% of the lactic acid measured for cultures at pH 7.4, suggesting attenuated lactate release. Conclusions / Discussion. Extracellular pH has a differential effect on the replenishment of NAD+ from NADH produced by glycolysis. This is not solely accomplished by LDH, but may also involve plasma membrane NADH oxidase activity. Furthermore, lactate may be converted to pyruvate and support the TCA-cycle, thereby enhancing mitochondrial NADH levels. The resistance of NADH oxidase activity to rotenone suggests that reduction equivalents, along with the respective protons, are shuttled from the mitochondria to the cytosol. A hypothesis is proposed to explain the changes in plasma membrane NADH oxidase activity with respect to glycolysis and the tricarbonic acid cycle. Acknowledgements Part of this work was supported by the German Research Council (DFG). References (1) Berridge, M.V. and Tan, A.S. (1998) Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1. Protoplasma, 205:74-82. Keywords: Tumor Cells, Cultured, nutrient deprivation, Glucose, Glutamine, extracellular pH, tumor cell metabolism, Pyruvate Kinase, Lactate Dehydrogenases, extracellular lactate, NADH-oxidase, cell growth analysis Conference: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer, Garching, Germany, 10 Oct - 12 Oct, 2013. Presentation Type: Abstract Topic: 4. pH and energy metabolism Citation: Otto AM and Janzon C (2014). Extracellular pH, lactate, and a hypothesis for NADH-associated proton dynamics in tumor cells. Front. Pharmacol. Conference Abstract: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer. doi: 10.3389/conf.fphar.2014.61.00031 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Dec 2013; Published Online: 07 Feb 2014. * Correspondence: Dr. Angela M Otto, Technische Universitaet Muenchen, Institute of Medical Engineering (IMETUM), Garching, 85748, Germany, otto@tum.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Angela M Otto Cornelia Janzon Google Angela M Otto Cornelia Janzon Google Scholar Angela M Otto Cornelia Janzon PubMed Angela M Otto Cornelia Janzon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.