Article The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect Jorgelindo da Veiga Moreira 1,*, Minoo Hamraz 2, Mohammad Abolhassani 3, Erwan Bigan 1, Sabine Pérès 4,5, Loïc Paulevé 4, Marcel Levy Nogueira 1,6, Jean-Marc Steyaert 1 and Laurent Schwartz 1,* 1 Laboratoire d’Informatique du l’X–Unité Mixte de Recherche 7161, Ecole Polytechnique, Palaiseau 91128, France; [email protected] (E.B.); [email protected] (M.L.N.); [email protected] (J.-M.S.) 2 Imen Pharmed Iranian Company (IPI), Tehran 1514633711, Iran; [email protected] 3 Nosco Pharmaceuticals, Paris 75015, France; [email protected] 4 Laboratoire de Recherche en Informatique (LRI), Université Paris-Sud, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8623, Orsay 91190, France; [email protected] (S.P.); [email protected] (L.P.) 5 MaIAGE, INRA, Université Paris-Saclay, Jouy-en-Josas 78350, France 6 Institute of Translationnal Neurosciences (IHU-A-Institut du Cerveau et de la Moelle Epinière), Pitié Salpêtrière Hospital, Paris 75013, France * Correspondence: [email protected] (J.V.M.); [email protected] (L.S.); Tel.: +33-1-80-35-07-20 (J.V.M.); + 33-1-77-57-80-49 (L.S.) Academic Editor: Peter Meikle Received: 12 August 2016; Accepted: 27 September 2016; Published: 3 October 2016 Abstract: To better understand the energetic status of proliferating cells, we have measured the intracellular pH (pHi) and concentrations of key metabolites, such as adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP) in normal and cancer cells, extracted from fresh human colon tissues. Cells were sorted by elutriation and segregated in different phases of the cell cycle (G0/G1/S/G2/M) in order to study their redox (NAD, NADP) and bioenergetic (ATP, pHi) status. Our results show that the average ATP concentration over the cell cycle is higher and the pHi is globally more acidic in normal proliferating cells. The NAD+/NADH and NADP+/NADPH redox ratios are, respectively, five times and ten times higher in cancer cells compared to the normal cell population. These energetic differences in normal and cancer cells may explain the well-described mechanisms behind the Warburg effect. Oscillations in ATP concentration, pHi, NAD+/NADH, and NADP+/NADPH ratios over one cell cycle are reported and the hypothesis addressed. We also investigated the mitochondrial membrane potential (MMP) of human and mice normal and cancer cell lines. A drastic decrease of the MMP is reported in cancer cell lines compared to their normal counterparts. Altogether, these results strongly support the high throughput aerobic glycolysis, or Warburg effect, observed in cancer cells. Keywords: redox oscillators; Warburg effect; cancer disease; mitochondria; central carbon metabolism 1. Introduction The central carbon metabolism (CCM) is a metabolic blueprint shared by almost all living organisms. It consists of a complex series of enzymatic reactions that convert carbon sources (carbohydrates, proteins, lipids) into biomass and energy [1]. The core of the CCM is composed of glycolysis, the pentose phosphate pathway (PPP), the tricarboxylic acid cycle (TCA), and the fatty- acid pathway (Figure 1). The shift either from oxidative catabolism (energy production) to reductive anabolism (biomass synthesis), or from anabolism to catabolism, appears to be controlled by bio- Metabolites 2016, 6, 33; doi:10.3390/metabo6040033 www.mdpi.com/journal/metabolites Metabolites 2016, 6, 33 2 of 12 oscillators, playing the role of co-enzymes [2]. These bio-oscillators include reductive and oxidative (redox) couples, such as nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH), the universal energy carrier, adenosine triphosphate (ATP), the transmembrane potential (TMP), and, finallyeast, the intracellular pH (pHi). The dynamics of these internal biological rhythms are shown to oscillate when eukaryotic cells enter proliferation [2–4]. These orchestrated oscillations ensure genome duplication and cell membrane synthesis prior to cell division [5,6]. Figure 1. The central carbon metabolism (CCM). The CCM combines enzymatic reactions that convert carbon sources into biomass precursors. This figure shows the five main pathways forming the CCM: glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle (TCA), lipogenesis, and beta-oxidation. Two opposite metabolic demands are at the core of the CCM: anabolic reactions, which consist in biomass synthesis, and catabolic reactions, leading to the breakdown of macromolecules for energetic use. These two aspects of cell metabolism are managed by biochemical oscillators, including redox couples, such as nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH), and the universal energy carrier, adenine triphosphate (ATP/ADP). Transitions in CCM are reported to depend on these bio-oscillators. The NAD+/NADH ratio measures the glycolytic flux (glycolysis, PPP, and oxidative phosphorylation (OXPHOS). A high NADP+/NADPH ratio rewires glucose oxidation to the pentose phosphate pathway, whereas a low NADP+/NADPH ratio triggers lipogenesis. The ATP/(ADP + Pi) ratio senses the metabolic state of the cell and may lead to metabolic switches in the CCM. The eukaryotic cell cycle has been shown to be a redox cycle [7–10]. When resting cells are committed to division, it enters the first growth phase (G1). Then follows genome duplication in the S phase and the second growth phase (G2), prior to mitosis (M). As opposed to proliferating cells, differentiated cells have a basal oxidative metabolism. Pyruvate converted from glucose or acetyl- CoA obtained from beta-oxidation of fatty acids are degraded by the TCA cycle (Figure 1) [11]. The oxidative phosphorylation of acetyl-CoA into mitochondria yields large amounts of ATP and releases carbon dioxide and water as waste products [2]. Thus, contrary to differentiated cells, rapidly proliferating cells, such as stem cells or cancer cells, use the aerobic glycolysis for ATP synthesis, even in the presence of oxygen. This is known as the “Warburg effect”, in reference to the Nobel laureate Otto Heinrich Warburg who first reported the high glycolytic flux in proliferating tumors [12–14]. In G1 of the cell cycle, the shift from pyruvate to lactate may be conditioned by the NAD+/NADH ratio and is thought to support the high glycolytic demand in cancer cells [5,12]. Thus, the Warburg effect may also be characterized by a high NAD+/NADH ratio. In the S phase, the CCM may shift to the Metabolites 2016, 6, 33 3 of 12 PPP, the main route to nucleotide synthesis [4,5]. High metabolic flux through the PPP supplies enough NADPH to protect proliferating cells against oxidative stresses and triggers lipid synthesis in G2 [15–17]. Moreover, mitochondria activity is key to cell decision-making and cell cycle progression [18]. These organelles were first considered as a mere “powerhouse” of the eukaryotic cell, before the pioneering studies that deciphered their key role in such processes as cell development, cell survival, cell division, and cell death [18]. Regarding cell division, investigations support and highlight the idea of intertwined relationships between morphological changes in mitochondria and their signaling activity [18–20]. Subsequently, it has been demonstrated that energetic transitions occurring in the cell cycle are intrinsically related to mitochondrial ability to sense state parameters, such as pHi and the ATP/ADP ratio [21–23]. These measurements of pHi, ATP/ADP, and redox species are reported in several species, in different environmental conditions, but lack consistencies (pHi variations are contradictory [3,4,22]) and comprehensive overview of bio-oscillators variation (in yeast or human cancer cells [4,11,22]). Therefore, there is a need for a comprehensive analysis of changes in pHi, ATP/ADP, NAD+/NADH, and NADP+/NADPH during the proliferating cell cycle for healthy and cancerous human cells. Such analysis is a first step towards a holistic understanding of the coupling between electrochemistry and metabolism, and could ultimately lead to a better understanding of the principles operating current metabolic therapies and to identify new ones [24]. In this work, we studied the redox and energetic variations in freshly isolated normal and cancer cells extracted from human patients and controls (n = 8). 2. Results Human normal and cancer cells have been extracted from their respective tissues and isolated in five populations by the method of elutriation. This segregation, based on cell density, resulted in suspensions of cells in G0, G1, G1/S transition, S and G2/M transition phase of the cell cycle. By applying fluorometric measurements in each cell suspension, both in the normal and cancerous population, we managed to quantify the amount of ATP, NAD+, NADH, NADP+, NADPH, as well as the intracellular pH (pHi). Figures 2 and 3 show experimental results obtained from eight patients (n = 8) for both the normal and the cancerous region of the colon. Figure 2. Comparison of the energetic status of normal and cancer cells. These histograms represent the average of ATP, pHi, NAD+/NADH, NADP+/NADPH cancer/normal ratios over the cell cycle. Raw data for all experiments are reported in the supplementary materials section. (A) In normal cells, the average ATP concentration over