The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect
<p>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<sup>+</sup>/NADH) and nicotinamide adenine dinucleotide phosphate (NADP<sup>+</sup>/NADPH), and the universal energy carrier, adenine triphosphate (ATP/ADP). Transitions in CCM are reported to depend on these bio-oscillators. The NAD<sup>+</sup>/NADH ratio measures the glycolytic flux (glycolysis, PPP, and oxidative phosphorylation (OXPHOS). A high NADP<sup>+</sup>/NADPH ratio rewires glucose oxidation to the pentose phosphate pathway, whereas a low NADP<sup>+</sup>/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.</p> "> Figure 2
<p>Comparison of the energetic status of normal and cancer cells. These histograms represent the average of ATP, pHi, NAD<sup>+</sup>/NADH, NADP<sup>+</sup>/NADPH cancer/normal ratios over the cell cycle. Raw data for all experiments are reported in the Appendix Section. (<b>A</b>) In normal cells, the average ATP concentration over the cell cycle is higher compared to the cancer cell population (ratio = 0.60 ± 0.19). The intracellular pH (pHi) of cancer cells is slightly higher than normal cells (ratio = 1.05 ± 0.04); and (<b>B</b>) the redox ratios (NAD<sup>+</sup>/NADH and NADP<sup>+</sup>/NADPH) are five and ten time higher in cancer cells compared to the normal cell population (5.31 ± 3.76 and 11.15 ± 11.71, respectively).</p> "> Figure 3
<p>Redox signatures of normal and cancer cells during cell cycle progression. (<b>A</b>) In normal cells, the ATP concentration oscillates during the cell cycle: it is high in G0 (9.25 ± 0.40 pmoles/µg of protein) and S (8.29 ± 0.62 pmoles/µg of protein) and lower in G1/S (5.85 ± 0.57 pmoles/µg of protein) and G2/M (4.18 ± 0.29 pmoles/µg of protein). In cancer cells, ATP concentration is about twice lower in G0 (3.49 ± 0.25 pmoles/µg of protein), slightly increases from G0 to G1/S (4.6 ± 0.19 pmoles/µg of protein), and decreases from G1/S to G2/M (3.25 ± 0.11 pmoles/µg of protein); (<b>B</b>) in normal cells, the NAD<sup>+</sup>/NADH ratio oscillates between 5 and 10. For the cancerous cell line, the ratio increases from G0 (31.03 ± 1.54) and reaches a maximum value of 55 in S. Then, it declines at the G2/M transition (14); (<b>C</b>) the NADP<sup>+</sup>/NADPH ratio slightly increases from G0 (0.13 ± 0.05) to G1 (0.32 ± 0.04) in the normal cell population and stays stable. For the cancer cell population, a major peak is observed in S (5.68 ± 2.68); and (<b>D</b>) the intracellular pH (pHi) of cancer cells is alkaline and globally more acidic for normal cells. It oscillates from G0 (pH = 6.87 ± 0.10) to G1/S (pH = 7.29 ± 0.13), and then shows a marked decrease in S (pH = 6.78 ± 0.10). The cancerous cell population loses the pHi drop in the S phase and is much more alkaline (pH = 7.50 ± 0.12).</p> "> Figure 4
<p>ATP, intracellular pH (pHi) and redox (NAD/NADH, NADP<sup>+</sup>/NADPH) concentrations and variations throughout colon normal and cancer cell cycle.</p> "> Figure 5
<p>MMP, ATP, pHi and redox species concentrations in normal and cancer cell lines. (<b>A</b>) MMP are measured in human and mouse normal and cancer cell lines. FCCP is a compound that carries protons across the IMM and dissipates the electrochemical gradient (membrane potential). In order to maintain the membrane potential, the mitochondria need to increase the flow of electrons and thus oxygen consumption. Optimizing the concentration of the uncoupler (FCCP) reveals the maximal respiration for the sample; (<b>B</b>) The ATP concentration is lower in HT-29 compared to normal primary cells; (<b>C</b>) The intracellular pH (pHi) is alkaline in HT-29; (<b>D</b>) The NAD<sup>+</sup>/NADH ratio is higher in HT-29 cancerous line compared to normal primary cells; (<b>E</b>) The NADP<sup>+</sup>/NADPH ratio is higher in HT-29 cancerous line. Abbreviations: MMP (mitochondrial membrane potential); FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); IMM (internal mitochondrial membrane); HPC (Hematopoietic progenitor cells), LL/2 (mouse Lewis lung cancer cell line); MPC (mouse normal pheochromocytoma cell line).</p> ">
Abstract
:1. Introduction
2. Results
2.1. ATP Concentration Is Reduced in Colon Cancer Cells
2.2. Colon Cancer Cells Have a Reductive Energetic Status
2.3. The Intracellular pH of Cancer Cells Is Alkaline
2.4. Reduced Mitochondrial Membrane Potential in Immortal Cancer Cell Lines
3. Discussion
4. Materials and Methods
4.1. Cell Suspension Preparation and Centrifugal Elutriation
4.2. Fluorescence and Luminescence Measurements
4.3. pHi Measurement
4.4. Reduced Mitochondrial Membrane Potential in Immortal Cancer Cell Lines
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Appendix
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Moreira, J.D.V.; Hamraz, M.; Abolhassani, M.; Bigan, E.; Pérès, S.; Paulevé, L.; Nogueira, M.L.; Steyaert, J.-M.; Schwartz, L. The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect. Metabolites 2016, 6, 33. https://doi.org/10.3390/metabo6040033
Moreira JDV, Hamraz M, Abolhassani M, Bigan E, Pérès S, Paulevé L, Nogueira ML, Steyaert J-M, Schwartz L. The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect. Metabolites. 2016; 6(4):33. https://doi.org/10.3390/metabo6040033
Chicago/Turabian StyleMoreira, Jorgelindo Da Veiga, Minoo Hamraz, Mohammad Abolhassani, Erwan Bigan, Sabine Pérès, Loïc Paulevé, Marcel Levy Nogueira, Jean-Marc Steyaert, and Laurent Schwartz. 2016. "The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect" Metabolites 6, no. 4: 33. https://doi.org/10.3390/metabo6040033