The Warburg Effect and Mass Spectrometry-based Proteomic Analysis

Abstract

Compared to normal cells, cancer cells have a unique metabolism by performing lactic acid fermentation in the presence of oxygen, also known as the Warburg effect. Researchers have proposed several hypotheses to elucidate the phenomenon, but the mechanism is still an enigma. In this review, we discuss three typical models, such as "damaged mitochondria", "adaptation to hypoxia", and "cell proliferation requirement", as well as contributions from mass spectrometry analysis toward our understanding of the Warburg effect. Mass spectrometry analysis supports the "adaptation to hypoxia" model that cancer cells are using quasi-anaerobic fermentation to reduce oxygen consumption in vivo. We further propose that hypoxia is an early event and it plays a crucial role in carcinoma initiation and development.

Keywords: Cancer metabolism; Warburg effect; mass spectrometry; review.

Figure 1. Oxidative phosphorylation in mammalian cell. Electrons are transferred from NADH and FADH2 to oxygen in redox reactions. These redox reactions are carried out by a series of protein complexes (NADH dehydrogenase or complex I, succinate dehydrogenase or complex II, cytochrome c reductase or complex III, cytochrome c oxidase or complex IV). The energy released by electrons flowing through the electron transport chain is used to transport protons across the inner mitochondrial membrane. This pH gradient generates an electrical potential energy across the membrane. When protons flow back across the membrane through ATP synthase, the energy is stored in ATP (1). Cytochrome c oxidase mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane (2, 3).

Figure 2. The link of glycolysis and glutaminolysis. Pyruvate and glutamate, products from glycolysis and glutaminolysis, are substrates for Krebs cycle. Separately, glutamine and fructose-6-phosphate (product from glycolysis) are used for hexosamine biosynthesis and glycosylation, catalyzed by glutamine-fructose-6-phosphate transaminase. Additionally, glutamine can be hydrolyzed to glutamate and ammonia (NH₃) by glutaminase. The resulted ammonia can react with lactic acid to form ammonium lactate to help the pH balance in cancer cells.

Figure 3. Heat production in mammalian cells. About 2,880 kJ mol^–1 of energy is released when burning glucose in air (C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O). The same amount of energy will be released by aerobic respiration in mammalian cells; however, the chemical energy is first stored in synthesized ATP by a series of biochemical reactions including glycolysis, Krebs cycle, and oxidative phosphorylation, and the energy stored in ATP can then be released in a controlled way to drive processes requiring energy such as biosynthesis and transportation of molecules. Surprisingly, mitochondrial oxidative phosphorylation as shown in Figure 1 is not perfectly coupled to ATP synthesis. Proton-leak catalyzed by uncoupling proteins in adipocytes accounts for a significant part of the resting metabolic rate (57). Separately, although the last destination for an electron along the electron transport chain is an oxygen molecule to form water catalyzed by cytochrome c oxidase in normal conditions, a certain percentageof electrons can be leaked before this reaction takes place and oxygen is prematurely and incompletely reduced to superoxide radical (O₂ + e– →O₂–) (58). In compliance with the relationship of chemical reaction rate and concentration, it is expected that less superoxide radical can be formed in hypoxia condition. Remarkably, it is well-established that mitochondrial superoxide dismutase (Mn-SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide (Mn³+-SOD + O₂− → Mn²+-SOD + O₂, Mn²+-SOD + O₂− + 2 H⁺ → Mn³+-SOD + H₂O₂). Catalase, which is concentrated in peroxisomes located next to mitochondria, reacts with the hydrogen peroxide to catalyze the formation of water and oxygen (2 H²O² → 2 H₂O + O₂). Consequently, the combined reaction catalyzed by superoxide dismutase and catalase is 4 O₂− + 4 H+ → 2 H₂O + 3 O₂, and energy stored in superoxide radical is released as heat

Figure 4. Mechanisms of cellular adaptation. Living organisms face a succession of environmental challenges as they grow and develop. In response to the imposed conditions, organisms are equipped with adaptive traits that are maintained and evolved by means of natural selection. Adaptation can be achieved by three typical mechanisms at the molecular and cellular level. First, reversible post-translational modifications (PTMs), such as phosphorylation, acetylation, and methylation, are involved in rapid adaption of cellular exposure to stimulus. At physiological pH, the side chains of Ser/Thr/Tyr are not charged, the side chain of lysine is cationic, and the side chains of Asp/Glu are negatively charged. Consequently, phosphorylation of Ser/Thr/Tyr will introduce negative charge to these amino acid residues; N-acetylation of lysine side chain will quench the positive charge; methylation on carboxylate side chain will cover up a negative charge and add hydrophobicity. PTMmediated adaptation can be quickly achieved by affecting of protein’s catalytic activity, localization in the cell, stability, and the ability to form complex with other molecules. Second, by gene regulation, cells can express protein when needed. For example, Jacob and Monod et al. discovered the lac operon regulation system in the genome of E. coli in which some enzymes involved in lactose metabolism are expressed in the presence of lactose and absence of glucose (64). Since this mechanism requires de novo mRNA and protein synthesis, it is a slower response compared to PTMs. Third, in extreme conditions, cells are prepared to resort to gene mutation for survival if the above two mechanisms fail. The mutant protein gains a new catalytic activity that helps the cell to overcome harsh conditions. Although the intrinsic mutation rate is quite low (65), the rate can be increased under selection pressure such as hypoxia. This mechanism is supported by reports that sub-lethal antibiotic treatment can increase bacteria’s mutation rate and drug resistance (66-68).