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Introduction
Activated T cells rely on aerobic glycolysis and glutaminolysis in order to proliferate and differentiate into effector T cells (1,2). Intervention in these metabolic pathways inhibits the proliferation of activated T cells and their differentiation to effector cells. For example, treatment of activated T cells with 2-deoxy-D-glucose abrogates their proliferation and differentiation into effector T cells by inhibiting glycolysis at the level of hexokinase II (HKII) (3,4). A similar effect is yielded by treatment with dichloroacetate, which activates pyruvate dehydrogenase (PDH) through the inhibition of pyruvate dehydrogenase kinase (PDK) (3,5). The PDH dehydrogenase regulates the influx of pyruvate into the mitochondria, increasing the ratio of glucose oxidation to glycolysis. Notably, indoleamine 2,3-dioxygenase, a key immunomodulatory enzyme, exerts its suppressive effect at least in part by inhibiting aerobic glycolysis and glutaminolysis in activated T cells (6–8).
To date, the effect of Krebs' cycle inhibition on the proliferation of activated T cells has not been fully evaluated. For this purpose, the present study was devised to investigate the activity of the 3-(aryloxyacetylamino)-4-hydroxybenzoic acid derivative LW6. LW6 was originally considered to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α) (9). However, a further study by the same investigators revealed that LW6 is a specific malate dehydrogenase-2 (MDH2) inhibitor (10). Furthermore, the inhibition of HIF-1α is secondary and results from its degradation due to increased intracellular O2 induced by LW6. By inhibiting the Krebs' cycle, LW6 decreases the production of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), which are electron donors required for oxidative phosphorylation, and thus reduce O2 consumption (10). The specific Krebs' cycle inhibitor LW6 was selected because suppression at the MDH2 level may prevent total cell energy collapse due to pyruvate-malate cycling. Accumulated malate exits the mitochondria and enters the cytoplasm, where it is converted by malic enzyme to pyruvate, which may re-enter the mitochondria and the Krebs' cycle (11).
The aim of the present study was to evaluate the effects of the MDH2 inhibitor LW6 on human activated T-cell proliferation and survival. In addition, the effects of LW6 on the levels of certain transcription factors involved in cell proliferation, apoptosis and metabolism, and certain enzymes associated with glucose metabolism and glutaminolysis were evaluated, and Krebs' cycle activity was...