Abstract
Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.
Highlights
Pyridine nucleotides, such as nicotinamide adenine dinucleotide or its ribose-2′-phosphorylated derivative, are universal electron donors/acceptors in cellular processes
The opposite action of thiamin and oxythiamin on cellular NAD(P)H:resazurin oxidoreductase in both cell lines, cannot be due to the changes in cellular protein, which is affected by both thiamin and oxythiamin, but in T98G cells only
We show that the inhibitors of cellular NAD(P)H producers, oxythiamin and phosphonate analogs of 2-oxo acids, may cause a paradoxical increase in cellular NAD(P)H:resazurin oxidoreductase (Figures 1 and 5)
Summary
Pyridine nucleotides, such as nicotinamide adenine dinucleotide or its ribose-2′-phosphorylated derivative, are universal electron donors/acceptors in cellular processes They undergo reversible oxidation-reduction in a number of biological reactions and are abundant in cells. Dysfunctional cells are characterized by a changed level of reduction and depletion of total pool of the pyridine nucleotides, which prime the cells to enter the death pathway. This phenomenon underlies existing methods of monitoring cellular viability through the NAD(P)H-dependent reduction of artificial dyes—a side reaction catalyzed by a number of cellular dehydrogenases [8,9]. Appropriate caution is required when using resazurin reduction as a measure of cellular viability
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