Metabolite modulation of HeLa cell response to ENOX2 inhibitors EGCG and phenoxodiol

BackgroundConstituents and inhibitors of intermediary metabolism resulting in alterations in levels of cytosolic NADH, stimulation of sphingomyelinase and inhibition of sphingosine kinase were evaluated for effects on growth inhibition and induction of apoptosis by the ENOX2 inhibitors EGCG, the principal catechin of green tea, and phenoxodiol, a naturally occurring isoflavone.MethodsResponses were evaluated from dose–response curves of the metabolites and metabolic inhibitors in which growth of HeLa cells, apoptosis based on DAPI fluorescence and cytosolic NADH levels were correlated with sphingomyelinase and spingosine kinase activities and levels of ceramide and sphingosine1-phosphate.ResultsGrowth inhibition correlated with the modulation of localized cytosolic NADH levels by metabolites and metabolic inhibitors, the response of sphingomyelinase and sphingosine kinase located near the inner surface of the plasma membrane, and apoptosis.ConclusionsBased on findings with metabolites, we conclude that apoptosis in cancer cell lines caused by ENOX2 inhibitors such as EGCG and phenoxodiol is a direct response to elevated levels of cytosolic NADH that result from ENOX2 inhibition.General significanceThe findings help to explain why increased NADH levels resulting from ENOX2 inhibition result in decreased prosurvival sphingosine-1-phosphate and increased proapoptotic ceramide, both of which may be important to initiation of the ENOX2 inhibitor-induced apoptotic cascade.

Research highlights
► ENOX2 is a cancer-specific terminal oxidase of mammalian plasma membrane redox.
► When blocked by inhibitors EGCG or phenoxodiol, cytosolic NADH levels increase.
► Increased NADH inhibits prosurvival sphingosine kinase.
► Increased cytosolic NADH levels activate proapoptotic sphingomyelinase.
► Metabolites induce apoptosis in direct proportion to NADH levels.