Cytosolic NADH as an intracellular modulator of sphingolipid-mediated apoptosis by inhibitors of a cell-surface tumor-associated NADH oxidase

Thomas De Luca, Purdue University

Abstract

ENOX2, formerly designated tNOX, is a tumor-associated cell surface ubiquinol (NADH) oxidase that functions in cells as the terminal oxidase for plasma membrane electron transport. Ubiquitous in all cancer cell lines studied thus far, its expression is believed to support abnormally high glycolytic metabolism of cancer cells by oxidizing pyridine nucleotides (NADH) at the inner leaflet of the plasma membrane. This activity correlates with the abnormal growth and division observed in cells displaying a malignant phenotype. ENOX2 has been proposed as the cellular target for various quinone site inhibitors that demonstrate anticancer activity such as the green tea constituent epigallocatechin-3-gallate (EGCg) and the pharmacologically designed isoflavone phenoxodiol (PXD). Here we present a possible mechanism that explains how these drugs cause apoptosis in cancer cells by ENOX2-mediated alterations of NAD+/NADH content in the cytosol. When ENOX2 is inhibited and plasma membrane electron transport is diminished, fluorescence measurements demonstrate that NADH accumulates at the cytosolic surface of the plasma membrane. Furthermore, both cell culture and cell-free in vitro assays indicate that the different redox states of NAD(H) modulate the activites of two pivotal enzymes of sphingolipid metabolism: sphingosine kinase 1 (SK1) and neutral sphingomyelinase (nSMase). The respective products sphingosine 1-phosphate (S1P) and ceramide (Cer) are key determinants of cell fate: S1P promotes cell survival and Cer promotes apoptosis. Using plasma membranes isolated from cervical adenocarcinoma (HeLa) cells as well as purified proteins of both bacterial and human origin, we demonstrate that NADH inhibits SK1 and stimulates nSMase, while NAD+ inhibits nSMase and has no noticeable effect on SK1. Both radiolabeled substrates and colorimetric determination of lipid products separated by thin layer chromatography confirm that whole cells treated with ENOX2 inhibitors demonstrate an increase in Cer and a decrease in S1P. Treatments that stimulate cytosolic NADH production potentiate the antiproliferative effects of ENOX2 inhibitors while those that attenuate NADH production or stimulate plasma membrane electron transport confer a survival advantage. Lastly, experiments with inhibitors of transcription, translation and protein phosphatases suggest these actions occur independently of gene expression and interfere with proliferative kinase cascades by preventing phosphorylation of Akt.

Degree

Ph.D.

Advisors

Morre, Purdue University.

Subject Area

Molecular biology|Biochemistry|Nutrition

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