Reciprocal relationship between cytosolic NADH and ENOX2 inhibition triggers sphingolipid-induced apoptosis in HeLa cells.
ENOX2 (tNOX), a tumor-associated cell surface ubiquinol (NADH) oxidase, functions as an alternative terminal oxidase for plasma membrane electron transport. Ubiquitous in all cancer cell lines studied thus far, ENOX2 expression correlates with the abnormal growth and division associated with the 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 isoflavone phenoxodiol (PXD). Here we present a possible mechanism that explains how these substances result in apoptosis in cancer cells by ENOX2-mediated alterations of cytosolic amounts of NAD(+) and NADH. When ENOX2 is inhibited, plasma membrane electron transport is diminished, and cytosolic NADH accumulates. We show in HeLa cells that NADH levels modulate the activities of two pivotal enzymes of sphingolipid metabolism: sphingosine kinase 1 (SK1) and neutral sphingomyelinase (nSMase). Their 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 effect on SK1. Additionally, intact HeLa cells treated with ENOX2 inhibitors exhibit 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.
De Luca
Thomas
T
Department of Foods and Nutrition, Purdue University, Stone Hall, 700 W. State Street, West Lafayette, Indiana 47907-2059, USA.
Morré
Dorothy M
DM
Morré
D James
DJ
D016588:Anticarcinogenic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology*; D008246:Lysophospholipids / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D000099157:Sphingosine Kinase
D016588:Anticarcinogenic Agents; D002392:Catechin; D002518:Ceramides; D007529:Isoflavones; D008246:Lysophospholipids; D009097:Multienzyme Complexes; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D017853:Phosphotransferases (Alcohol Group Acceptor); D013107:Sphingolipids; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate; C471183:phenoxodiol; C045651:epigallocatechin gallate; C023107:NADH oxidase