Reciprocal Relationship Between Cytosolic NADH and ENOX2 Inhibition Triggers Sphingolipid‐Induced Apoptosis in HeLa Cells

Abstract

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.

The novel antiangiogenic VJ115 inhibits the NADH oxidase ENOX1 and cytoskeleton-remodeling proteins

“…The pathway analysis of the down-regulated proteins identified c-Jun as a common regulator and immunoblotting confirmed that phosphorylation of c-Jun was suppressed by VJ115 (Supplemental Figure 2). Recently, De Luca et al [35] have shown that inhibition of ENOX activity and the subsequent increase in NADH resulted in sphingosine kinase inhibition. Su et al [36] found that sphingosine 1-phosphate positively regulates cJun.…”

Cancer prevention trial of a synergistic mixture of green tea concentrate plus Capsicum (CAPSOL-T) in a random population of subjects ages 40-84

“…The approach to early intervention is based on previous work in cell culture models showing that ENOX2 proteins are required to support the unregulated growth that typifies cancer cells [7]. If the growth function of ENOX2 is blocked for 48 to 72 h, the cancer cells cannot enlarge following division, cannot pass the checkpoint in G1 that monitors cell size and eventually undergo programmed cell death (apoptosis) [22,37,43]. As an early intervention strategy targeted to ENOX2, the herbal mixture of green tea and a powdered efficacious source of chili peppers ( Capsicum species) with levels of capsaicin sufficiently low so as to not cause discomfort (Capsol-T®), emerges as being effective in reducing ENOX2 levels below the limits of detection in greater than 90% of the subjects thus far evaluated when combined with early detection based on ONCOblot® Tissue of Origin Cancer Test results.…”

Early Detection: An Opportunity for Cancer Prevention Through Early Intervention

“…The approach to early intervention is based on previous work in cell culture models showing that ENOX2 proteins are required to support the unregulated growth that typifies cancer cells. If the growth function of ENOX2 is blocked for 48 to 72 h, the cancer cells cannot enlarge following division, cannot pass the checkpoint in G 1 that monitors cell size and eventually undergo programmed cell death (apoptosis) (Morré and Morré, 2003b;De Luca et al, 2010) as diagrammed in Figure 1. Among the early intervention strategies under investigation are several targeted to ENOX2, production of ENOX2-directed vaccines being one promising example.…”