Cell Surface NADH Oxidase Research Articles

The Cell Surface NADH Oxidase ENOX2 Is Highly Expressed in Chronic Myeloid Leukemia (CML): a Redox Protein As a Secreted Surrogate Biomarker and Druggable Target

Ecto-nicotinamide Adenine Dinucleotide Oxidase 2 (ENOX2) or Tumor-Associated Nicotinamide Adenine Dinucleotide Oxidase (tNOX), plays a major role as a membrane hydroxyquinone oxidase catalyzing the conversion of reduced NADH to the oxidized NAD+ .Cellular NAD+/NADH ratio plays a major role in the regulation of several metabolic pathways such cell cycle and apoptosis. The membrane NAD+/NADH ratio has been shown to be involved membrane lipid and sphingomyelin metabolism. Several lines of experimental data indicate that ENOX2, as compared to ENOX1 isoform, is preferentially expressed in cancer cells and ENOX2 is also secreted in the serum of patients with cancer and representing a surrogate marker for cancer progression as well as a drug target. The expression of ENOX2 in CML has not been studied so far. To this end, we have analyzed the expression of ENOX2 by Western blots in 40 patients with CML at diagnosis and compared this expression to that of control cells from healthy donors (n= 30). ENOX2 expression was found to be highly increased ( x 6.6 fold) in primary leukemic cells and this was highly significant ( p= 0.0081). To determine if ENOX2 expression is related directly to BCR-ABL expression, we have analyzed the expression of ENOX2 in UT7 cell line and its BCR-ABL-expressing counterparts UT7-11. As compared to parental UT7, ENOX2 expression was highly increased in UT7 cells expressing either native or T315I-mutated BCR-ABL. This increase was also documented in DOX- inducible cell line BaF/p210 sin1.55 in which activation of BCR-ABL expression correlated with ENOX2 expression. The expression of ENOX2 in CML cells was a tyrosine kinase-dependent event as demonstrated by Western blot experiments showing that ENOX2 expression was reduced UT7/11 cell line treated with Imatinib mesylate (1 microM) for 6, 18 and 24 hours whereas there was no change in ENOX2 expression in similarly treated parental UT7 cell line. As ENOX2 is a protein secreted from tumor cells, we have analyzed the levels of ENOX2 in the plasma of CML patients at diagnosis as compared to controls. A series of 41 patients with CML as compared to plasma from 28 healthy donors were analyzed by ELISA. This analysis showed a highly significant increase of ENOX2 protein levels in the plasma of patients with CML (mean levels of 800 pg/ml in CML versus 500 pg/ml, Mann-Whitney U-Test, p < 0.0001). There was no correlation between ENOX2 levels and leukocyte numbers at diagnosis. In order to determine the potential clinical value of ENOX2 expression in different phases of the disease, ENOX2 expression in CML CD34+ cells was compared to healthy donor samples in microarray dataset GSE 4170. CML patients in chronic phase overexpressed ENOX2 in CD34+ cells as compared to control (two-sided test with Welch correction p-value=0.00054). Gene expression pattern matching correlating ENOX2 in CML CD34+ cells was determined in three phases of the disease. Pavlidis template matching algorithm used with ENOX2 as predictor allowed to discover 301 genes correlated with CML in chronic phase. Unsupervised principal component analysis performed with ENOX2 pattern matching gene expression profile allowed us to discriminate the 3 phases of CML in CD34+ cell compartment in a highly significant manner (p-value 6.75E-15). Functional enrichment performed with ENOX2 pattern matching on Gene Ontology Biological Process database revealed implication of different pathways of cell signaling such as: Rho GTPase, MAPKs, GPCR, RAS and NOTCH pathways, the latter being connected to stem cell biology. This analysis also showed implication of metabolic functions such as carbohydrate homeostasis. Other functionalities that could act on hematopoiesis have been also highlighted by this analysis such as proliferation, integrin-mediated signaling, and circadian rhythm. Finally genes related to angiogenesis have been also found to be implicated in ENOX2 signalling such as placental growth factor (PLGF) also EPHB3 which is a receptor tyrosine kinase implicated in cell migration. Overall these results suggest that ENOX2 pathway plays a major role in the pathogenesis of CML and represents, to the best of our knowledge, the first surrogate secreted tumor marker in CML. ENOX2 expression correlates with disease progression and experiments are underway to determine the use of ENOX2 as a drug target in CML and T315I-mutated CML stem cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aging-Related Nicotinamide Adenine Dinucleotide Oxidase Response to Dietary Supplementation: The French Paradox Revisited

Aging-related cell-surface NADH oxidase (arNOX)-specific activities increase with age between age 30 and ages 50-65. The protein is shed and circulates. Activity correlates with a number of aging-related disorders including low-density lipoprotein (LDL) oxidation as a precondition to atherosclerosis as well as oxidation of collagen and elastin as a major contributor to skin aging. arNOX inhibitors formulated for sustained release are capable of maintaining circulating arNOX at low levels with regular use as food supplements formulated with natural compounds. Among the best sources are certain culinary seasonings, all of which are ingredients used extensively in the French kitchen. Their regular use may contribute to an understanding of the nutritional basis for the French Paradox.

Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells.

Full length mRNA to a drug-inhibited cell surface NADH oxidase, tNOX or ENOX2, is present in both non-cancer and cancer cells but is translated only in cancer cells as alternatively spliced variants. ENOX2 is a growth-related protein of the external plasma membrane surface that is shed into the circulation and is inhibited by a series of quinone site inhibitors with anticancer activity. To test the possibility that ENOX2 expression might be important to early stages of non-cancer cell development, the expression of the protein was monitored in chicken embryos during their development. Polyclonal antisera to a 34 kDa human serum form of ENOX2 cross-immunoreactive with the drug-responsive NADH oxidase of chicken hepatoma cells was used. The protein was identified based on drug-responsive enzymatic activities and analyses by western blots. The drug-responsive activity was associated with plasma membranes and sera of early chicken embryos and with chicken hepatoma plasma membranes but was absent from plasma membranes prepared from livers or from sera of normal adult chickens and from late embryo stages. The findings suggest that ENOX2 may fulfill some functions essential to the growth of early embryos which are lost in late embryo stages and absent from normal adult cells but which then reappear in cancer.

Mouse embryonic fibroblast cells from transgenic mice overexpressing tNOX exhibit an altered growth and drug response phenotype

Mouse embryonic fibroblast (MEF) cells prepared from transgenic mice overexpressing a cancer-specific and growth-related cell surface NADH oxidase with protein disulfide-thiol interchange activity grew at rates approximately twice those of wild-type embryonic fibroblast cells. Growth of transgenic MEF cells overexpressing tNOX was inhibited by low concentrations of the green tea catechin (-)-epigallocatechin-3-gallate (EGCg) or the synthetic isoflavene phenoxodiol. Both are putative tNOX-targeted inhibitors with anti-cancer activity. With both EGCg and phenoxodiol, growth inhibition was followed after about 48 h by apoptosis. Growth of wild-type mouse fibroblast cells from the same strain was unaffected by EGCg and phenoxodiol and neither compound induced apoptosis even at concentrations 100-1,000-fold higher than those that resulted in apoptotic death in the transgenic MEF cells. The findings validate earlier reports of evidence for tNOX presence as contributing to unregulated growth of cancer cells as well as the previous identification of the tNOX protein as the molecular target for the anti-cancer activities attributed to both EGCg and phenoxodiol. The expression of tNOX emerges as both necessary and sufficient to account for the cancer cell-specific growth inhibitions by both EGCg and phenoxodiol.

Anticancer activity of grape and grape skin extracts alone and combined with green tea infusions

Grapes and grape extracts were compared for inhibition of a growth-related and cancer-specific form of cell surface NADH oxidase with protein disulfide-thiol interchange activity designated tNOX from human cervical carcinoma (HeLa) cells and growth of HeLa and mouse mammary 4T1 cells in culture and transplanted tumors in mice. Grapes and grape extracts of several varieties had activity. With an extracted grape preparation provided by the California Table Grape Commission, an active fraction was eluted with methanol from a Diaion HP-20 column after removal of inactive water-soluble materials. Grape skins were a much more potent source than either grape pulp, juice or seeds. Ethanol extracts of the ground freeze-dried pomace was an excellent source. The grape extracts interacted, often synergistically, with decaffeinated green tea extracts both in the inhibition of tNOX activity and in the inhibition of cancer cell growth. Intratumoral injections of a 25:1 mixture of a green tea extract plus ground freeze-dried pomace was nearly as effective as standard synergistic green tea– Capsicum mixtures in inhibiting growth of 4T1 mammary tumors in situ in mice.

NAD+/NADH and/or CoQ/CoQH2 ratios from plasma membrane electron transport may determine ceramide and sphingosine‐1‐phosphate levels accompanying G1 arrest and apoptosis

To elucidate possible biochemical links between growth arrest from antiproliferative chemotherapeutic agents and apoptosis, our work has focused on agents (EGCg, capsaicin, cis platinum, adriamycin, anti-tumor sulfonylureas, phenoxodiol) that target tNOX. tNOX is a cancer-specific cell surface NADH oxidase (ECTO-NOX protein), that functions in cancer cells as the terminal oxidase for plasma membrane electron transport. When tNOX is active, coenzyme Q(10) (ubiquinone) of the plasma membrane is oxidized and NADH is oxidized at the cytosolic surface of the plasma membrane. However, when tNOX is inhibited and plasma membrane electron transport is diminished, both reduced coenzyme Q(10) (ubiquinol) and NADH would be expected to accumulate. To relate inhibition of plasma membrane redox to increased ceramide levels and arrest of cell proliferation in G(1) and apoptosis, we show that neutral sphingomyelinase, a major contributor to plasma membrane ceramide, is inhibited by reduced glutathione and ubiquinone. Ubiquinol is without effect or stimulates. In contrast, sphingosine kinase, which generates anti-apoptotic sphingosine-1-phosphate, is stimulated by ubiquinone but inhibited by ubiquinol and NADH. Thus, the quinone and pyridine nucleotide products of plasma membrane redox, ubiquinone and ubiquinol, as well as NAD(+) and NADH, may directly modulate in a reciprocal manner two key plasma membrane enzymes, sphingomyelinase and sphingosine kinase, potentially leading to G(1) arrest (increase in ceramide) and apoptosis (loss of sphingosine-1-phosphate). As such, the findings provide potential links between coenzyme Q(10)-mediated plasma membrane electron transport and the anticancer action of several clinically-relevant anticancer agents.

Mitochondrial gene‐knockout (ρ0) cells: A versatile model for exploring the secrets of trans‐plasma membrane electron transport

Plasma membrane electron transport (tPMET) pathways have been identified in all living cells, and a wide variety of tools have been used to study these processes. In our laboratory we have used the cell-impermeable tetrazolium dye WST-1, together with the mitochondrial gene knockout (rho0) cell model, to investigate one of these pathways. We have shown that growth of HL60rho0 cells is dependent on oxygen, and that these cells consume oxygen at the cell surface. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and WST-1 reduction suggest that both systems share a common tPMET pathway. In support of this, oxygen was shown to compete with the intermediate electron acceptor that mediates WST-1 reduction, for reducing electrons. The observation that tPMET activity is higher in rho0 cells compared to their mitochondrially-competent counterparts was shown to be the result of competition between the mitochondrial and plasma membrane electron transport systems for intracellular reducing equivalents. Elevated rates of dye reduction appear to be mediated through increased expression of the key components of tPMET, which include the cell surface NADH oxidase, CNOX. These findings have played a critical role in shaping our current understanding of the mechanisms of this particular pathway of tPMET.

Adriamycin tolerance in human mesothelioma lines and cell surface NADH oxidase

Adriamycin tolerant human mesothelioma cell lines derived from a single tumor prior to either chemotherapy or radiation therapy and a susceptible cell line were investigated. Not only was growth resistant to low doses of adriamycin but an unusual pattern of resistance was encountered in which cells seemed to better tolerate high adriamycin doses than intermediate doses. The differential growth susceptibility of the tolerant lines compared to A549 lung carcinoma and the bimodal dose response correlated with differences in the specific activity of a plasma membrane-associated NADH oxidase (NOX). Plasma membrane fractions of high purity were isolated by aqueous two-phase partition and assayed directly. The NADH oxidase activity of the plasma membranes for the susceptible cell line was maximally inhibited by 1 μM adriamycin whereas the NADH oxidase activity of the tolerant lines was less and was maximally inhibited by 0.1 μM adriamycin with 1 and 10 μM adriamycin being less inhibitory than 0.1 μM adriamycin. The findings suggest a relationship between the growth response to adriamycin of the adriamycin tolerant mesothelioma lines and the activity of the plasma membrane-associated NADH oxidase activity of the cell surface in these cell lines.

Specificity of coenzyme Q inhibition of an aging-related cell surface NADH oxidase (ECTO-NOX) that generates superoxide.

Our laboratories have described a novel class of ectoproteins at the cell surface with both NADH or hydroquinone oxidase (NOX) and protein disulfide-thiol interchange activities (ECTO-NOX proteins). The two activities exhibited by these proteins alternate to generate characteristic patterns of oscillations where the period length is independent of temperature. The period length for the constitutive ECTO-NOX is 24 min. Here we describe a distinctive age-related ECTO-NOX (arNOX) whose activity is blocked by coenzyme Q10. arNOX occurs exclusively in aged cells and tissues. The period length of the oscillations is 26 min. Rather than reducing 1/2 O2 to H2O, electrons are transferred to O2 to form superoxide. Superoxide formation was demonstrated by superoxide dismutase-sensitive reduction of ferricytochrome c and by reduction of a superoxide-specific tetrazolium salt. Quinone inhibition was given by coenzymes Q8, 9 and Q10 but not by Q0, Q2, Q4, Q6 or 7. The arNOX provides a mechanism to propagate reactive oxygen species generated at the cell surface to surrounding cells and circulating lipoproteins of importance to atherogenesis. Inhibition of arNOX by dietary coenzyme Q10 provides a rational basis for dietary coenzyme 10 use to retard aging-related arterial lesions.

An aging-related cell surface NADH oxidase (arNOX) generates superoxide and is inhibited by coenzyme Q.

This report describes a novel ECTO-NOX protein with an oscillating activity having a period length of ca. 26 min encountered with buffy coat fractions and sera of aged individuals (70-100 years) that generates superoxide as measured by the reduction of ferricytochrome c. The oscillating, age-related reduction of ferricytochrome c is sensitive to superoxide dismutase, is inhibited by coenzyme Q and is reduced or absent from sera of younger individuals (20-40 years). An oscillating activity with a regular period length is a defining characteristic of ECTO-NOX proteins (a group of cell surface oxidases with enzymatic activities that oscillate). The period length of ca. 26 min is longer than the period length of 24 min for the usual constitutive (CNOX) ECTO-NOX proteins of the cell surface and sera which neither generate superoxide nor reduce ferricytochrome c. The aging-related ECTO-NOX protein (arNOX) provides a mechanism to transmit cell surface oxidative changes to surrounding cells and circulating lipoproteins potentially important to atherogenesis. Additionally, the findings provide a rational basis for the use of dietary coenzyme Q to retard aging-related arterial lesions.

Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor.

The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

Cancer isoform of a tumor-associated cell surface NADH oxidase (tNOX) has properties of a prion.

We have described a drug-responsive form of a cell surface NADH oxidase (hydroquinone oxidase) of cancer cells (tNOX) that exhibits unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristics of PrP(sc) (PrP(res)), the presumed infective and proteinase K resistant particle of the scrapie prion. The tNOX protein from the HeLa cell surface copurified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH). Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant. In this paper, we show that combination of authentic rabbit muscle GAPDH with tNOX renders the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions.

CHO cell enlargement oscillates with a temperature-compensated period of 24 min

The rate of increase in cell area of CHO cells when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a minimum period of about 24 min. The pattern of oscillations paralleled those of the 24 min period observed with the oxidation of NADH by an external cell surface or plasma membrane NADH oxidase. The increase in cell area was non-linear. Intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the 24 min period was temperature-compensated (approximately the same when measured at 14°C, 24°C or 34°C) while the rate of cell enlargement increased with temperature over this same range of temperatures.

Preferential inhibition by (−)-epigallocatechin-3-gallate of the cell surface NADH oxidase and growth of transformed cells in culture

A drug-responsive and cancer-specific NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa and human mammary adenocarcinoma by the naturally-occurring catechin of green tea, (−)-epigallocatechin-3-gallate (EGCg). With cells in culture, EGCg preferentially inhibited growth of HeLa and mammary adenocarcinoma cells compared with growth of mammary epithelial cells. Inhibited cells became smaller, and cell death was accompanied by a condensed and fragmented appearance of the nuclear DNA as revealed by fluorescence microscopy with 4′,6-diamidino-2-phenylindole, suggestive of apoptosis. Mammary epithelial cells recovered from EGCg treatment even at 50 μM, whereas growth of HeLa and mammary adenocarcinoma cells was inhibited by EGCg at concentrations as low as 1 μM with repeated twice-daily additions and did not recover from treatment with 50 μM EGCg. The findings correlate inhibition of cell surface NADH oxidase activity and inhibition of growth with EGCg-induced apoptosis.

Cell-surface NAD(P)H-oxidase: relationship to trans-plasma membrane NADH-oxidoreductase and a potential source of circulating NADH-oxidase.

The surface of mammalian cells faces an oxidizing environment that has the potential to damage proteins, lipids, and carbohydrates to which it is exposed. In contrast, the cytoplasm is reducing and its redox state is tightly regulated. Trans-plasma membrane oxidoreductases that shift electrons from cytosolic NADH to external electron acceptors such as oxygen are widely involved in cellular redox control. They reduce oxygen to water and may generate reactive oxygen species such as superoxide and hydrogen peroxide. In addition, external NAD(P)H-oxidases have been demonstrated on intact cells and as eluted proteins, but the relationship between trans-plasma membrane NADH-oxidoreductases and cell-surface NAD(P)H-oxidases is not known. To investigate further the relationship between plasma membrane NAD(P)H-oxidoreductases, and to gain insight into the physiological functions of these redox active membrane proteins, we have adapted a simple colorimetric assay for measuring the trans-plasma membrane NADH-oxidoreductase activity of viable cells to measure NAD(P)H-oxidase at the cell surface in real time. Using the cell-impermeable tetrazolium salt WST-1 in the presence of NADH or NADPH, but in the absence of an intermediate electron acceptor, we show that cell-surface NAD(P)H-oxidase is widely expressed on mammalian cells, being more abundant on rapidly proliferating cells than on resting neutrophils and spleen cells. The ratio of cofactor dependence of NAD(P)H-oxidase (NADH:NADPH) varied widely between different cells (0.7-5.2), suggesting a family of cell surface oxidases or that the activity of these enzymes may be modulated in various ways. Comparison of NAD(P)H-oxidase on the surface of viable cells with trans-membrane NADH-oxidoreductase, measured with WST-1 in the presence of 1-methoxy PMS, showed that cell-surface NAD(P)H-oxidase was differentially inhibited by the cell-impermeable thiol-blocking agent pCMBS, but was unaffected or stimulated by other thiol blocking agents. Capsaicin, which inhibits trans-plasma membrane NADH-oxidoreductase activity, stimulated surface NAD(P)H-oxidase. Metabolic inhibitors had little effect on surface NAD(P)H-oxidase activity but inhibited trans-plasma membrane activity. These results do not support the view the surface NAD(P)H-oxidase is a terminal oxidase for trans-plasma membrane NADH-oxidoreductase.

High-capacity redox control at the plasma membrane of mammalian cells: trans-membrane, cell surface, and serum NADH-oxidases.

The high capacity of proliferating mammalian cells to transfer electrons from cytosolic NADH to extracellular electron acceptors like oxygen is poorly understood and not widely recognized. Nevertheless, trans-plasma membrane electron transport (plasma membrane redox control) probably ranks alongside the Na+/H+ antiport system (pH control) and glucose transport in facilitating cellular responses to physiological stimuli. These plasma membrane transport systems are acutely responsive to receptor ligation by growth factors, polypeptide hormones, and other cell activators. A novel tetrazolium-based cell proliferation assay that we have shown to measure an NADH-oxidoreductase component of the trans-plasma membrane electron transport system has allowed direct comparisons with NADH:ferricyanide-oxidoreductase and respiratory burst NADPH-oxidoreductase. In addition, an NAD(P)H-oxidase at the cell surface and an NADH-oxidase activity in body fluids can be measured by modifying the basic cell proliferation assay. As determined by reduction of the cell-impermeable tetrazolium reagent, WST-1, electron transfer across the plasma membrane of dividing cells can exceed that of fully activated human peripheral blood neutrophils. Cellular reduction of WST-1 is dependent on the presence of an intermediate electron acceptor and is inhibited by superoxide dismutase (SOD) and by oxygen, implying indirect involvement of superoxide in WST-1 reduction. Cell-surface NAD(P)H-oxidase and serum NADH-oxidase are shown to be distinct from trans-plasma membrane NADH-oxidoreductase by their differential sensitivity to capsaicin and pCMBS. The glycolytic metabolism of cancer cells may be linked to changes in trans-plasma membrane NADH:WST-1-oxidoreductase activity and to increased serum NADH-oxidase in cancer.

A light-responsive and periodic NADH oxidase activity of the cell surface of Tetrahymena and of human buffy coat cells.

Oxidation of external NADH (NADH is an impermeant substrate) by cells of Tetrahymena pyriformis oscillated with a period of 24-26 min. The period length in darkness (25.6 min) appeared to be slightly longer than the period in light (approximately 24 min). When Tetrahymena were placed in darkness for 30-50 min and then returned to light, a new maximum in the rate of NADH oxidation was observed 36-38 min (13 + 24) min after the beginning of the light treatment. The cell-surface NADH oxidase of human buffy coats (a mixture of white cells and platelets) also was periodic and light responsive.

Cell surface NADH oxidase activity of brine shrimp oscillates with a period of 25 min and is entrained by light

Plants have a surface NADH oxidase that measures time by oscillating with a 24-min period. The period is synchronized by light. With plants, a new maximum is observed exactly 12 min after the beginning of the light exposure. These experiments were to determine if animals exhibited a cell surface NADH oxidase having a similar periodicity and to answer the question, does the periodicity in animals respond to light? Using brine shrimp as a model, the findings show that plants and animals exhibit similar oscillating NADH oxidase activity and that the periodicity in this invertebrate animal does respond to light. Brine shrimp were grown for two to three days and transferred to darkness for 45 min. After return to light for one min, NADH was added and measurements of NADH oxidation were recorded over 50 min. The brine shrimp exhibited a cell surface NADH oxidase that oscillated with a period of 25 min. After being subjected to light, the brine shrimp showed a new maximum in NADH oxidation between 12 to 13 min after the beginning of the light exposure and again at 37 min and at 25 min intervals thereafter. The findings demonstrate that the periodic oscillations in NADH oxidation of brine shrimp are light entrainable.

Cell Surface NADH Oxidase Activity of Brine Shrimp Oscillates With a Period of 25 Min and is Entrained by Light

Cell Surface NADH Oxidase Activity of Brine Shrimp Oscillates With a Period of 25 Min and is Entrained by Light

Response of a cell-surface NADH oxidase to the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenylurea) (LY181984) modulated by redox

In previous reports, our laboratory has described a drug-responsive NADH oxidase activity of the external surface of the plasma membrane of HeLa and other cancer cells, but not from normal cells, that was shed into media conditioned by the growth of cancer cells such as HeLa and also into sera of cancer patients. The sulfonylurea-altered activity was found in sera of a wide variety of cancer patients but the activity was either inhibited or stimulated by 1 μM LY181984. In this report, we demonstrate that one basis for whether or not the activity was stimulated or inhibited may be the redox environment of the protein. If plasma membrane vesicles from HeLa cells were first treated with dithiothreitol (DTT) or with reduced glutathione (GSH) and then assayed for NADH oxidase activity, the sulfonylurea inhibited the activity in a concentration-dependent manner. In contrast, if the plasma membrane vesicles were first treated with diluted hydrogen peroxide or oxidized glutathione (GSSG) and then assayed for NADH oxidase activity, the antitumor sulfonylurea stimulated the activity. Growth experiments were conducted in parallel. LY181984 administered to HeLa cells in the presence of GSH was approximately 2 log orders more effective than LY181984 administered to HeLa cells in the presence of GSSG. Similar results were found in the sera of cancer patients. With sera from normal individuals or with plasma membranes of rat liver, the oxidizing or reducing conditions were without effect. The findings suggest that the response of the cell surface NADH oxidase of HeLa cells to the antitumor sulfonylurea LY181984 is influenced by the redox environment which may determine whether the drug will stimulate or inhibit the activity and that the degree of response may be reflected in the ability of LY181984 to inhibit HeLa cell growth.