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Background: Malignant mesothelioma is an aggressive, almost uniformly fatal tumor, caused primarily by exposure to asbestos. In this study, serum presence of mesothelioma-specific protein transcript variants of ecto-nicotinamide adenine dinucleotide oxidase disulfide-thiol exchanger 2 (ENOX2), a recently identified marker of malignancy, were investigated using the ONCOblot tissue of origin cancer detection test., Methods: Sequential serum samples collected from asbestos-exposed individuals prior to the development of frank mesothelioma were assayed for ENOX2 presence by 2-D gel immunoblot analysis to determine how long in advance of clinical symptoms mesothelioma-specific ENOX2 transcript variants could be detected., Results: Two mesothelioma-specific ENOX2 protein transcript variants were detected in the serum of asbestos-exposed individuals 4-10 years prior to clinical diagnosis of malignant mesothelioma (average 6.2 years). Either one or both ENOX2 protein transcript variants indicative of malignant mesothelioma were absent in 14 of 15 subjects diagnosed with benign pleural plaques either with or without accompanying asbestosis., Conclusions: In a population of asbestos-exposed subjects who eventually developed malignant mesothelioma, ENOX2 protein transcript variants characteristic of malignant mesothelioma were present in serum 4-10 years in advance of clinical symptoms. As with all biomarker studies, these observations require validation in a larger, independent cohort of patients and should include prospective as well as retrospective sampling.

Age-related NADH oxidase (arNOX), a cell surface-located hydroquinone oxidase capable of superoxide generation, appears at age 30 and increases with age thereafter. The ectodomain of arNOX is shed from the cell surface into body fluids including sera and saliva where its activity was measured spectrophotometrically using a reduction of ferricytochrome c as a measure of superoxide generation. The autofluorescence of advanced glycation end products correlates with epidermal arNOX activity as well. To demonstrate protein cross-linking, a fluorescence-labeled analog of tyrosine, tyramine, that would react with proteins carrying arNOX-generated tyrosyl radicals was used. The assay demonstrated the potential for arNOX-induced oxidative damage (dityrosine formation) to human collagen and elastin and to other surface proteins of intact human embryo fibroblasts and frozen sections from epidermal punch biopsies. The findings support a role for arNOX as a major source of oxidative damage leading to cross-linking of skin proteins.

Background: Experts agree that one of the more promising strategies in cancer management is early detection coupled with early intervention. In this study, we evaluated an early cancer detection strategy of cancer presence based on serum levels of the cancer-specific transcript variants of ENOX2 in serum coupled with an ENOX2-targeted nutraceutical preparation of green tea concentrate plus Capsicum (Capsol-T®) as a strategy of Curative Prevention® involving early detection coupled with early intervention in early stage cancer when in its most susceptible and manageable stages., Experimental Design: One hundred ten (110) subjects were tested for cancer presence using the ONCOblot® Tissue of Origin 2-D gel/western blot protocol for detection of serum presence of transcript variants of the ENOX2 protein. Subjects testing positive for ENOX2 received 350 mg of Capsol-T® in capsule form every 4 h including during the night for periods of at least 3 to 6 months or longer after which they were again tested for ENOX2 presence using the ONCOblot® Tissue of Origin Cancer Test protocol., Results: Of the 110 subjects, both male and female, ages 40 to 84, with no evidence of clinical symptoms of cancer, 40% were positive for ENOX2 presence in the ONCOblot® Tissue of Origin Cancer Test. After completion of 3 to 17 months of Capsol-T® use, 94% of subjects subsequently tested negative for ENOX2 presence., Conclusions: Oral Capsol-T® is well tolerated and, for ENOX2 presence in serum in the absence of clinical cancer symptoms, is consistently effective in reducing the serum ENOX2 levels to below detectable limits.

ME-143 (NV-143), a synthetic isoflavone under clinical evaluation for efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated ENOX2 (tNOX) and inhibited the growth of cultured cancer cells with EC50s in the range of 20-50 nM. Purified recombinant ENOX2 also bound ME-143 with a Kd of 43 (40-50) nM. Both the oxidative and protein disulfide-thiol interchange activities of ENOX proteins that alternate to generate a complex set of oscillations with a period length of 22 min compared to 24 min for the constitutive counterpart ENOX1 (CNOX) that characterizes ENOX proteins responded to ME-143. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked. In contrast, the protein disulfide-thiol interchange activity measured from the cleavage of dithiodipyridine (EC50 of ca. 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ENOX1 (CNOX) forms of either cancer or noncancer cells were unaffected by ME-143 over the range of concentrations inhibiting ENOX2. Taken together, the findings show that ME-143 binds to ENOX2 with an affinity 4 to 10 times greater than that reported previously for the related anticancer isoflavone, phenoxodiol.

Reactive oxygen species that are produced by aerobic metabolism and signaling cascades have the potential to play important roles in maintaining homeostatic redox and cell proliferation. When the balance between the production and elimination of reactive oxygen species is perturbed toward production, the result is oxidative stress. High levels of oxidative stress are a general characteristic of cancer. The altered redox state within a tumor microenvironment confers a growth advantage through increased proliferation rates, evasion of apoptosis, and increased resistance to therapeutic compounds. We have tested a synergistic combination of green tea-Camellia sinensis-concentrate and powdered Capsicum powder (TeaFense™/Capsol-T™) as a dietary supplement to reduce oxidative stress as an approach to elimination of malignant cells. Here, we demonstrate that the green tea-powdered Capsicum mixture effectively reduces levels of oxidative stress in both cancer (HeLa) and noncancer (MCF-10A) cells as determined from measurements of levels of the oxidative stress indicator Nrf-2 by western blot analysis. Nrf-2 is a transcription factor that controls an antioxidant response element. Increased expression of Nrf-2 is linked to high levels of oxidative stress and vice versa. Based on levels of Nrf-2, the mixture of green tea concentrate plus powdered Capsicum reduced oxidative stress by more than 50% compared with 15% by the green tea concentrate alone.

HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.

The key role of coenzyme Q (ubiquinone or Q) is in mitochondrial and prokaryotic energetics. Less well investigated is the basis for its presence in eukaryotic membrane locations other than mitochondria and in plasma where both antioxidant and potentially more targeted roles are indicated. Included in the latter is that of a lipid-soluble electron transfer intermediate that serves as the transmembrane component of plasma membrane and Golgi apparatus electron transport, which regulates cytosolic NAD(+) /NADH ratios and is involved in vectorial membrane displacements and in the regulation of cell growth. Important protective effects on circulating lipoproteins and in the prevention of coronary artery disease ensue not only from the antioxidant role of CoQ(10) but also from its ability to directly block protein oxidation and superoxide generation of the TM-9 family of membrane proteins known as age-related NADH oxidase or arNOX (ENOX3) and their shed forms that appear after age 30 and some of which associate specifically with low-density lipoprotein particles to catalyze protein oxidation and crosslinking., (Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.)

Background: Constituents 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., Methods: Responses 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., Results: Growth 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., Conclusions: Based 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 Significance: The 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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46 kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.

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., ((c) 2010 Wiley-Liss, Inc.)

Epidemiological and laboratory studies suggest that dietary fatty acids (oleic acid (in olive oil), eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) (in fish oil)) play important roles in carcinogenesis. The most potent antitumor effects of all fatty acids are given by fatty acid conjugated linoleic acid (CLA). The antitumor effects of CLA may be mediated through enhanced apoptosis. While CLA, EPA, and DHA (omega-3 polyunsaturated fatty acids) have inhibitory effects on cancer cells, omega-6 fatty acids have often shown negative or potentiating effects on cancer cells. Linoleic acid (an omega-6) is desaturated in the cell by delta 6 and 5 destaturases to form arachidonic acid. COX 1 and 2 isoforms then act on arachidonic acid to form prostaglandins and other related regulatory molecules. It is normally thought that what is important to the development of the cancerous phenotype is some balance of these various metabolites. In experiments with surface NOX proteins released from HeLa cells, spectrophotometric measurements of the oxidation of NADH revealed inhibition of the cancer-specific ENOX2 activity by CLA and the omega-3 fatty acids, eicosapentaenoic, docosahexaenoic, and α-linolenic acids. The constitutive ENOX1 activity was not inhibited. In contrast, the omega-6 fatty acids, linoleic acid, and arachidonic acid inhibited neither ENOX1 nor ENOX2. The findings indicate the possibility that a direct effect of CLA and omega-3 fatty acids on ENOX2 may be responsible for the potent activity of CLA and omega-3 fatty acids in cancer prevention and therapy.

An aging-related cell-surface oxidase (aging-related NADH oxidase, arNOX) generating superoxide and other reactive oxygen species is shed from the cell surface and is found in saliva, urine, perspiration, and interstitial fluids that surround the collagen and elastin matrix underlying dermis. arNOX activity correlates with age and reaches a maximum at about age 65 in males and 55 in females. arNOX activities are highly correlated with values of human skin where a causal relationship is indicated. Ongoing efforts focus on cloning arNOX proteins and development of antiaging formulas based on arNOX inhibition (intervention).

Golgi apparatus from rat liver contain an ascorbate free radical oxidoreductase that oxidizes NADH at neutral pH with monodehydroascorbate as acceptor to generate a membrane potential. At pH 5.0, the reverse reaction occurs from NAD(+). The electron spin resonance signal of the ascorbate-free radical and its disappearance upon the addition of NADH (pH 7) or NAD(+) (pH 5.0) confirms monodehydroascorbate involvement. Location of monodehydroascorbate both external to and within Golgi apparatus compartments is suggested from energization provided by inward or outward flux of electrons across the Golgi apparatus membranes. The isolated membranes are sealed, oriented cytoplasmic side out and impermeable to NAD(+) and ascorbate. NAD(+) derived through the action of Golgi apparatus beta-NADP phosphohydrolase is simultaneously reduced to NADH with monodehydroascorbate present. The response of the NADH- (NAD(+)-) ascorbate free radical oxidoreductase system to pH in Golgi apparatus provides a simple regulatory mechanism to control vesicle acidification.

Activity of an age-related, superoxide-forming, cell-surface oxidase (arNOX) comparing dermis, epidermis, serum, and saliva from female and male subjects ages 28-72 years measured spectrophotometrically using reduction of ferricytochrome c correlated with oxidative skin damage as estimated from autofluoresence of skin using an Advanced Glycation End products Reader (AGE-Reader; DiagnOptics B.V., Netherlands). By reducing arNOX activity in skin with arNOX-inhibitory ingredients (NuSkin's ageLOC technology), skin appearance was improved through decreased protein cross-linking and an accelerated increase in collagen.

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.

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.

The Purdue-UAB Botanicals Research Center for Age Related Disease uses multidisciplinary and innovative technologies to investigate the bioavailability of bioactive polyphenolic constituents from botanicals and their relationship to human health. Many age-related diseases are associated with oxidative stress and tissue damage. One of the research goals of the Purdue-UAB Center is to investigate the bioavailability of bioactive natural compounds from a complex botanical mixture to the organ affected by the disease, determine the uptake and metabolism of these compounds and relate these data to a protective mechanism. Equally important is to screen commercially available botanicals for their safety and efficacy. The central aims of the Center include the investigation of botanicals and their relationship to bone antiresorptive capacity, cognitive function, vascular effects, and cancer prevention.

Phenoxodiol is an experimental anticancer drug under development as a chemosensitizer intended to reverse multidrug resistance mechanisms in ovarian and prostate cancer cells to most standard cytotoxics. The putative molecular target of phenoxodiol is a cell-surface, tumor-specific NADH oxidase, ENOX2 (tNOX), with phenoxodiol having no apparent effect on the constitutive form of this enzyme ENOX1 (CNOX). Using ENOX2 as the target, this study was conducted to explore the temporal relationship between phenoxodiol and paclitaxel or cisplatin in achieving chemosensitization in HeLa cells which are relatively resistant to both paclitaxel and cisplatin. Sequential addition of phenoxodiol and paclitaxel or phenoxodiol and cisplatin showed greater inhibition of HeLa cell ENOX1 activity and growth compared to adding the drugs simultaneously or individually. In parallel, a similar chemosensitizing response of phenoxodiol for cisplatin was observed. ENOX1 was not affected and trans-platinum had no effect. With spent media from phenoxodiol-treated cells sensitivity was enhanced to both paclitaxel and cisplatin if the cells were first pretreated with phenoxodiol. Similar results were obtained with ENOX2-enriched preparations stripped from the surfaces of phenoxodiol-treated cells. In keeping with a speculative prion model, it seems as though the ENOX2 "remembers" the phenoxodiol and "teaches" other ENOX2 molecules to respond to paclitaxel and cisplatin as if phenoxodiol were still present.

A single case of carcinoma in situ (actinic keratosis) was treated topically with a patch consisting of an aqueous paste of a commercially available mixture (50:1) of green tea concentrate plus Capsicum (Capsol-T®) for approximately 14 days. The carcinoma responded by the formation of apoptotic blisters whereas surrounding normal tissue showed no response. A second untreated carcinoma 17 cm distant from the treated area also responded indicative of a systemic action of the substance.

ENOX (ECTO-NOX) proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide-thiol interchange and exhibit both prion-like and time-keeping (clock) properties. The two enzymatic activities they catalyze alternate to generate a regular period of 24 min in length. Here we report the cloning, expression, and characterization of a human candidate constitutive ENOX (CNOX or ENOX1) protein. The gene encoding this 643 amino acid long protein is located on chromosome 13 (13q 14.11). Functional motifs previously identified by site-directed mutagenesis in a cancer-associated ENOX (tNOX or ENOX2) as adenine nucleotide or copper binding along with essential cysteines are present, but the drug-binding motif (EEMTE) sequence of ENOX2 is absent. The activities of the recombinant protein expressed in Escherichia coli were not affected by capsaicin, EGCg, and other ENOX2-inhibiting substances. The purified recombinant protein bound ca. 2 mol of copper/mol of protein. Bound copper was necessary for activity. H260 and H579 were required for copper binding as confirmed by site-directed mutagenesis, loss of copper-binding capacity, and resultant loss of enzymatic activity. Addition of melatonin phased the 24 min period such that the next complete period began exactly 24 min after the melatonin addition as appears to be characteristic of ENOX1 activities in general. Oxidative activity was exhibited with both NAD(P)H and reduced coenzyme Q as substrate. Concentrated solutions of the purified candidate ENOX1 protein irreversibly formed insoluble aggregates, devoid of enzymatic activity, resembling amyloid.

tNOX (ENOX2), a cancer-specific and growth-related cell surface protein with protein disulfide-thiol interchange and hydroquinone (NADH) oxidase activities was overexpressed in a transgenic mouse model. Female transgenic mice grew faster than wild type as did embryonic fibroblast cells prepared from the transgenic mice. The tissue expression of tNOX mRNA was greatest in heart, lung and liver. When these tissues were analyzed for cell size, the cells from the tissues of transgenic animals were, on average, 20% larger in surface area than cells from corresponding wild-type tissues. Also analyzed were cells of intestine, spleen and kidney in which tNOX overexpression was observed but to a lesser extent. Cell size was increased as well with intestine and kidney but less so with spleen. At the end of the study, carcass weights of the transgenic animals were greater than those of wild type. This increase in carcass weight was reflected in an increase in femur weight and thickness in both male and female transgenic mice but not in femur length. Other carcass parameters such as skin weight and body fat or body fluids were unchanged or changes were insufficient to account for the increased carcass weight. The findings are consistent with the property of tNOX observed in studies with cultured cells as contributing to the enlargement phase of cell growth.

A family of cell surface and growth-related proteins, designated ECTO-NOX proteins, carry out both copper-dependent NADH and hydroquinone oxidation and protein disulfide-thiol interchange. The two activities they catalyze alternate to generate a regular period of 24min in length for the constitutive CNOX. Unexpectedly, Cu(II) salts alone in solution catalyze NADH (or hydroquinone) oxidation with a similar oscillatory pattern. Both patterns consist of five maxima, two of which at physiological temperatures are separated by an interval of 6min and three of which are separated by intervals of 4.5min [6min+4 (4.5min)]. EXAFS and infrared spectroscopic measurements on pure water have shown previously that the ratios of ortho and para isomers of the hydrogen atoms of water occur on a similar time scale and produce regular patterns of unequally spaced oscillations similar to those observed with ECTO-NOX proteins and Cu(II)Cl(2) solutions. Here, we provide results from Cu(II)Cl(2) solutions that demonstrate that ECTO-NOX-/Cu(II)-catalyzed oscillations in NADH oxidation are phased by exposure to low frequency electromagnetic fields.

Lithium has been used widely both as a clinical agent to treat manic depressive disorders and as a substance targeted to the regulation of the circadian cycle. In this study, we show that lithium at physiological concentrations of less than 1microM uniquely induces an ECTO-NOX activity previously inactive from plant (soybean), murine (3T3 cells) and human (HUVEC and HeLa cells) sources and resets the period of the constitutive CNOX. The average period length of the new oscillation set induced by the presence of lithium of 23.5min was slightly less than the period length in the absence of lithium (24min). The constitutive period was retained in the presence of lithium but the period length was increased on an average by 4% to about 25min. Targeting circadian rhythm abnormalities may be a particularly useful strategy in management of bipolar disorder and related illnesses since circadian cycles appear to be an inherent function conserved through evolution in all organisms and consistently implicated in the pathophysiology of bipolar disorder.

The Purdue-University of Alabama Botanicals Research Center for Age Related Disease joins novel technologies to study the bioavailability of bioactive polyphenolic constituents and their relation to health. Many diseases that manifest with age relate to oxidative stress and tissue damage. Our goal is to follow the fate of bioactive constituents from a complex mixture to the organ affected by the disease and relate that to a protective mechanism. Equally important is to screen commercially available botanicals for their efficacy and safety. Botanicals and their relation to bone antiresorptive capacity, cognitive function, vascular effects, and cancer are principal themes in our center.

Phenoxodiol, an ENOX2 inhibitor, alters cytosolic NADH levels to initiate a regulatory cascade linking sphingolipid metabolism and the PI3K/Akt pathway to programmed cell death. Specifically, the pyridine nucleotide products of plasma membrane redox, NAD+ and NADH, directly modulate in a recriprocal manner two key plasma membrane enzymes. NADH stimulation of sphingomyelinase and NADH inhibition of sphingosine kinase potentially lead to G1 arrest (increase in ceramide) and apoptosis (loss of sphingosine-1-phosphate). The findings link plasma membrane electron transport and the anticancer action of several clinically-relevant anticancer agents targeted to ENOX2 such as phenoxodiol. Growth inhibition by phenoxodiol is unaffected by inhibitors of protein or mRNA synthesis. Findings with okadiaic acid, an inhibitor of serine/threonine phosphatases, suggest that hyperphosphorylation of intracellular substrates does not affect the action of phenoxodiol on ENOX2. Our findings and those of others are consistent with operation of the FAS signaling pathway of apoptosis and its suppression by sphingosine-1-phosphate. The prevailing hypothesis is that products of Akt activation, c-FLIP and XIAP, which exhibit anticaspase activities to block FAS signaling when sphingosine-1-phospate is elevated, are down regulated to permit apoptosis when sphingosine-1-phosphate is decreased by inhibition of sphingosine kinase under conditions of elevated cytosolic NADH associated with anticancer drug inhibition of ENOX2.

Our work has identified an aging-related ECTO-NOX activity (arNOX), a hydroquinone oxidase which is cell surface located and generates superoxide. This activity increases with increasing age beginning >30 y. Because of its cell surface location and ability to generate superoxide, the arNOX proteins may serve to propagate an aging cascade both to adjacent cells and to oxidize circulating lipoproteins as significant factors determining atherogenic risk. The generation of superoxide by arNOX proteins is inhibited by Coenzyme Q10 as one basis for an anti-aging benefit of CoQ10 supplementation in human subjects. In a preliminary pilot study, 25 female subjects between 45 and 55 y of age were recruited at Stanford University from the Palo Alto, CA area. Informed consent was obtained. Ten of the subjects received Coenzyme Q10 supplementation of 180 (3 x 60 mg) per day for 28 days. Serum, saliva and perspiration levels of arNOX were determined at 7, 14 and 28 days of CoQ10 supplementation and compared to the initial baseline value. Activity correlated with subject age up to a maximum between age 50 and 55 years of age for saliva and perspiration as well and then declined. With all three sources, the arNOX activity extrapolated to zero at about age 30. Response to Coenzyme Q10 also increased with age being least between ages 45 and 50 and greatest between ages 60 and 65. With all three biofluids, arNOX activity was reduced between 25 and 30% by a 3 x 60 mg daily dose Coenzyme Q10 supplementation. Inhibition was the result of Coenzyme Q10 presence.

arNOX is a coenzyme Q10-inhibited, aging-related ECTO-NOX protein of the cell surface also present in sera. It is capable of superoxide generation measured as superoxide dismutase-inhibited reduction of ferricytochrome c and is a potential contributor to atherogenic risk. Here, we report an arNOX activity of saliva of older individuals also inhibited by coenzyme Q10. The activity first appears after age 30 to a near maximum at about age 55. Those surviving beyond age 55 usually have reduced arNOX activities. Our studies demonstrate significant (25 to 30%) reduction of arNOX levels with coenzyme Q10 supplementation of 60 mg (2 x 30 mg) per day for 28 days. Activity correlated with age. Response to coenzyme Q10 increased with age being greatest between ages 60 and 65. Saliva arNOX levels varied in a regular pattern throughout the day so it was important that samples be collected at approximately the same time each day for comparative purposes. The coenzyme Q10 response was reversible and within 12 h after the last intake of coenzyme Q10, the salivary arNOX levels returned to base line. The findings suggest that salivary arNOX provides a convenient and non-invasive method to monitor arNOX levels in clinical coenzyme Q10 intervention trials with the response levels paralleling those seen with serum and cellular arNOX.

ENOX proteins with an oscillatory pattern of production of superoxide (measured by ferricytochrome c reduction) and with a period length of 26 min increase linearity with age beginning at about 30 y to a maximum of about age 60. The proteins are shed and appear in serum, saliva and urine. Enhanced arNOX activity correlates with age and with oxidative changes contributing to skin aging. Topical cosmetic preparations containing substances that block arNOX activity are under evaluation to reduce visible symptoms of skin aging.

A proteomics approach with detection on western blots using an S-peptide tagged pan-tNOX (ENOX2) recombinant (scFv) antibody followed by alkaline phosphatase-linked anti S has revealed a family of more than 20 ENOX2 isoforms of varying molecular weights (34 to 94 kDa) and mostly of low isoelectric points (4.6 +/- 0.7) based on serum analysis. Different isoforms characterize cancers of different tissue origins indicative of both cancer presence and tissue site of origin. ENOX2 proteins are cancer-associated and differ from constitutive (CNOX or ENOX1) proteins primarily by the absence of a drug binding site to which the cancer-specific scFv is directed. All are located on the cell surface where they function both as terminal oxidases for plasma membrane electron transport and carry out protein disulfide-thiol interchange. These proteins are shed into the blood and can also be found in urine. The tNOX isoform technology is under development as a clinical aid to identify unknown or uncertain primary cancers, evaluation of metastatic spread in post surgery patients, monitoring remission following cessation of therapy and for early diagnosis in at-risk populations.

Catechins are a major constituent of green tea. For green tea to have cancer therapeutic benefit, catechin concentrations in the range of 100 nM are required continuously until apoptosis (programmed cell death) is induced. To prolong elevated plasma and interstitial concentrations of catechins, a sustained-release formulation of green tea extract was tested and compared to a commercial green tea extract (Tegreen97®). Sustained-release formulations are usually developed in the pharmaceutical industry to slowly deliver the compound over a period of time and increase the dosing interval. Plasma and interstitial fluid (ISF) pharmacokinetics of catechins were determined following an oral dose in the rat. The sustained-release formulation profile included multiple smaller peaks of total catechins in both plasma and ISF. Interstitial fluid profiles of green tea extract indicate that higher catechins concentration and longer duration in tissue than in blood may make a sustained-release form unnecessary.

A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera of healthy individuals. Full-length tNOX mRNA is present in both normal and tumor cells but appears not to be expressed in either. Our research suggests alternative splicing as the basis for the cancer specificity of tNOX expression at the cell surface. Four splice variants were found. Of these, the exon 4 minus and exon 5 minus forms present in cancer cell lines were absent in noncancer cell lines. In contrast to full-length tNOX cDNA, transfection of COS cells with tNOX exon 4 minus cDNA resulted in overexpression of mature 34 kDa tNOX protein at the plasma membrane. The exon 4 minus form resulted in initiation of translation at a downstream M231 initiation site distinct from that of full-length mRNA. With replacement of M231 by site-directed mutagenesis, no translation of exon 4 minus cDNA or cell surface expression of 34 kDa mature tNOX was observed. The unprocessed molecular mass of 47 kDa of the exon 4 minus cDNA translated from methionine 231 corresponded to that of the principal native tNOX form of the endoplasmic reticulum. Taken together, the molecular basis of cancer-cell-specific expression of 34 kDa tNOX appears to reside in the cancer-specific expression of exon 4 minus splice variant mRNA.

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.

Cell surface ECTO-NOX proteins exhibit a clock-related, temperature-independent entrainable pattern of periodic (24 min) oscillations in the rate of oxidation of NAD(P)H. Aqueous solutions of copper salts also oxidize NAD(P)H with a similar temperature-independent pattern. For both, five maxima are observed, two of which are separated by 6 min and the remaining three are separated by 4.5 min. In D2O, the pattern is retained but the period length is proportionately increased to 30 min in direct relationship to the 30 h circadian day observed with D2O-grown organisms. With copper solutions, periodic changes in redox potential correlate precisely with the periodic changes in the rates of NAD(P)H oxidation. Consequently, the local environment of the Cu2+ ion in copper chloride solutions was investigated by X-ray absorption spectroscopy. Detailed extended X-ray absorption fine structure (EXAFS) analyses revealed a pattern of oscillations closely resembling those of the copper-catalyzed oxidation of NADH. With CuCl2 in D2O, a pattern with a period length of 30 min was observed. The findings suggest a regular pattern of distortion in the axial and/or equatorial oxygen atoms of the coordinated water molecules which correlate with redox potential changes sufficient to oxidize NADH. A metastable equilibrium condition in the ratio of ortho to para nuclear spin orientation of the water associated hydrogen atoms would be kinetically consistent with a 24-30 min timeframe. The temperature independence of the biological clock can thus be understood as the consequence of a physical rather than a chemical basis for the timing events.

A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera from healthy individuals. Transfection of HeLa (human cervical carcinoma) cells with antisense oligonucleotides and measurement of mRNA levels by real-time quantitative PCR and growth and drug response by in vitro cytotoxicity assays were combined to demonstrate encoding of a cancer-specific and growth-related cell surface protein, tNOX, via an exon 4 minus splice variant. tNOX mRNA levels of HeLa cells were determined following transfection with antisense relative to control cells transfected with Lipofectamine using the cycle threshold method normalized for GAPDH mRNA. Antisense to tNOX exon 4 mRNA blocked generation of full-length tNOX mRNA but not of exon 4 minus mRNA. Antisense to exon 5 mRNA inhibited the production of exon 4 minus mRNA and full-length tNOX mRNA. Scrambled antisense to exon 5 mRNA was without effect. Antisense to exon 5 mRNA decreased the amount of tNOX protein on the surface of cancer cells. As a control, antisense-mediated downregulation of exon 5 minus mRNA of tNOX also was demonstrated as detected using exon 4/exon 6 primers. Exon 5 antisense blocked the cell surface expression of tNOX whereas exon 4 antisense was without effect. In contrast to nontransfected HeLa cells, cells transfected with exon 5 antisense were not inhibited by the green tea catechin, (-)-epigallocatechin-3-gallate. A relationship of tNOX to unregulated growth of cancer cells was provided by data where growth of HeLa cells was inhibited by transfection with the exon 5 antisense oligonucleotides. Growth inhibition was followed by apoptosis in greater than 70% of the transfected cells.

Phenoxodiol, a synthetic isoflavene with clinical efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated tNOX. Purified recombinant tNOX bound phenoxodiol with high affinity (Kd of 50 nM). The tNOX protein appeared to be both necessary and sufficient for the cancer-specific cytotoxicity of phenoxodiol. Growth inhibition of fibroblasts from embryos of mice expressing a tNOX transgene, but not from wild-type mice, was inhibited by phenoxodiol followed by apoptosis. Both the oxidative and protein disulfide-thiol interchange activities that alternate to generate the complex set of oscillations with a period length of 22 min (24 min for the constitutive counterpart CNOX) that characterize ECTO-NOX proteins respond to phenoxodiol. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked by phenoxodiol. In contrast, the protein disulfidethiol interchange activity measured either by the restoration of activity to scrambled and inactive RNase or from the cleavage of dithiodipyridine (EC50 of 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ECTO-NOX (CNOX) forms of either cancer or noncancer cells were unaffected by phenoxodiol to help explain how the cytotoxic effects of phenoxodiol may be restricted to cancer cells.

A family of cell surface and growth related proteins that oxidize both NADH and hydroquinones and carry out protein disulfide-thiol interchange (ECTO-NOX proteins) exhibits unique characteristics. The two activities they catalyze, hydroquinone or NADH oxidation and protein disulfide-thiol interchange, alternate in CNOX (the constitutive ECTO-NOX), to generate a regular period length of 24min. For NADH or hydroquinone oxidation each period is defined by maxima that recur at intervals of 24min. Here, we report that bound Cu(II) is required to sustain the 24min oscillation cycle of CNOX. CNOX preparations from plasma membranes of soybean, when unfolded in the presence of the copper chelator bathocuproine and refolded, lose activity. When refolded in the presence of copper, activity is restored. Unexpectedly, however, the released copper is capable of catalyzing NADH (or hydroquinone) oxidation in the absence of protein. Solvated Cu(II) as the chloride or other salts alone is capable of catalyzing NADH oxidation and the oxidation rates oscillate with an overall period length of 24min. With Cu(II)Cl(2) the pattern consists of five maxima, two of which are separated by an interval of 6min and three of which are separated by intervals of 4.5min [6min+4 (4.5min)]. The period length is independent of temperature and pH. The asymmetry of the oscillatory pattern is retained after solvation of the Cu(II) salts in D(2)O but the overall period length is increased to 30min. The findings suggest that the bound copper of CNOX and perhaps of ECTO-NOX proteins in general, is essential to maintain the structural changes that underlie the periodic alternations in activity that define the 24min time-keeping cycle of the protein.

tNOX, a novel cell surface protein related to unregulated growth and drug response of cancer cells, has been proposed as the cellular target for the anticancer action of various quinone site inhibitors with anticancer activity including the polyphenol (-)-epigallocatechin-3-gallate (EGCg). A transgenic mouse line overexpressing tNOX was generated to determine its overall growth phenotype and susceptibility to EGCg. Cultured noncancer cells lack tNOX and are unresponsive to EGCg. Overexpression of tNOX in cultured noncancer cells through transfection resulted in both enhanced growth and an acquired inhibitory response to EGCg. The tNOX transgenic mouse line was developed using a phCMV2 vector with the hemagglutinin (HA) tag. Transgenic mice exhibited both an enhanced growth rate and a response to EGCg not observed with wild-type mice. Female transgenic mice grew twice as fast as wild type, and growth was reflected in an overall increased carcass weight. Administration of EGCg in the drinking water [500 mg/kg body weight (BW)] reduced the growth rate of the transgenic mice to that of wild-type mice. The findings provide in situ validation of the hypothesis that tNOX represents a necessary and sufficient molecular target as the basis for the protective and potential cancer therapeutic benefits of EGCg.

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.

A recurring pattern of spectral changes indicative of periodic changes in the proportion of beta-structure and a-helix of a recombinant ECTO-NOX fusion protein of tNOX, with a cellulose binding domain peptide, was demonstrated by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic analyses. The pattern of structural changes correlated with oscillatory patterns of enzymatic activities exhibited by the protein previously interpreted as indicative of a clock function. The pattern consisted of a repeating pattern of oscillations with a period length of 21 min with five maxima (two separated by 5 min and 3 separated by 4 to 4.5 min) within each 21 min repeat. Oscillatory patterns were not obvious in comparable FTIR or CD spectra of albumin, ribonuclease or concanavalin A. The period length was constant at 5, 15, 25, 35 and 45 degrees C (temperature compensated) and oscillations occurred independently of substrate presence. Spectra obtained in deuterium oxide yielded a longer period length of 26 min both for oscillations in enzymatic activity and absorbance ratios determined by FTIR. Taken together the findings suggest that the regular patterns of oscillations exhibited by the ECTO-NOX proteins are accompanied by recurrent global changes in the conformation of the protein backbone that directly modulate enzymatic activity.

Currently there is wide interest in the medicinal benefits of green tea (Camellia sinensis). Tea is one of the most widely consumed beverages in the world, and extracts of tea leaves are also sold as dietary supplements. Green tea extracts contain a unique set of catechins that possess biologic activity in antioxidant, antiangiogenesis, and antiproliferative assays that are potentially relevant to the prevention and treatment of various forms of cancer. With the increasing interest in the health properties of tea and a significant rise in their scientific investigation, it is the aim of this review to summarize recent findings on the anticancer and medicinal properties of green tea, focusing on the biologic properties of the major tea catechin, (-)-epigallocatechin and its antitumor properties.

An in vitro coculture model of prostate cancer cells (LNCaP) with human osteoblasts (hFOB) was utilized to define the efficacy of the tNOX inhibitors EGCg, capsaicin, Capsibiol-T and phenoxodiol against bone metastasis of prostate cancer alone and in combination with Taxol and cisplatin. In general, the LNCaP cells were more resistant to treatment with EGCg, capsaicin, phenoxodiol and Taxol when grown in coculture than when grown in monoculture. Only with Capsibiol-T (50 microM) was growth of LNCaP cells in coculture inhibited comparable with monoculture. Pretreatment with Capsibiol-T followed by the treatment with Taxol had an additive effect on reduction of viability of LNCaP cells in monoculture. In contrast, an antagonistic effect of cisplatin was observed following capsaicin pretreatment.

Tea, in the form of green or black tea, is one of the most widely consumed beverages in the world. Extracts of tea leaves also are sold as dietary supplements. However, with the increasing interest in the health properties of tea and a significant rise in scientific investigation, this review covers recent findings on the medicinal properties and noncancer health benefits of both green and black tea. In Part II, a review of anticancer properties of green tea extracts is presented. Green tea contains a unique set of catechins that possess biological activity in antioxidant, anti-angiogenesis, and antiproliferative assays potentially relevant to the prevention and treatment of various forms of cancer. Although there has been much focus on the biological properties of the major tea catechin epigallocatechin gallate (EGCg) and its antitumor properties, tea offers other health benefits; some due to the presence of other important constituents. Characteristics unrelated to the antioxidant properties of green and black teas may be responsible for tea's anticancer activity and improvement in cardiac health and atherosclerosis. Theanine in green tea may play a role in reducing stress. Oxidized catechins (theaflavins in black tea) may reduce cholesterol levels in blood. Synergistic properties of green tea extracts with other sources of polyphenolic constituents are increasingly recognized as being potentially important to the medicinal benefits of black and green teas. Furthermore, due to presumed antioxidant and antiaging properties, tea is now finding its way into topical preparations. Each of these aspects is surveyed.

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD+ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.

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.