Elevated NAD Research Articles

Exogenous NAD+ supplementation protects H9c2 cardiac myoblasts against hypoxia/reoxygenation injury via Sirt1‐p53 pathway

Nicotinamide adenine dinucleotide (NAD(+) ) not only transfers electrons in mitochondrial respiration, but also acts as an indispensable cosubstrate for Sirt1, the class III histone/nonhistone deacetylase. However, NAD(+) is depleted in myocardial ischemia/reperfusion (IR) injury. The objective of this study was to investigate the role of exogenous NAD(+) supplementation in hypoxia/reoxygenation (HR)-stressed H9c2 cardiac myoblasts. Firstly, the effects of distinct treating time points and doses of NAD(+) supplementation on the viability of HR-stressed H9c2 cells were detected. Secondly, intracellular NAD(+) levels in HR-stressed H9c2 cells at various extracellular NAD(+) concentrations were determined. Thirdly, the role of NAD(+) supplementation in HR-induced cell apoptosis and its relevance to Sirtuin 1-p53 pathway were investigated. Exogenous NAD(+) supplementation elevated intracellular NAD(+) level and reduced HR-induced cell death in both time- and concentration-dependent manners. It appeared that NAD(+) supplementation exerted the greatest protection when extracellular concentration ranged from 500 to 1000μm and when NAD(+) was added immediately after reoxygenation began. NAD(+) replenishment restored Sirt1 activity, reduced the acetylation level of p53 (Lys373 & 382), and attenuated cell apoptosis in HR-stressed H9c2 cells, whereas inhibition of Sirt1 activity alleviated the effects of NAD(+) replenishment. These results indicated that exogenous NAD(+) supplementation attenuated HR-induced cell apoptosis, which was at least partly mediated by restoring Sirt1 activity and subsequently inhibiting p53 activity via deacetylating p53 at lysine 373 and 382.

PhoY2 of Mycobacteria Is Required for Metabolic Homeostasis and Stress Response

The ability of pathogenic mycobacteria to adapt to diverse environments is essential for their success as pathogens. Here we describe a transposon-inactivated phoY2 mutant of Mycobacterium marinum. PhoY2 of mycobacteria is a functional homologue of PhoU in Escherichia coli and an important component of the Pho regulon. We found that PhoY2 is required for maintaining intracellular inorganic phosphate (P(i)) homeostasis and balanced energy and redox states. Disruption of phoY2 resulted in elevated levels of intracellular poly-P(i) and ATP and an elevated NAD(+)/NADH ratio, and the mutant strain exhibited increased sensitivity to environmental stress conditions, including nutrient deprivation as well as SDS and antibiotic treatments. Taken together, our results suggest that PhoY2 is required for maintaining metabolic homeostasis and adaptation to stress conditions, which may provide an explanation for the suggested role of PhoY2 in drug tolerance.

Chronic cranberry juice consumption restores cholesterol profiles and improves endothelial function in ovariectomized rats

Postmenopausal women experience higher risks for cardiovascular diseases than age-matched men and pre-menopausal women. There is a need for better treatment strategy for estrogen-deficient-related cardiovascular complications. We and others have recently reported that activated renin-angiotensin system and the associated oxidative stress impaired endothelium-dependent relaxation in ovariectomized rat, while angiotensin receptor blocker rescues endothelial dysfunction. Dietary supplements and lifestyle modifications provide an alternative way to improve cardiovascular health. The present study tests the hypothesis that chronic cranberry juice consumption improves cholesterol profiles and vascular functions in estrogen-deficient animal model. The effect of cranberry consumption on expression and activity of renin-angiotensin system in the vasculature will be determined. Ovariectomized rats were treated daily with commercial cranberry juice at 7 mg/kg for 8 weeks, a dosage comparable to recent clinical studies. Serum was collected for measuring cholesterol levels while aorta was isolated for isometric force assay and expression studies. Cranberry juice consumption reduced circulating levels of total cholesterol, triacylglycerols, HDL, nHDL, and nHDL/HDL ratio. Meanwhile, cranberry juice consumption improved endothelium-dependent relaxation in aorta of ovariectomized rats by restoring p-eNOS level (endothelial nitric oxide synthase phosphorylated at ser-1177), reversing the up-regulated levels of renin-angiotensin system markers (angiotensin-converting enzyme, angiotensin II, and angiotensin II type 1 receptor), and normalizing the elevated NAD(P)H oxidase expression and oxidative stress. Our data demonstrate the novel cardiovascular benefits of cranberry juice consumption in improving both vascular functions and cholesterol profiles, providing insight into developing cranberry products into useful dietary supplements for postmenopausal women.

Altered behavioral and metabolic circadian rhythms in mice with disrupted NAD+ oscillation

The Intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) are rhythmic and controlled by the circadian clock. However, whether NAD(+) oscillation in turn contributes to circadian physiology is not fully understood. To address this question we analyzed mice mutated for the NAD(+) hydrolase CD38. We found that rhythmicity of NAD(+) was altered in the CD38-deficient mice. The high, chronic levels of NAD(+) results in several anomalies in circadian behavior and metabolism. CD38-null mice display a shortened period length of locomotor activity and alteration in the rest-activity rhythm. Several clock genes and, interestingly, genes involved in amino acid metabolism were deregulated in CD38-null livers. Metabolomic analysis identified alterations in the circadian levels of several amino acids, specifically tryptophan levels were reduced in the CD38-null mice at a circadian time paralleling with elevated NAD(+) levels. Thus, CD38 contributes to behavioral and metabolic circadian rhythms and altered NAD(+) levels influence the circadian clock.

NAD+-dependent SIRT1 Deacetylase Participates in Epigenetic Reprogramming during Endotoxin Tolerance

Gene-selective epigenetic reprogramming and shifts in cellular bioenergetics develop when Toll-like receptors (TLR) recognize and respond to systemic life-threatening infections. Using a human monocyte cell model of endotoxin tolerance and human leukocytes from acute systemic inflammation with sepsis, we report that energy sensor sirtuin 1 (SIRT1) coordinates the epigenetic and bioenergy shifts. After TLR4 signaling, SIRT1 rapidly accumulated at the promoters of TNF-α and IL-1β, but not IκBα; SIRT1 promoter binding was dependent on its co-factor, NAD(+). During this initial process, SIRT1 deacetylated RelA/p65 lysine 310 and nucleosomal histone H4 lysine 16 to promote termination of NFκB-dependent transcription. SIRT1 then remained promoter bound and recruited de novo induced RelB, which directed assembly of the mature transcription repressor complex that generates endotoxin tolerance. SIRT1 also promoted de novo expression of RelB. During sustained endotoxin tolerance, nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for endogenous production of NAD(+), and SIRT1 expression increased. The elevation of SIRT1 required protein stabilization and enhanced translation. To support the coordination of bioenergetics in human sepsis, we observed elevated NAD(+) levels concomitant with SIRT1 and RelB accumulation at the TNF-α promoter of endotoxin tolerant sepsis blood leukocytes. We conclude that TLR4 stimulation and human sepsis activate pathways that couple NAD(+) and its sensor SIRT1 with epigenetic reprogramming.

GPCR Ligands Differentially Modulate Islet Metabolism and Ca 2+ Signaling

Glucose homeostasis is a tightly regulated process coordinated by insulin and glucagon secretion. In α- and β-cells, G-protein coupled receptor (GPCR) activation facilitates the optimization of insulin and glucagon secretion. Glucagon-like peptide-1 (GLP-1), a ligand activating the Gs pathway, increases insulin secretion, but inhibits glucagon secretion. Neuropeptide-Y (NPY) and somatostatin (SST), whose receptors are coupled to Gi, inhibit insulin secretion; however, NPY stimulates glucagon release, while SST inhibits glucagon secretion. Here, we question whether and how these GPCR ligands impact cellular metabolism and oscillations of intracellular Ca2+ activity ([Ca2+]i) to alter secretion in the pancreatic islet. We utilized two-photon excitation microscopy to measure the combined autofluorescence of NADH and NADPH (NAD(P)H, a cellular redox state indicator) upon application of NPY, SST, or GLP-1. Under untreated conditions, an increase in extracellular glucose results in elevated NAD(P)H fluorescence intensities. Treatment with NPY and SST produces a supplementary increase in NAD(P)H autofluorescence at glucose concentrations above 5 mM compared to untreated control. NAD(P)H autofluorescence is not impacted significantly (relative to untreated control) by GLP-1 treatment, which is consistent with a previous report that found that GLP-1 does not alter β-cell metabolism. At glucose concentrations greater than ∼7 mM, pancreatic islets display synchronous oscillations in [Ca2+]i, leading to the pulsatile release of insulin. The [Ca2+]i oscillation frequency increases significantly in the presence of NPY. SST treatment decreases the oscillation frequency in β-cells, but does not affect α-cells. Addition of GLP-1 does not significantly alter the [Ca2+]i oscillation frequency. Thus, GLP-1 likely modulates insulin and glucagon secretion downstream of Ca2+ signaling. NPY may inhibit insulin secretion downstream of Ca2+ signaling, but stimulates glucagon secretion upstream of Ca2+ activity. Islet hormone secretion may be inhibited by SST through alteration of pathways up- and downstream of Ca2+ signaling.

Mouse embryonic fibroblasts from CD38 knockout mice are resistant to oxidative stresses through inhibition of reactive oxygen species production and Ca2+ overload

CD38 is a multifunctional enzyme that has both ADP-ribosyl cyclase and cADPR hydrolase activities, being capable of cleaving NAD+ to cyclic ADP ribose (cADPR) and hydrolyzing cADPR to ADPR. It has been reported that there is markedly a reduction of cADPR and elevation of NAD in many tissues from CD38 knockout (CD38−/−) mice. Cyclic ADPR is a potent second messenger for intracellular Ca2+ mobilization, and NAD is a key cellular metabolite for cellular energetic and a crucial regulator for multiple signaling pathways in cells. We hypothesize that CD38 knockout may have a protective effect in oxidative stresses through elevating NAD and decreasing cADPR. In the present study, we observed that the mouse embryonic fibroblasts (MEFs) from CD38−/− mice were significantly resistant to oxidative stress such as H2O2 injury and hypoxia/reoxygenation compared with wild type MEFs (WT MEFs). We further found that production of reactive oxygen species (ROS) and concentrations of intracellular Ca2+ ([Ca2+]i) in CD38−/− MEFs were markedly reduced compared with WT MEFs during hypoxia/reoxygenation. Coincidence with these results, a remarkably lower mRNA level of Nox1, one of the enzymes responsible for ROS generation, was observed in CD38−/− MEFs. Furthermore, we found that transcription of Nox1 mRNA in WT MEFs could be elevated by calcium ionophore ionomycin in a dose-dependent manner, indicating that the expression of Nox1 mRNA can be regulated by elevation of intracellular [Ca2+]. Therefore we concluded that CD38−/− MEFs are resistant to oxidative stresses through inhibiting intracellular Ca2+ overload and ROS production which may be regulated by Ca2+-mediated inhibition of Nox1 expression. Our data should provide an insight for elucidating the roles of CD38 in oxidative stresses and a novel perspective of dealing with the ischemia/reperfusion-related diseases.

Redirecting Reductant Flux into Hydrogen Production via Metabolic Engineering of Fermentative Carbon Metabolism in a Cyanobacterium

Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H(2)), a carbon-neutral, renewable product derived from water, in low yields during autofermentation (anaerobic metabolism) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have constructed a mutant (the ldhA mutant) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this AMOP. Both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolomic methods have shown that autofermentation by the ldhA mutant resulted in no D-lactate production and higher concentrations of excreted acetate, alanine, succinate, and hydrogen (up to 5-fold) compared to that by the wild type. The measured intracellular NAD(P)(H) concentrations demonstrated that the NAD(P)H/NAD(P)(+) ratio increased appreciably during autofermentation in the ldhA strain; we propose this to be the principal source of the observed increase in H(2) production via an NADH-dependent, bidirectional [NiFe] hydrogenase. Despite the elevated NAD(P)H/NAD(P)(+) ratio, no decrease was found in the rate of anaerobic conversion of stored carbohydrates. The measured energy conversion efficiency (ECE) from biomass (as glucose equivalents) converted to hydrogen in the ldhA mutant is 12%. Together with the unimpaired photoautotrophic growth of the ldhA mutant, these attributes reveal that metabolic engineering is an effective strategy to enhance H(2) production in AMOPs without compromising viability.

Effects of chronic antioxidant treatment on AT‐1 receptor, NAD(P)H oxidase subunits and main antioxidant enzymes gene expression in the kidneys of 2K‐1C rats

Previous studies showed that chronic treatment with an antioxidant: vitamin C (vC) improves hypertension and sympathetic vasomotor hyperactivity in an Ang II‐dependent model of renovascular arterial hypertension. To test the hypothesis that oxidative stress is associated with NAD(P)H oxidase activation by Ang II, gene expressions of AT‐1 receptor (AT1R), NAD(P)H oxidase subunits (p47phox and gp91phox) and main antioxidant enzymes were evaluated in the renal cortex of 2 kidney‐1 clip model (2K1C, 6 weeks after clipping) rats before and after vC (150mg/kg/day). Gene expression of AT1R was elevated (51%) in the clipped kidney (CK) and reduced (47%) in the nonclipped kidney (NK) of 2K1C group. vC reduced AT1R expression only in the CK (31%). Elevated gene expression of p47phox and gp91phox was observed only in the CK of 2K‐1C group (184% and 132%, respectively) and vC produced no changes in these expressions. Gene expression of catalase was reduced in the CK (70%) and NK (83%) of 2K1C rats. vC increased gene expression of glutathione peroxidase (GPx) in the CK (185%) and NK (212%) of 2K1C. The present study suggests that renal oxidative stress may be related to elevated AT1R and NAD(P)H oxidase subunits gene expression in the CK of 2K1C rats and vitC treatment may produce beneficial effects by AT1R downregulation in the CK and increase in GPx gene expression in the kidneys of 2K1C rats.Supported by FAPESP (07/56925‐1)

Metabolism-Excitation Coupling in a Model of KATP Channel Neonatal Diabetes Mellitus

The KATP channel is critical to pancreatic beta cells, linking glucose metabolism with insulin secretion. KATP channel mutations that reduce ATP-sensitivity lead to neonatal diabetes mellitus (NDM). Expression of these KATP channel mutations in mice also leads to severe diabetes. Beta cell mass and insulin content are initially preserved, but both decrease with time following the prolonged hyperglycemia. When glycemic control is imposed by transplantation of exogenous islets, this secondary loss of beta-cell mass and insulin content is avoided.We have examined various steps of the glucose stimulated insulin secretion pathway in islets that express ATP-insensitive KATP channels, which are either unprotected from hyperglycemia or protected by islet transplantation. In protected islets, expression of mutant KATP channels leads to a severe blunting of glucose stimulated [Ca2+] activity. There remains some sulfonylurea stimulated [Ca2+] and normal KCl stimulated [Ca2+], with insulin secretion following a similar pattern. In unprotected islets, isolated from severely diabetic mice, very similar patterns of [Ca2+] activity are measured but insulin secretion is markedly decreased.Using two-photon microscopy of NAD(P)H we find glucose metabolism is unaltered in protected mutant islets. This suggests that the reduced [Ca2+] elevation and thus mitochondrial Ca2+ uptake has little effect on mitochondrial metabolism. However in unprotected islets, there is elevated basal NAD(P)H and an absence of glucose stimulated NAD(P)H increase, suggesting a disruption of glycolysis and/or mitochondrial metabolism.Finally the low level of glucose stimulated [Ca2+] in mutant islets is partially synchronized, indicating coupling is present. Reduction of gap junction conductance via chemical inhibitors or islet dispersal leads to elevated glucose stimulated Ca2+. This indicates an important role for gap junctions in regulating Ca2+ triggering in the presence of KATP channel mutations.

Contiguous gene deletion of ELOVL7, ERCC8 and NDUFAF2 in a patient with a fatal multisystem disorder

Contiguous gene syndromes affecting the mitochondrial oxidative phosphorylation system have been rarely reported. Here, we describe a patient with apparent mitochondrial encephalomyopathy accompanied by several unusual features, including dysmorphism and hepatopathy, caused by a homozygous triple gene deletion on chromosome 5. The deletion encompassed the NDUFAF2, ERCC8 and ELOVL7 genes, encoding complex I assembly factor 2 (also known as human B17.2L), a protein of the transcription-coupled nucleotide excision repair (TC-NER) machinery, and a putative elongase of very long-chain fatty acid synthesis, respectively. Detailed evaluation of cultured skin fibroblasts revealed disturbed complex I assembly, depolarization of the mitochondrial membrane, elevated cellular NAD(P)H level, increased superoxide production and defective TC-NER. ELOVL7 mRNA was not detectable in these cells and no alterations in fatty acid synthesis were found. By means of baculoviral complementation we were able to restore the aberrations, thereby establishing causative links between genotype and cell-physiological phenotype. This first chromosomal microdeletion illustrates that beside primary defects in mitochondrial genes also additional genes possibly contribute to the disease phenotype, providing an additional explanation for the broad clinical symptoms associated with these disorders.

Nicotinamide Prevents NAD+ Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia: NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons

Neurons require large amounts of energy to support their survival and function, and are therefore susceptible to excitotoxicity, a form of cell death involving bioenergetic stress that may occur in several neurological disorders including stroke and Alzheimer's disease. Here we studied the roles of NAD(+) bioenergetic state, and the NAD(+)-dependent enzymes SIRT1 and PARP-1, in excitotoxic neuronal death in cultured neurons and in a mouse model of focal ischemic stroke. Excitotoxic activation of NMDA receptors induced a rapid decrease of cellular NAD(P)H levels and mitochondrial membrane potential. Decreased NAD(+) levels and poly (ADP-ribose) polymer (PAR) accumulation in nuclei were relatively early events (<4 h) that preceded the appearance of propidium iodide- and TUNEL-positive cells (markers of necrotic cell death and DNA strand breakage, respectively) which became evident by 6 h. Nicotinamide, an NAD(+) precursor and an inhibitor of SIRT1 and PARP1, inhibited SIRT1 deacetylase activity without affecting SIRT1 protein levels. NAD(+) levels were preserved and PAR accumulation and neuronal death induced by excitotoxic insults were attenuated in nicotinamide-treated cells. Treatment of neurons with the SIRT1 activator resveratrol did not protect them from glutamate/NMDA-induced NAD(+) depletion and death. In a mouse model of focal cerebral ischemic stroke, NAD(+) levels were decreased in both the contralateral and ipsilateral cortex 6 h after the onset of ischemia. Stroke resulted in dynamic changes of SIRT1 protein and activity levels which varied among brain regions. Administration of nicotinamide (200 mg/kg, i.p.) up to 1 h after the onset of ischemia elevated brain NAD(+) levels and reduced ischemic infarct size. Our findings demonstrate that the NAD(+) bioenergetic state is critical in determining whether neurons live or die in excitotoxic and ischemic conditions, and suggest a potential therapeutic benefit in stroke of agents that preserve cellular NAD(+) levels. Our data further suggest that, SIRT1 is linked to bioenergetic state and stress responses in neurons, and that under conditions of reduced cellular energy levels SIRT1 enzyme activity may consume sufficient NAD(+) to nullify any cell survival-promoting effects of its deacetylase action on protein substrates.

Pnc1p-Mediated Nicotinamide Clearance Modifies the Epigenetic Properties of rDNA Silencing in Saccharomyces cerevisiae

The histone deacetylase activity of Sir2p is dependent on NAD(+) and inhibited by nicotinamide (NAM). As a result, Sir2p-regulated processes in Saccharomyces cerevisiae such as silencing and replicative aging are susceptible to alterations in cellular NAD(+) and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD(+) concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss-Handler NAD(+) salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD(+) concentration. This growth condition alters the epigenetics of rDNA silencing, such that repression of a URA3 reporter gene located at the rDNA induces growth on media that either lacks uracil or contains 5-fluoroorotic acid (5-FOA), an unusual dual phenotype that is reminiscent of telomeric silencing (TPE) of URA3. Despite the similarities to TPE, the modified rDNA silencing phenotype does not require the SIR complex. Instead, it retains key characteristics of typical rDNA silencing, including RENT and Pol I dependence, as well as a requirement for the Preiss-Handler NAD(+) salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level.

Effect of altered AMP‐activated protein kinase activity and exercise training on muscle PBEF‐1/visfatin levels.

Pre‐B‐cell enhancing factor 1 (PBEF‐1; also known as visfatin) is a stress‐inducible regulator of intracellular NAD+. Elevated NAD+ levels are associated with increased Sirtuin activity, which can contribute to life span extension in some organisms. Caloric restriction may serve to extend life span via this mechanism. Since AMP‐activated protein kinase (AMPK) serves an important role as a cellular fuel gauge, we investigated the role of AMPK in regulating muscle PBEF‐1 levels. PBEF‐1 was significantly increased (50%) in tibialis anterior muscle from mice expressing an activating mutation of AMPK (γ1R70Q) in skeletal muscle (p&lt;0.01). PBEF‐1 was significantly decreased in the muscles of two models of impaired AMPK activity, muscle‐specific AMPKα2 inactive transgenic mice (13%; P&lt;0.05) and muscle‐specific LKB1 knockout mice (30%; p&lt;0.01). In addition to skeletal muscle, PBEF‐1 was also decreased in cardiac tissue from the AMPKα2 inactive transgenic mice and LKB1 knockouts, and this effect was relatively greater in the heart compared to skeletal muscle. We next investigated the effects of seven weeks of voluntary wheel cage running on PBEF‐1 protein expression in skeletal muscle and heart from rats. Training resulted in 26% increased PBEF‐1 levels in cardiac muscle (p&lt;0.05), but had no effect in triceps muscle despite clearly increased citrate synthase activity in this muscle. In conclusion, AMPK may be an important regulator of the purported anti‐aging protein, PBEF‐1 in skeletal and cardiac muscles. PBEF‐1 levels are significantly elevated after training in cardiac muscle, suggesting a previously unidentified potential aging‐protective role of chronic exercise in the heart.

An NQO1- and PARP-1-mediated cell death pathway induced in non-small-cell lung cancer cells by β-lapachone

Lung cancer is the number one cause of cancer-related deaths in the world. Patients treated with current chemotherapies for non-small-cell lung cancers (NSCLCs) have a survival rate of approximately 15% after 5 years. Novel approaches are needed to treat this disease. We show elevated NAD(P)H:quinone oxidoreductase-1 (NQO1) levels in tumors from NSCLC patients. beta-Lapachone, an effective chemotherapeutic and radiosensitizing agent, selectively killed NSCLC cells that expressed high levels of NQO1. Isogenic H596 NSCLC cells that lacked or expressed NQO1 along with A549 NSCLC cells treated with or without dicoumarol, were used to elucidate the mechanism of action and optimal therapeutic window of beta-lapachone. NSCLC cells were killed in an NQO1-dependent manner by beta-lapachone (LD50, approximately 4 microM) with a minimum 2-h exposure. Kinetically, beta-lapachone-induced cell death was characterized by the following: (i) dramatic reactive oxygen species (ROS) formation, eliciting extensive DNA damage; (ii) hyperactivation of poly(ADP-ribose)polymerase-1 (PARP-1); (iii) depletion of NAD+/ATP levels; and (iv) proteolytic cleavage of p53/PARP-1, indicating mu-calpain activation and apoptosis. Beta-lapachone-induced PARP-1 hyperactivation, nucleotide depletion, and apoptosis were blocked by 3-aminobenzamide, a PARP-1 inhibitor, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a Ca2+ chelator. NQO1- cells (H596, IMR-90) or dicoumarol-exposed NQO1+ A549 cells were resistant (LD50, >40 microM) to ROS formation and all cytotoxic effects of beta-lapachone. Our data indicate that the most efficacious strategy using beta-lapachone in chemotherapy was to deliver the drug in short pulses, greatly reducing cytotoxicity to NQO1- "normal" cells. beta-Lapachone killed cells in a tumorselective manner and is indicated for use against NQO1+ NSCLC cancers.

Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast

Consumers of higher levels of Brassica vegetables, particularly those of the genus Brassica (broccoli, Brussels sprouts and cabbage), reduce their susceptibility to cancer at a variety of organ sites. Brassica vegetables contain high concentrations of glucosinolates that can be hydrolyzed by the plant enzyme, myrosinase, or intestinal microflora to isothiocyanates, potent inducers of cytoprotective enzymes and inhibitors of carcinogenesis. Oral administration of either the isothiocyanate, sulforaphane, or its glucosinolate precursor, glucoraphanin, inhibits mammary carcinogenesis in rats treated with 7,12-dimethylbenz[a]anthracene. In this study, we sought to determine whether sulforaphane exerts a direct chemopreventive action on animal and human mammary tissue. The pharmacokinetics and pharmacodynamics of a single 150 mumol oral dose of sulforaphane were evaluated in the rat mammary gland. We detected sulforaphane metabolites at concentrations known to alter gene expression in cell culture. Elevated cytoprotective NAD(P)H:quinone oxidoreductase (NQO1) and heme oxygenase-1 (HO-1) gene transcripts were measured using quantitative real-time polymerase chain reaction. An observed 3-fold increase in NQO1 enzymatic activity, as well as 4-fold elevated immunostaining of HO-1 in rat mammary epithelium, provides strong evidence of a pronounced pharmacodynamic action of sulforaphane. In a subsequent pilot study, eight healthy women undergoing reduction mammoplasty were given a single dose of a broccoli sprout preparation containing 200 mumol of sulforaphane. Following oral dosing, sulforaphane metabolites were readily measurable in human breast tissue enriched for epithelial cells. These findings provide a strong rationale for evaluating the protective effects of a broccoli sprout preparation in clinical trials of women at risk for breast cancer.

Calcium-dependent Modulation of Poly(ADP-ribose) Polymerase-1 Alters Cellular Metabolism and DNA Repair

After genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) can be hyperactivated, causing (ADP-ribosyl)ation of nuclear proteins (including itself), resulting in NAD(+) and ATP depletion and cell death. Mechanisms of PARP-1-mediated cell death and downstream proteolysis remain enigmatic. beta-lapachone (beta-lap) is the first chemotherapeutic agent to elicit a Ca(2+)-mediated cell death by PARP-1 hyperactivation at clinically relevant doses in cancer cells expressing elevated NAD(P)H:quinone oxidoreductase 1 (NQO1) levels. Beta-lap induces the generation of NQO1-dependent reactive oxygen species (ROS), DNA breaks, and triggers Ca(2+)-dependent gamma-H2AX formation and PARP-1 hyperactivation. Subsequent NAD(+) and ATP losses suppress DNA repair and cause cell death. Reduction of PARP-1 activity or Ca(2+) chelation protects cells. Interestingly, Ca(2+) chelation abrogates hydrogen peroxide (H(2)O(2)), but not N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation and cell death. Thus, Ca(2+) appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage, which alters cellular metabolism and DNA repair.

Effect of selenium-containing compounds on hepatic chemoprotective enzymes in mice

Selenite and organoselenium compounds have been examined at supranutritional levels for their ability to influence the activity and mRNA levels of chemoprotective enzymes in the livers of selenium-sufficient mice and the changes compared to those elicited by oltipraz. Compounds investigated included novel selenocysteine prodrugs that have previously been evaluated for their ability to reduce the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. Following seven daily doses (i.g.), all compounds except 2-methylselenazolidine-4( R)-carboxylic acid (MSCA) increased thioredoxin reductase activity (43–92%) but only for 2-oxoselenazolidine-4( R)-carboxylic acid (OSCA) was there an accompanying increase in mRNA. No compound enhanced glutathione peroxidase activity, although sodium selenite significantly elevated the mRNA of this enzyme. Oltipraz was an efficacious inducer of both thioredoxin reductase and glutathione peroxidase mRNAs. Sodium selenite, selenazolidine-4( R)-carboxylic acid (SCA), and OSCA elevated NAD(P)H-quinone oxidoreductase mRNA but only for OSCA was the elevation in mRNA accompanied by an increase in enzyme activity. l-Selenocystine significantly increased this activity without increasing mRNA levels. Sodium selenite, l-selenocystine, l-selenomethionine, and Se-methyl- l-selenocysteine all enhanced glutathione S-transferase activity. The increased activity with sodium selenite was accompanied by increases in mRNAs of Gst α, Gst μ and Gst π classes, while for l-selenocystine and Se-methyl- l-selenocysteine, only an elevation in the mRNA for the Gst α class was observed. Gst α and Gst μ class mRNAs were elevated by OSCA without a significant elevation in enzyme activity. SCA and MSCA both elevated a Gst π mRNA and MSCA elevated Gst μ in addition. By comparison, oltipraz only significantly elevated the mRNA of Gst μ, adding to the conclusion that across the entire study, no selenium compound appears to be acting purely through the antioxidant response typified by oltipraz. Despite their chemical similarity, the three cysteine prodrugs, SCA, MSCA, and OSCA, each produced its own unique pattern of effects on protective enzymes and none was identical to the pattern elicited by sodium selenite, l-selenocystine, l-selenomethionine, and Se-methyl- l-selenocysteine. The study also shows that after 7 days of administration, there was only occasional concordance between elevations in mRNA and enzyme activity for any selenium compound and for any protective enzyme, there was no response in common for all selenium compounds.

Development of β-Lapachone Prodrugs for Therapy Against Human Cancer Cells with Elevated NAD(P)H:Quinone Oxidoreductase 1 Levels

beta-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces beta-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to beta-lapachone. Thus, beta-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non-small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of beta-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to beta-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to beta-lapachone. Once converted, beta-lapachone derivatives caused NQO1-dependent, mu-calpain-mediated cell death in human cancer cells identical to that caused by beta-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect beta-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of beta-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to beta-lapachone. The use of beta-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

Extremely potent triterpenoid inducers of the phase 2 response: Correlations of protection against oxidant and inflammatory stress

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-gamma of inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 in mouse macrophages. Here, we show that these analogues are also extremely potent inducers of the phase 2 response [e.g., elevation of NAD(P)H-quinone oxidoreductase and heme oxygenase 1], which is a major protector of cells against oxidative and electrophile stress. Moreover, like previously identified phase 2 inducers, the TP analogues use the antioxidant response element-Nrf2-Keap1 signaling pathway. Thus, induction of the phase 2 response and suppression of the iNOS induction was abrogated in nrf2(-/-) and keap1(-/-) mouse embryonic fibroblasts. The high potency of TP analogues in inducing the phase 2 response and blocking inflammation depends on the presence of activated Michael reaction (enone) functions at critical positions in rings A and C. The most potent TP doubles NAD(P)H-quinone oxidoreductase in murine hepatoma cells at 0.28 nM and has an IC(50) for suppression of iNOS induction in primary mouse macrophages of 0.0035 nM. The direct interaction of this TP with thiol groups of the Keap1 sensor for inducers is demonstrated spectroscopically. The antiinflammatory and phase 2 inducer potencies of 18 TP are closely linearly correlated (r(2) = 0.91) over 6 orders of magnitude of concentration. Thus, in addition to blocking inflammation and promoting differentiation, these TP exhibit another very important protective property: the induction of the phase 2 response.