Inhibition Of NAD Research Articles

Histone Deacetylase Sirtuin 1 Promotes Loss of Primary Cilia in Cholangiocarcinoma.

Sirtuin 1 (SIRT1) is a complex NAD+ -dependent protein deacetylase known to act as a tumor promoter or suppressor in different cancers. Here, we describe a mechanism of SIRT1-induced destabilization of primary cilia in cholangiocarcinoma (CCA). A significant overexpression of SIRT1 was detected in human CCA specimens and CCA cells including HuCCT1, KMCH, and WITT1 as compared with normal cholangiocytes (H69 and NHC). Small interfering RNA (siRNA)-mediated knockdown of SIRT1 in HuCCT1 cells induced cilia formation, whereas overexpression of SIRT1 in normal cholangiocytes suppressed ciliary expression. Activity of SIRT1 was regulated by presence of NAD+ in CCA cells. Inhibition of NAD -producing enzyme nicotinamide phosphoribosyl transferase increased ciliary length and frequency in CCA cells and in SIRT1-overexpressed H69 cells. Furthermore, we also noted that SIRT1 induces the proteasomal mediated degradation of ciliary proteins, including α-tubulin, ARL13B, and KIF3A. Moreover, overexpression of SIRT1 in H69 and NHC cells significantly induced cell proliferation and, conversely, SIRT1 inhibition in HuCCT1 and KMCH cells using siRNA or sirtinol reduced cell proliferation. In an orthotopic transplantation rat CCA model, the SIRT1 inhibitor sirtinol reduced tumor size and tumorigenic proteins (glioma-associated oncogene 1, phosphorylated extracellular signal-regulated kinase, and IL-6) expression. In conclusion, these results reveal the tumorigenic role of SIRT1 through modulation of primary cilia formation and provide the rationale for developing therapeutic approaches for CCA using SIRT1 as a target.

Activation of Coronary Arteriolar PKCβ2 Impairs Endothelial NO-Mediated Vasodilation: Role of JNK/Rho Kinase Signaling and Xanthine Oxidase Activation.

Protein kinase C (PKC) activation can evoke vasoconstriction and contribute to coronary disease. However, it is unclear whether PKC activation, without activating the contractile machinery, can lead to coronary arteriolar dysfunction. The vasoconstriction induced by the PKC activator phorbol 12,13-dibutyrate (PDBu) was examined in isolated porcine coronary arterioles. The PDBu-evoked vasoconstriction was sensitive to a broad-spectrum PKC inhibitor but not affected by inhibiting PKCβ2 or Rho kinase. After exposure of the vessels to a sub-vasomotor concentration of PDBu (1 nmol/L, 60 min), the endothelium-dependent nitric oxide (NO)-mediated dilations in response to serotonin and adenosine were compromised but the dilation induced by the NO donor sodium nitroprusside was unaltered. PDBu elevated superoxide production, which was blocked by the superoxide scavenger Tempol. The impaired NO-mediated vasodilations were reversed by Tempol or inhibition of PKCβ2, xanthine oxidase, c-Jun N-terminal kinase (JNK) and Rho kinase but were not affected by a hydrogen peroxide scavenger or inhibitors of NAD(P)H oxidase and p38 kinase. The PKCβ2 protein was detected in the arteriolar wall and co-localized with endothelial NO synthase. In conclusion, activation of PKCβ2 appears to compromise NO-mediated vasodilation via Rho kinase-mediated JNK signaling and superoxide production from xanthine oxidase, independent of the activation of the smooth muscle contractile machinery.

Parp mutations protect from mitochondrial toxicity in Alzheimer\u2019s disease

Alzheimer’s disease is the most common age-related neurodegenerative disorder. Familial forms of Alzheimer’s disease associated with the accumulation of a toxic form of amyloid-β (Aβ) peptides are linked to mitochondrial impairment. The coenzyme nicotinamide adenine dinucleotide (NAD+) is essential for both mitochondrial bioenergetics and nuclear DNA repair through NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Here we analysed the metabolomic changes in flies overexpressing Aβ and showed a decrease of metabolites associated with nicotinate and nicotinamide metabolism, which is critical for mitochondrial function in neurons. We show that increasing the bioavailability of NAD+ protects against Aβ toxicity. Pharmacological supplementation using NAM, a form of vitamin B that acts as a precursor for NAD+ or a genetic mutation of PARP rescues mitochondrial defects, protects neurons against degeneration and reduces behavioural impairments in a fly model of Alzheimer’s disease. Next, we looked at links between PARP polymorphisms and vitamin B intake in patients with Alzheimer’s disease. We show that polymorphisms in the human PARP1 gene or the intake of vitamin B are associated with a decrease in the risk and severity of Alzheimer’s disease. We suggest that enhancing the availability of NAD+ by either vitamin B supplements or the inhibition of NAD+-dependent enzymes such as PARPs are potential therapies for Alzheimer’s disease.

Linking altered microglial metabolism to an impaired inflammatory response in Leigh syndrome

Leigh syndrome is a progressive neurodegenerative mitochondrial disease stemming from genetic defects in the mitochondrial electron transport chain, such as Complex I, which leads to lactic acidosis, bilateral necrotizing brain lesions, respiratory failure, and early death. The NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUFS4, a Complex I assembly factor) knockout (KO) mouse is an established mouse model of Leigh Syndrome. The Ndufs4 KO mouse manifests many of the biochemical and clinical aspects of Leigh syndrome, including microglial accumulation in lesioned brain regions. However, the specific contribution of altered microglial metabolism in the neuropathology of Leigh Syndrome remains understudied. The inhibition of Complex I function following loss of Ndufs4 reduces the electron transport chain activity leading to mitochondrial dysfunction and increased reductive stress, manifest as an elevation of NADH:NAD+ levels. Reductive stress and the consequent inhibition of NAD+-dependent Krebs cycle enzymes facilitates the accumulation of intermediary metabolites, including the dicarboxylate fumarate. Fumarate can irreversibly modify protein thiols, a modification known as protein succination, and we have previously demonstrated increased protein succination in the pathologically lesioned regions of the Ndufs4 KO brain. Activated immune cells produce another dicarboxylic acid, itaconate, through the action of immunoresponsive gene 1 (Irg1). Itaconate acts as part of a negative feedback loop to suppress pro-inflammatory activity. I propose that altered levels of Krebs cycle intermediates may interfere with the capacity to mount a normal anti-inflammatory response in Complex I deficient Ndufs4 KO microglia; specifically as a result of impaired itaconate production. This impairment is predicted to limit microglial phagocytic function and may alter the capacity to respond to infections. Real time PCR confirms increased expression of microglial genes (Iba1, Trem2, CD68), a ~2 fold increase in pro- (IL-1β) and anti-inflammatory (IL-10 and HO-1) factors, but a decrease in Irg1 in late stage Ndufs4 KO olfactory bulb samples. Ndufs4 KO immune cells show decreased itaconate levels, particularly in response to lipopolysaccharide (LPS, a pro-inflammatory stimulus). LPS stimulation enhanced glycolytic metabolism, but with a lower metabolic capacity in Ndufs4 KO vs. WT cells. Inflammatory challenge also resulted in an impaired induction of several pro- and anti-inflammatory cytokines signaling factors and phagocytic uptake. KO of Ndufs4 increased metabolite-induced protein modification. Treatment of Ndufs4 KO mice with 4-octyl itaconate, a derivative of itaconate, enhanced the expression of pro-phagocytic TREM2 in Ndufs4 KO brain, but did not improve fine motor behavior. Overall, our data suggests that altered mitochondrial metabolism and reduced itaconate production may impair microglial ability to resolve inflammation in the Ndufs4 KO model of Leigh Syndrome.

Pathogen induced subversion of NAD+ metabolism mediating host cell death: a target for development of chemotherapeutics

Hijacking of host metabolic status by a pathogen for its regulated dissemination from the host is prerequisite for the propagation of infection. M. tuberculosis secretes an NAD+-glycohydrolase, TNT, to induce host necroptosis by hydrolyzing Nicotinamide adenine dinucleotide (NAD+). Herein, we expressed TNT in macrophages and erythrocytes; the host cells for M. tuberculosis and the malaria parasite respectively, and found that it reduced the NAD+ levels and thereby induced necroptosis and eryptosis resulting in premature dissemination of pathogen. Targeting TNT in M. tuberculosis or induced eryptosis in malaria parasite interferes with pathogen dissemination and reduction in the propagation of infection. Building upon our discovery that inhibition of pathogen-mediated host NAD+ modulation is a way forward for regulation of infection, we synthesized and screened some novel compounds that showed inhibition of NAD+-glycohydrolase activity and pathogen infection in the nanomolar range. Overall this study highlights the fundamental importance of pathogen-mediated modulation of host NAD+ homeostasis for its infection propagation and novel inhibitors as leads for host-targeted therapeutics.

Dicoumarol, an NQO1 inhibitor, blocks cccDNA transcription by promoting degradation of HBx

Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.

Herpetosiphon Secondary Metabolites Inhibit Amyloid-β Toxicity in Human Primary Astrocytes.

The accumulation of extracellular plaques containing amyloid-β protein (Aβ) in the brain is one of the main pathological hallmarks of Alzheimer's disease (AD). Aβ peptide can promote the production of highly volatile free radicals and reactive oxygen species (ROS) that can induce oxidative damage to neurons and astrocytes. At present, numerous studies have investigated the neuroprotective and glioprotective effects of natural products derived from plants, animals, and microorganisms. We investigated the glioprotective effect of secondary metabolites obtained from Herpetosiphon sp. HM 1988 against Aβ40-induced toxicity in human primary astrocytes. The protective effect of bacterial secondary metabolites against Aβ40-induced inducible nitric oxide synthase (iNOS) activity was evaluated using the citrulline assay. To confirm the iNOS activity, nitrite production was assessed using the fluorometric Griess diazotization assay. Intracellular NAD+ depletion and lactate dehydrogenase (LDH) release in human primary astrocytes were also examined using well-established spectrophotometric assays. Our results indicate that Aβ40 can induce elevation in iNOS and LDH activities, nitrite production, and cellular energy depletion. Importantly, extract of Herpetosiphon sp. HM 1988 decreased iNOS activity, nitrite production, and LDH release. In addition, metabolites of the strain were able to restore cellular energy deficits through inhibition of NAD+ depletion mediated by Aβ40. These findings suggest that Herpetosiphon metabolites may represent a promising, novel source for the prevention of Aβ toxicity in AD.

Root isoprene formation alters lateral root development

Isoprene is a C5 volatile organic compound, which can protect aboveground plant tissue from abiotic stress such as short-term high temperatures and accumulation of reactive oxygen species (ROS). Here, we uncover new roles for isoprene in the plant belowground tissues. By analysing Populus x canescens isoprene synthase (PcISPS) promoter reporter plants, we discovered PcISPS promoter activity in certain regions of the roots including the vascular tissue, the differentiation zone and the root cap. Treatment of roots with auxin or salt increased PcISPS promoter activity at these sites, especially in the developing lateral roots (LR). Transgenic, isoprene non-emitting poplar roots revealed an accumulation of O2 - in the same root regions where PcISPS promoter activity was localized. Absence of isoprene emission, moreover, increased the formation of LRs. Inhibition of NAD(P)H oxidase activity suppressed LR development, suggesting the involvement of ROS in this process. The analysis of the fine root proteome revealed a constitutive shift in the amount of several redox balance, signalling and development related proteins, such as superoxide dismutase, various peroxidases and linoleate 9S-lipoxygenase, in isoprene non-emitting poplar roots. Together our results indicate for isoprene a ROS-related function, eventually co-regulating the plant-internal signalling network and development processes in root tissue.

Angiotensin Type 1 Receptors and Superoxide Anion Production in Hypothalamic Paraventricular Nucleus Contribute to Capsaicin-Induced Excitatory Renal Reflex and Sympathetic Activation.

Chemical stimulation of the kidney increases sympathetic activity and blood pressure in rats. The hypothalamic paraventricular nucleus (PVN) is important in mediating the excitatory renal reflex (ERR). In this study, we examined the role of molecular signaling in the PVN in mediating the capsaicin-induced ERR and sympathetic activation. Bilateral PVN microinjections were performed in rats under anesthesia. The ERR was elicited by infusion of capsaicin into the cortico-medullaryborder of the right kidney. The reflex was evaluated as the capsaicin-induced changes in left renal sympathetic nerve activity and mean arterial pressure. Blockade of angiotensin type 1 receptors with losartan or inhibition of angiotensin-converting enzyme with captopril in the PVN abolished the capsaicin-induced ERR. Renal infusion of capsaicin significantly increased NAD(P)H oxidase activity and superoxide anion production in the PVN, which were prevented by ipsilateral renal denervation or microinjection of losartan into the PVN. Furthermore, either scavenging of superoxide anions or inhibition of NAD(P)H oxidase in the PVN abolished the capsaicin-induced ERR. We conclude that the ERR induced by renal infusion of capsaicin is mediated by angiotensin type 1 receptor-related NAD(P)H oxidase activation and superoxide anion production within the PVN.

Subcellular Characterization of Nicotinamide Adenine Dinucleotide Biosynthesis in Metastatic Melanoma by Using Organelle-Specific Biosensors.

Aim: Nicotinamide adenine dinucleotide (NAD+) plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD+ biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD+ levels in live cells and to address the issue of the compartmentalization of NAD+ biosynthesis, we exploited a recently described genetically encoded NAD+ biosensor (LigA-circularly permutated Venus), which was targeted to the cytosol, mitochondria, and nuclei of BRAF-V600E A375 melanoma cells, a model of metastatic melanoma (MM). Results: FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD+-producing enzyme in MM cells, was used to monitor NAD+ depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD+ precursors, including nicotinamide, nicotinic acid, nicotinamide riboside, and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression of NAD+ biosynthetic enzymes was then biochemically studied in isolated organelles, revealing the presence of NAMPT in all three cellular compartments, whereas nicotinate phosphoribosyltransferase was predominantly cytosolic and mitochondrial, and nicotinamide riboside kinase mitochondrial and nuclear. In keeping with biosensor data, quinolinate phosphoribosyltransferase was expressed at extremely low levels. Innovation and Conclusions: Throughout this work, we validated the use of genetically encoded NAD+ biosensors to characterize subcellular distribution of NAD+ production routes in MM. The chance of real-time monitoring of NAD+ fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD+ pool manipulation in cancer and metabolic diseases.

Proteome analysis of Candida albicans cells undergoing chlamydosporulation

Chlamydospore though considered as a unique and rare morphological form of Candida albicans, regulation and significance of chlamydosporulation is not very clear. SWATH-MS analysis of chlamydosporulation specific proteins revealed that 319 (137-Up regulated and 182-Down regulated) proteins expressed differentially. Functional annotation showed significant modulations in proteins involved in cellular architecture (30), carbohydrate (29), amino acid (17), fatty acid (3), Nucleic acid (14), vitamins (1) metabolism as well as signaling (6), stress response (26), transport (cytoplasmic-21, mitochondrial-6 and nuclear-1), gene expression (transcription-12, RNA processing-6, translation-53, PTM-18), proteolysis (15) etc. Enhanced mannan, β1, 3-glucan and chitin contribute in thickening of cell wall while Hyr1 (218-fold) and Als3 (38.16-fold) dominates the cell surface chemistry of chlamydospores. In addition to ergosterol, enhanced sphingolipids, phospholipids and fatty acids make chlamydospore membrane more sturdy and rigid. Up-regulation of maltase (64-fold) followed by enhanced glycolysis and tricarboxylic acid cycle under nutrient-limiting condition is indicative of chlamydosporulation. Glyoxylate and fermentative pathway reported to facilitate survival of C. albicans under glucose limiting and microaerophilic condition was up-regulated. Enhanced biosynthesis of glutathione, trehalose homeostasis, and inhibition of NAD+ generation ,etc., potentiate oxidative, osmotic and nitrosative stress tolerance. Up regulation of Rsr1 (8.83-fold) and down regulation of Bcy1 (4.20-fold), Tfs1 (negative regulator of RAS) indicates cAMP-PKA pathway activates chlamydosporulation through Efg1 (a morphogenic regulator) in our study. In general, morpho-physiological modulations in C. albicans is a result of different sets of transcriptional programs that facilitate survival under nutrient and oxygen limiting condition.

Multi-targeted Effect of Nicotinamide Mononucleotide on Brain Bioenergetic Metabolism.

Dysfunctions in NAD+ metabolism are associated with neurodegenerative diseases, acute brain injury, diabetes, and aging. Loss of NAD+ levels results in impairment of mitochondria function, which leads to failure of essential metabolic processes. Strategies to replenish depleted NAD+ pools can offer significant improvements of pathologic states. NAD+ levels are maintained by two opposing enzymatic reactions, one is the consumption of NAD+ while the other is the re-synthesis of NAD+. Inhibition of NAD+ degrading enzymes, poly-ADP-ribose polymerase 1 (PARP1) and ectoenzyme CD38, following brain ischemic insult can provide neuroprotection. Preservation of NAD+ pools by administration of NAD+ precursors, such as nicotinamide (Nam) or nicotinamide mononucleotide (NMN), also offers neuroprotection. However, NMN treatment demonstrates to be a promising candidate as a therapeutic approach due to its multi-targeted effect acting as PARP1 and CD38 inhibitor, sirtuins activator, mitochondrial fission inhibitor, and NAD+ supplement. Many neurodegenerative diseases or acute brain injury activate several cellular deathpathways requiring a treatment strategy that will targetthese mechanisms. Since NMN demonstrated the ability to exert its effect on several cellular metabolic pathways involved in brain pathophysiology it seems to be one of the mostpromising candidates to be used for successful neuroprotection.

Erratum: A small compound spindlactone A sensitizes human endometrial cancer cells to TRAIL-induced apoptosis via the inhibition of NAD(P)H dehydrogenase quinone 1 [Corrigendum

[This corrects the article on p. 3609 in vol. 11, PMID: 29950865.].

Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β.

Transforming growth factor-β (TGF-β) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] generators), ROS, and ROS-sensitive p38 mitogen-activated protein kinase (p38MAPK) in this signaling pathway in cultured human vascular smooth muscle cells (VSMCs). The present in vitro study was performed on human VSMCs. Proteins were detected by western blotting utilizing an anti-phospho-Smad2 (Ser245/250/255) rabbit polyclonal antibody and a horseradish peroxidase-labeled secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The phospho-Smad2 linker region (pSmad2L) was detected in all the experimental groups: a control group (untreated group), a group treated with TGF-β (2 ng/mL), and a group treated with TGF-β plus different inhibitors. The data were normalized and presented as mean±SEM. The statistical analyses were performed using SPSS, version 16.0, and the nonparametric Kruskal-Wallis test. A P value smaller than 0.05 was considered statistically significant. The VSMCs treated with TGF-β (2 ng/mL) showed a time-dependent increase in the pSmad2L level. The highest level was observed at 15 minutes (P=0.03). The inhibitors of NAD(P)H oxidases (diphenyleneiodonium and apocynin) (P=0.04), ROS scavenger (N-acetylcysteine) (P=0.04), and p38MAPK inhibitor (SB-202190) (P=0.04) were able to reduce the increased level of the pSmad2L by TGF-β. Our results suggested that NAD(P)H oxidases played an important role in the Smad2L phosphorylation in the human VSMCs. Furthermore, our results confirmed that ROS and p38MAPK were involved in this signaling pathway. Thus, TGF-β via a ROS-dependent mechanism can transmit its signals to the pSmad2L.

A small compound spindlactone A sensitizes human endometrial cancer cells to TRAIL-induced apoptosis via the inhibition of NAD(P)H dehydrogenase quinone 1.

IntroductionSpindlactone A (SPL-A) is a novel small molecule inhibitor of TACC3 that selectively inhibits the nucleation of centrosome microtubules and induces mitotic arrest in ovarian cancer cells. SPL-A is derived from dicoumarol which inhibits the activity of NAD(P)H dehydrogenase quinone oxidoreductase 1 (NQO1). This study aimed to investigate the mechanism by which SPL-A enhances TRAIL-induced apoptosis in endometrial carcinoma cells.Materials and methodsEndometrial carcinoma cells were treated with SPL-A and/or TRAIL, and the apoptosis and protein expression in the treated cells were examined.ResultsCombined treatment with SPL-A and TRAIL significantly induced apoptosis in various human endometrial carcinoma cells, but not in normal human endometrial stromal cells and endometrial epithelial cells. Notably, both NQO1 inhibitor ES936 and NQO1 siRNA enhanced TRAIL-induced apoptosis of endometrial carcinoma cells. Furthermore, SPL-A downregulated the expression of c-FLIP, Bcl-2, Bcl-xl, and Mcl-1, while increasing p53 expression.ConclusionIn particular, luciferase assay showed that SPL-A inhibited Bcl-2 promoter activity, and p53 inhibitor PFT-α could reverse the effect of SPL-A on Bcl-2 expression. Moreover, Bcl-2 overexpression inhibited the apoptosis induced by SPL-A and TRAIL. Taken together, our results suggest that SPL-A sensitizes endometrial cancer cells to TRAIL-induced apoptosis via the regulation of apoptosis-related proteins and the inhibition of NQO1 activity.

A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline

Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD+) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction.

β-aminoisobutyric acid accelerates the proliferation and differentiation of MC3T3-E1 cells via moderate activation of ROS signaling

BackgroudOsteoporosis is one of the bone-metabolic diseases associated with decreased bone renewal and bone mineral density. β-aminoisobutyric acid (BAIBA), a natural thymine catabolite, can reduce inflammation in skeletal muscle and alleviate hepatic endoplasmic reticulum stress. However, the roles of BAIBA in osteoblast proliferation and differentiation remain largely unknown. MethodsThe cultured MC3T3-E1 cells received various treatments in this study, including BAIBA alone, H2O2 alone, BAIBA plus N-acetyl-l-cysteine and BAIBA plus apocynin. Cell proliferation was determined by CCK-8 assay and 3H-Thymidine incorporation. Cell differentiation was evaluated by determining mRNA level of differentiation makers and ALP, and ALP activity. Reactive oxygen species (ROS) were determined by DHE staining while superoxide anion level and NAD(P)H oxidase activity were determined by the lucigenin-derived chemiluminescence method. The content of hydrogen peroxide (H2O2) was detected using a commercial kit. The level of NOX1, NOX2 and NOX4 was determined by Western-blot or qRT-PCR. ResultsWe show that treatment of BAIBA stimulated the proliferation of MC3T3-E1 osteoprogenitor cells and enhanced the gene expression of osteoblast differentiation markers. Incubation of MC3T3-E1 cells with BAIBA evoked increases in NAD(P)H oxidase-derived reactive oxygen species (ROS). Scavenging of reactive oxygen species (N-acetyl-l-cysteine) or inhibition of NAD(P)H oxidase (apocynin) abolished the BAIBA-elicited proliferation and differentiation of MC3T3-E1 cells. ConclusionOur results provide the first evidence that BAIBA stimulates proliferation and differentiation of osteoprogenitor cells via activation of NAD(P)H oxidase/ROS signaling.

Glucose impairs aspirin inhibition in platelets through a NAD(P)H oxidase signaling pathway

Hyperglycemia has been suggested to play a role in the increased platelet resistance to antiplatelet therapy in patients with diabetes mellitus. Exposure to high glucose impairs platelet inhibition by aspirin. It has been found that antioxidant agents reduce the effect of glucose, confirming the involvement of reactive oxygen species (ROS) in the effect of glucose. The aim of the study was to examine the mechanism of ROS increase by high glucose in aspirin-treated platelets. Platelet aggregation was measured by the optical method, and the production of ROS was detected using luminol-dependent horseradish peroxidase-enhanced chemiluminescence. We found that glucose did not affect ADP-induced platelet aggregation. However, it reduced the effect of aspirin on platelet aggregation, which was accompanied by an increase in ROS generation. The inhibition of NAD(P)H oxidase (NOX) prevented the glucose effect and ROS generation. The same result was recorded after the inhibition of p38 mitogen-activated protein kinases (p38 MAPK), phospholipase A2 (PLA2) or 12-lipoxygenase (12-LOX). The inhibition of TxA2 receptor did not decrease the effect of glucose indicating that the effect was not caused by activation of TxA2 receptors.

Retraction notice to: Resveratrol attenuates lipopolysaccharide-induced dysfunction of blood-brain barrier in endothelial cells via AMPK activation.

[This retracts the article on p. 325 in vol. 20, PMID: 27382348.].

Abstract 3016: KPT-9274 inhibits cellular NAD and synergizes with NAD depleting enzymes to induce cancer cell death

Abstract Nicotinamide adenine dinucleotide (NAD) is an essential metabolite and important cofactor of several biological processes (i.e. metabolism and genomic stability) that undergo significant alterations during malignant transformation. In cancer cells, NAD undergoes quick turnover through the high metabolic demands of rapidly proliferating cells and increased activity of NAD consuming enzymes, such as sirtuin 1 (SIRT1) and poly ADP ribose polymerase 1 (PARP1). NAD can be generated de novo from tryptophan or regenerated by nicotinamide phosphoribosyl transferase (NAMPT) or nicotinate phosphoribosyl transferase 1 (NAPRT1) in NAD salvage pathways. However, cancer cells do not utilize the de novo or NAPRT1 pathways effectively and instead rely on the NAMPT-dependent salvage pathway to generate NAD, making NAD depletion a promising anti-cancer therapy. We have previously described the PAK4 allosteric modulator, KPT-9274, which can also inhibit the enzymatic activity of NAMPT. KPT-9274 and other NAMPT inhibitors rapidly deplete cellular NAD levels, ultimately leading to ATP depletion and cell death. Similarly, several studies have shown that hyper activation of NAD consuming enzymes can lead to apoptosis. The purpose of this study is to determine whether NAD inhibition by KPT-9274 can synergize with NAD depleting enzymes to enhance the cytotoxic effects of KPT-9274 in cancer cells. Methods: Celltiter-Glo was used to measure ATP levels and viability of cells. NAD/NADH-Glo was used to measure total NAD levels in cells. Gene and protein expression was measured using quantitative PCR and western blot analysis, respectively. Protein knockdown was accomplished using RNAi. Results: We have identified an orally bioavailable dual inhibitor of PAK4 and NAMPT, which demonstrated potent anti-cancer activity in a variety of cell lines both in vitro and in vivo. We have identified a mechanistic combination that increase the antitumor activity of KPT-9274, through the activation of NAD consuming enzymes such as SIRT1 and PARP1. Specifically, we found that SRT1720 (an activator of SIRT1) synergizes with KPT-9274 to increase cancer cell death while RNAi of SIRT1 diminishes the efficacy of KPT-9274. In addition, activation of PARP1 by DNA damaging agents (e.g. gemcitabine) significantly enhances the effectiveness of KPT-9274 mediated cell death. Finally, we show that PARP activation through DNA-damaging agents enhances the cytotoxicity and anti-tumor properties of KPT-9274 in xenograft models. Conclusions: Here we report that KPT-9274 synergizes with NAD depleting enzymes to induce cancer cell death in vitro and in vivo. This noteworthy enhancement to the anticancer activity of KPT-9274, together with its previously described PAK4 inhibition, support the continued development of this orally bioavailable small molecule in combination with current therapies. Citation Format: Christian Argueta, Trinayan Kashyap, Margaret Lee, Yosef Landesman, Sharon Shacham, William Senapedis, Erkan Baloglu. KPT-9274 inhibits cellular NAD and synergizes with NAD depleting enzymes to induce cancer cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3016.