Regeneration Of Nicotinamide Adenine Dinucleotide Research Articles

Shining light on green chemistry: BODIPY-infused porphyrin nanocomposite-enzymatic photocatalyst boosts selective aldehydes hydrogenation and organic transformation under solar spectrum

Photo-bio enzyme coupled biotransformation solar radiation driven processes grasp promising in the production of solar driven organic chemicals. Here we reported the synthesis and development of (NH2)4TPP@BODIPY photocatalyst, which was constructed from 5,10,15,20 tetrakis (4 amino phenyl) porphyrin and BODIPY {5,5-difluoro-3,7-bis((E)-4-nitrobenzylidene)-10-(4-nitrophenyl)-5,7-dihydro-3H-4l4,5l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinine} molecule through poly-condensation mechanism resulting in the composite formation which manifest excellent light harvesting properties and photocatalytic reactions with suitable satisfactory band gap and formation of huge electrons channels which bears the organic transformation (98 %) and photo-regeneration of NADH (66.92 %) in 60 min which further enhances reduction of aldehyde resulting alcohol production. The highly selective aldehyde hydrogenation process is proposed via coordination of alcohol dehydrogenase with (NH2)4TPP@BODIPY assistant reduces from the coupling process; regeneration of nicotinamide adenine dinucleotide (NADH). Our results show an excellent benchmark for the NAD+ co-factor coupled solar-driven alcohol production via (NH2)4TPP@BODIPY photocatalyst.

Immobilization of cross-linked enzymes aggregates on hierarchical covalent organic frameworks: Highly stable chemoenzymatic nanoreactor for asymmetric synthesis of optically active halohydrins

Organometallic catalyst is extensively applied for the non-enzymatic regeneration of nicotinamide adenine dinucleotide (phosphate) cofactors, but suffering from the mutual inactivation with the enzymes in one pot. The spatially separated immobilization of organometallic catalyst and enzymes on suitable carriers not only can reduce their mutual inhabitation but also can enhance their reusability. Here in this work, we present a hierarchical porous COFs (HP-TpBpy) that incorporated with [(Cp*RhCl2]2 to generate the metalized COF, Rh-HP-TpBpy. The obtained Rh-HP-TpBpy exhibited superior performance in nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration using formate as the hydride donor, significantly outperforming the natural formate dehydrogenases in cofactor preference toward NADP+. Subsequently, the Lactobacillus fermentum short-chain dehydrogenase/reductase 1 (LfSDR1) was then cross-linked into enzyme aggregates (CLEA) and immobilized on hierarchical Rh-HP-TpBpy, achieving the integrated chemoenzymatic catalyst, LfSDR1@Rh-HP-TpBpy, which can catalyze the chemoenzymatic reduction of halogenated aryl ketones and give the corresponding optically active halohydrins with high conversion and enantiomeric excess (ee) value up to 99 %. The LfSDR1@Rh-HP-TpBpy also exhibits largely enhanced stability compared with the free LfSDR1 and the CLEAs-LfSDR1, enabling its excellent reusability.

Mesoporous graphitic carbon nitride for photocatalytic coenzyme regeneration

Efficient regeneration of nicotinamide adenine dinucleotide (NADH) is of great significance for the application of oxidoreductase-catalyzed reactions. Photocatalytic regeneration is advantageous for the utilization of cheap, abundant, and clean solar energy. Graphitic carbon nitride (g-C3N4) has attracted wide attention due to its non-toxicity, wide range of precursors, suitable band gap, and superior chemical and thermal stability. In this work, mesoporous g-C3N4 (MPCN) was prepared using silica with different size as the template. The preparation process of MPCN was optimized and the effect of the template size on the structure and properties of MPCN were investigated. The results demonstrate that the smaller the template size, the larger the specific surface area of MPCN, and thus the better the photocatalytic performance. After optimization, the catalytic activity of MPCN with 7 nm silica as the template (MPCN-7) was 17.4 mM/(g min) and the NADH yield after 40 min was 70.6 %. MPCN-7 was also modified by 3-aminothiophene-2-carbonitrile (ATCN) and polyvinylpyrrolidone (PVP). Under same conditions, the catalytic activity of ATCN-MPCN, PVP-MPCN, and ATCN/PVP-MPCN was upgraded from 10.8 to 49.5, 16.4, 29.2 mM/(g min), respectively. The final yield of NADH rose from 66.9 % (40 min) to 76.3 % (12 min), 72.8 % (25 min) and 82.0 % (15 min).

Efficient Nicotinamide Adenine Dinucleotide Regeneration with a Rhodium-Carbene Catalyst and Isolation of a Hydride Intermediate.

Regeneration of nicotinamide adenine dinucleotide (NADH) has been the primary interest in the field of enzymatic transformation, especially associating oxidoreductases given the stoichiometric consumption. The synthesized carbene-ligated rhodium complex [(η5-Cp*)Rh(MDI)Cl]+ [Cp* = pentamethylcyclopentadienyl; MDI = 1,1'-methylenebis(3,3'-dimethylimidazolium)] acts as an exceptional catalyst in the reduction of NAD+ to NADH with a turnover frequency of 1730 h-1, which is over twice that of the higher catalytic activity of the commercially available catalyst [Cp*Rh(bpy)Cl]+ (bpy = 2,2'-bipyridine). Offsetting the contentious atmosphere currently taking place over the specific intermediate of the NADH regeneration, this study presents pivotal evidence of a metal hydride intermediate with a bis(carbene) ligand: a stable form of the rhodium hydride intermediate, [(η5-Cp*)Rh(MDI)H]+, was isolated and fully characterized. This enables thorough insight into the possible mechanism and exact intermediate structure in the NAD+ reduction process.

Versatile selective evolutionary pressure using synthetic defect in universal metabolism

The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.

Repurposing NAD Salvage Inhibitors for GCB Diffuse Large-B Cell Lymphoma

Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of diseases in terms of cell of origin, genetics and clinical outcome. About 30% of all DLBCL patients represent an unmet clinical need as they either do not respond to the standard first line chemo-immunotherapy or recur after initial remission. DLBCL classifications based on the NMF (Non-Negative Matrix Factorization) and LymphGen algorithms have led to the identification of genetic subtypes based on the co-occurrence of specific lesions. Repositioning drugs that are approved or in active clinical development for other indications can be a powerful strategy to match these newly uncovered DLBCL subtypes to targeted therapies.We have previously shown that the screening of large panels of DLBCL cell lines representative of the genetics of the disease can facilitate the repositioning of approved drugs for biomarker-selected populations of DLBCL patients. Repositioned drugs can then be rapidly translated to the clinical use, as they have already been extensively characterized for their safety profile. As a proof of concept, we have demonstrated that Dasatinib, a Src/Abl inhibitor approved for B-cell Acute Lymphoblastic Leukemia and Chronic Myelogenous Leukemia, is highly effective in PTEN-positive DLBCLs, irrespective of their COO class (Scuoppo et al., PNAS 2019).Here we present the results of a new screening that was performed on a panel of eight cell lines (4 ABC- and 4 GCB-DLBCLs) to test the activity of 212 drugs, either approved or in advanced clinical development, for repositioning in DLBCL, followed by validation in a larger panel of 34 genetically characterized cell lines. Our results point to inhibitors of the Nicotinamide Phosphoribosyl Transferase (NAMPT) as potently active in 63% of GCB-DLBCLs. NAMPT catalyzes the conversion of Nicotinamide (NAM) to Beta-Nicotinamide Mononucleotide (Beta-NMN). This reaction is the rate-limiting step of the Nicotinamide Adenine Dinucleotide (NAD) salvage pathway, the major metabolic route of NAD regeneration in mammalian cells. We validated the on-target activity of NAMPT inhibitors by multiple genetic and pharmacological approaches. First, we found that the activities of three chemically distinct drugs (FK-866, STF-118804 and KPT-9274) were highly correlated across the 34 lines DLBCL panel. Second, we were able to abolish the activity of all three NAMPT inhibitors by supplementing cells with Beta-NMN. Third, we showed that transduction of the drug-resistant mutant NAMPT H191R offsets the activity of the drugs.Dose-response assays on the full DLBCL cell line panel confirmed ABC-DLBCL resistance and also highlighted the presence of sensitive (GCB-S) and resistant (GCB-R) GCB subsets that can be separated by a subnanomolar IC50 threshold. To generate biomarkers capable of predicting GCB-S patients, we examined the RNA-seq profiles and genetic make-ups of the DLBCL cell lines collection and observed that the GCB-S subset was associated to the LymphGen EZB subtype, characterized by the presence of EZH2 mutations and BCL2 translocations. Conversely, the GCB-R subtype was linked to a 5-gene expression signature for the Kynurenine De Novo pathway, an alternative route for NAD synthesis. Accordingly, expression of each of the five De Novo Kynurenine pathway genes induced resistance to NAMPT inhibitors. These results were validated in xenotransplants of luciferized DLBCL lines and Patient Derived Xenotransplant models (PDXs) that were transcriptionally classified for the status of the Kynurenine De Novo signature. Together, these data support the repositioning of NAMPT inhibitors as a therapeutically relevant strategy for EZB-type GCB-DLBCLs. DisclosuresPasqualucci: Astra Zeneca: Research Funding; Sanofi: Research Funding.

One-Pot Highly Efficient Synthesis of N-Enrich Graphene Quantum Dots as a Photocatalytic Platform for NAD+/NADP+ Reduction.

An efficient photocatalytic regeneration of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) has been carried out by two-electron reduction and protonation of NAD+ /NADP+ , induced by photons in the visible light region. This functional artificial photosynthetic counterpart of the complete energy-trapping occurring in natural photosystem I (PS I) is achieved with nitrogen-enrich graphene quantum dot (N-EGQD) as the light-harvesting photocatalyst. In buffer aqueous solution, this compound photo catalytically recycles a rhodium hydride complex of the type [Cp*Rh(bpy)H]+ (Cp* = pentamethylcyclopentadienyl, bpy = 2,2'-bipyridine) which can mediate hydride transfer processes leading to nucleotide co-factor reduction. Very promising yields of 73.31%/78.45% of NADH/NADPH with the excellent thermal stability of N-EGQD photocatalyst is observed. Thus, in this work, an efficient light-harvesting photocatalyst is synthesized for the regeneration of nicotinamide cofactor that has pharmaceuticals application.

Enzyme-Loaded Nanoreactors Enable the Continuous Regeneration of Nicotinamide Adenine Dinucleotide in Artificial Metabolisms.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme for numerous biocatalytic pathways. While in nature, NAD+ is continuously regenerated from NADH by enzymes, all synthetic NAD+ regeneration strategies require a continuous supply of expensive reagents and generate byproducts, making these strategies unattractive. In contrast, we present an artificial enzyme combination that produces NAD+ from oxygen and water continuously; no additional organic substrates are required once a minimal amount pyruvate is supplied. Three enzymes, i.e., LDH, LOX, and CAT, are covalently encapsulated into a substrate‐permeable silica nanoreactor by a mild fluoride‐catalyzed sol–gel process. The enzymes retain their activity inside of the nanoreactors and are protected against proteolysis and heat. We successfully used NAD+ from the nanoreactors for the continuous production of NAD+ i) to sense glucose in artificial glucose metabolism, and ii) to reduce the non‐oxygen binding methemoglobin to oxygen‐binding hemoglobin. This latter conversion might be used for the treatment of Methemoglobinemia. We believe that this versatile tool will allow the design of artificial NAD+‐dependent metabolisms or NAD+‐mediated redox‐reactions.

Enzyme‐Loaded Nanoreactors Enable the Continuous Regeneration of Nicotinamide Adenine Dinucleotide in Artificial Metabolisms

AbstractNicotinamide adenine dinucleotide (NAD) is an essential coenzyme for numerous biocatalytic pathways. While in nature, NAD+ is continuously regenerated from NADH by enzymes, all synthetic NAD+ regeneration strategies require a continuous supply of expensive reagents and generate byproducts, making these strategies unattractive. In contrast, we present an artificial enzyme combination that produces NAD+ from oxygen and water continuously; no additional organic substrates are required once a minimal amount pyruvate is supplied. Three enzymes, i.e., LDH, LOX, and CAT, are covalently encapsulated into a substrate‐permeable silica nanoreactor by a mild fluoride‐catalyzed sol–gel process. The enzymes retain their activity inside of the nanoreactors and are protected against proteolysis and heat. We successfully used NAD+ from the nanoreactors for the continuous production of NAD+ i) to sense glucose in artificial glucose metabolism, and ii) to reduce the non‐oxygen binding methemoglobin to oxygen‐binding hemoglobin. This latter conversion might be used for the treatment of Methemoglobinemia. We believe that this versatile tool will allow the design of artificial NAD+‐dependent metabolisms or NAD+‐mediated redox‐reactions.

Furfuryl Alcohol Production with High Selectivity by a Novel Visible-Light Driven Biocatalysis Process

Enzymatically upgrading furfural (FAL), the crude platform chemical derived from biomass, to the value-added furfuryl alcohol (FOL) has attracted considerable interest because of its high conversion and selectivity. However, the conventional biotransformation of FAL to FOL was limited by the difficulties in coenzyme regeneration. Herein, visible light-driven selective hydrogenation of FAL was realized on the basis of a coupled process that consisted of a photocatalyst and alcohol dehydrogenase (ADH). The QDs/g-C₃N₄ nanosheet was prepared in a “one-pot” process using graphitic carbon nitride (g-C₃N₄) and carboxyl-modified quantum dots (QDs) (CdSe/ZnS). The as-prepared material exhibited synergetically intensified charge generation and transfer properties, implementing the regeneration of nicotinamide adenine dinucleotide (NADH). As expected, the QDs/g-C₃N₄ exhibits an equilibrium NADH yield of 95.7%, which was two times higher than that using g-C₃N₄. The reusability of the QDs/g-C₃N₄ was also acceptable. After nine batches of operation, the conversion rate of NADH was still above 70%. Adopting the nanosheet in the selective hydrogenation by the photobiocatalytic process could reach excellent FOL yield (0.6 mM) and selectivity (∼100%).

Mimic of the Cellular Antioxidant Defense System for a Sustainable Regeneration of Nicotinamide Adenine Dinucleotide (NAD).

The prolonged use of enzymes under oxidative stress is a major challenge in enabling effective enzymatic reaction pathways. Herein, we report a biomimetic antioxidant defensive strategy capable of providing adequate protection of enzymes against superoxide-mediated oxidation. Superoxide dismutase (SOD) and catalase (CAT) were chosen as scavengers and covalently encapsulated into silica nanoreactors, together with glucose dehydrogenase (GDH), which simultaneously should produce the coenzyme nicotinamide adenine dinucleotide (NADH, reduced form). By the enzymatic reactions of SOD and CAT, the interior of silica nanoreactors becomes a “ROS safe zone” to protect the glucose-dependent NADH production of coencapsulated GDH. We further combined this protected NADH-producing module with photocatalytic nanoparticles that enable the light-triggered oxidation of NADH back to NAD+ (oxidized form). In combination, these two modules allow interconversion between NAD+ and NADH by the addition of glucose or by light irradiation (LED lamp or sunlight). This protection and regeneration strategy is a versatile tool for enzyme applications for biological reactors, catalysis, or prototypes of artificial organelles or building blocks that contains fragile biomolecules and rely on the coenzyme NAD+/NADH.

Antioxidant Alternatives in the Treatment of Amyotrophic Lateral Sclerosis: A Comprehensive Review.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that produces a selective loss of the motor neurons of the spinal cord, brain stem and motor cortex. Oxidative stress (OS) associated with mitochondrial dysfunction and the deterioration of the electron transport chain has been shown to be a factor that contributes to neurodegeneration and plays a potential role in the pathogenesis of ALS. The regions of the central nervous system affected have high levels of reactive oxygen species (ROS) and reduced antioxidant defenses. Scientific studies propose treatment with antioxidants to combat the characteristic OS and the regeneration of nicotinamide adenine dinucleotide (NAD+) levels by the use of precursors. This review examines the possible roles of nicotinamide riboside and pterostilbene as therapeutic strategies in ALS.

Flavin Oxidoreductase‐Mediated Regeneration of Nicotinamide Adenine Dinucleotide with Dioxygen and Catalytic Amount of Flavin Mononucleotide for One‐Pot Multi‐Enzymatic Preparation of Ursodeoxycholic Acid

AbstractUrsodeoxycholic acid (UDCA), a pharmaceutical ingredient widely used in clinics, can be prepared from chenodeoxycholic acid (CDCA) by the epimerization of the 7α‐OH group. In this study, a nicotinamide adenine dinucleotide (NAD+) regeneration system was developed by using flavin oxidoreductase (FR) and flavin mononucleotide (FMN). Only catalytic amount of FMN is required for the effective NAD+ recycling. FR/FMN system was then applied in the oxidation of CDCA to 7‐ketolithocholic acid (7‐keto‐LCA) by NAD+‐dependent 7α‐hydroxysteroid dehydrogenase (Bs‐7α‐HSDH) from Brevundimonas sp., which showed extremely high enzyme activity toward CDCA (kcat/Km=8050 s−1 ⋅ mM−1). When Escherichia coli whole cells coexpressing Bs‐7α‐HSDH and FR genes were used as biocatalyst, CDCA (50 mM) was completely converted to 7‐keto‐LCA with the turnover number of FMN being 227 and 58.8 g ⋅ L−1 ⋅ d−1 space‐time yield of 7‐keto‐LCA. For the reduction of 7‐keto‐LCA, nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent 7‐β‐hydroxysteroid dehydrogenase (Cm‐7β‐HSDH) from Clostridium sp. Marseille was employed with alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) and iso‐propanol as co‐factor regeneration system. When E. coli whole cells coexpressing Cm‐7β‐HSDH and TbADH genes were used as biocatalyst, 40 mM 7‐keto‐LCA was reduced to UDCA with 26.8 g ⋅ L−1 ⋅ d−1 space‐time yield. The oxidation and reduction were then carried in a one‐pot concurrent mode, 12.5 mM CDCA was completely converted to UDCA. The epimerization of CDCA to UDCA proceeded to completion at the substrate concentration of 30 mM in the one‐pot sequential process. Therefore, the complete conversion of CDCA to UDCA in one‐pot has been realized by employing 7α‐HSDH and 7β‐HSDH of different co‐factor specificities with independent co‐factor recycling systems. The cholic acids, especially UDCA, exert inhibitive effect on the activities of these enzymes, preventing the complete epimerization of 7α‐OH at higher substrate loading. This inhibition issue should be solvable by engineering the involved enzymes, that is currently pursued in our laboratory.magnified image

NAD+ Cofactor Regeneration by TMB‐Mediated Horseradish‐Peroxidase‐Catalyzed Reactions

AbstractThe regeneration of nicotinamide adenine dinucleotide (NAD+) has attracted substantial interest in the enzymatic chemical transformations. In this work, we report the use of a traditional horseradish peroxidase (HRP)/H2O2 system for the catalytic transformation of reduced nicotinamide adenine dinucleotide (NADH) into NAD+, in the presence of a common HRP substrate, 3,3′,5,5′‐tetramethylbenzidine (TMB), as an electron mediator. The effect of H2O2, HRP or TMB concentration on the oxidization rate of NADH was systematically investigated. The results show that a small amount of HRP and TMB resulted in high efficient oxidization of NADH into NAD+, through the switching between the oxidized and reduced states of TMB. The enzymatic system also exhibited pH‐dependent catalytic properties, so that a XOR logic gate was constructed with H2O2 and H+ as the gate inputs, and the generated TMB charge transfer complex as the output signals. The addition of glucose dehydrogenase (GDH)/glucose allows the cycling between the NADH and NAD+ and the coupled activation of HRP and GDH. This work provides a strategy for efficient regeneration of the NAD+ cofactor and enzyme‐driven transformations.

NAMPT Is a Potent Oncogene in Colon Cancer Progression that Modulates Cancer Stem Cell Properties and Resistance to Therapy through Sirt1 and PARP.

Purpose: Colorectal cancer is the second most common cancer in women and the third most common in men worldwide. However, despite current progress, many patients with advanced and metastatic tumors still die from the malignancy. Refractory disease often relies on nicotinamide adenine dinucleotide (NAD)-dependent mechanisms. NAD metabolism and a stable NAD regeneration circuit are required to maintain tissue homeostasis and metabolism. However, high levels of NAD confer therapy resistance to tumors.Experimental Design: Ectopic overexpression of nicotinamide phosphoribosil transferase (NAMPT) and shRNAs in colorectal cancer cell lines, tumorigenic and stemness properties and transcription measurement in culture and in vivo Transcriptional analysis in public databases. Therapeutic approaches.Results: NAMPT, the rate-limiting enzyme responsible for the highest source of physiologic NAD biosynthesis, increases tumorigenic properties and induces cancer stem cell-like properties through pathways that control stem cell signaling, thus enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of CIC-like cells in colon tumors directly extracted from patients, and transcription meta-analysis revealed that NAMPT is also a key factor that induces cancer stem pathways in colorectal cancer tumors. This effect is mediated by PARP and SIRT1. In addition, we report a novel NAMPT-driven signature that stratifies prognosis from high to low expression groups. The NAMPT signature contained SIRT1 and PARP1 levels as well as other cancer stem cell-related genes. Finally, NAMPT inhibition increased the sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres.Conclusions: NAMPT represents a novel therapeutic target in colon cancer progression and relapse, particularly the CIC subset of human colon cancers. Clin Cancer Res; 24(5); 1202-15. ©2017 AACR.

NAMPT overexpression induces cancer stemness and defines a novel tumor signature for glioma prognosis.

Gliomas are the most prevalent primary malignant brain tumors associated with poor prognosis. NAMPT, a rate-limiting enzyme that boosts the nicotinamide adenine dinucleotide (NAD) regeneration in the salvage pathway, is commonly expressed in these tumors. NAD metabolism is required to maintain tissue homeostasis. To maintain metabolism, cancer cells require a stable NAD regeneration circuit. However, high levels of NAD confer resistance to therapy to these tumors, usually treated with Temozolomide (TMZ). We report that NAMPT overexpression in glioma cell lines increases tumorigenic properties controlling stem cell pathways and enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of Nanog, CD133 and CIC-like cells in glioblastoma directly extracted from patients. Meta-analysis reveals that NAMPT is also a key factor inducing cancer stem pathways in glioma cells. Furthermore, we report a novel NAMPT-driven signature which stratify prognosis within tumor staging. NAMPT signature also correlates directly with EGFR positive and IDH negative tumors. Finally, NAMPT inhibition increases sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres. Therefore, NAMPT represents a novel therapeutic target in Glioma progression and relapse.

Regeneration of NAD keeps mouse muscles young

Aging Evidence for a critical role of nicotinamide adenine dinucleotide (NAD) metabolism in aging is accumulating. Frederick et al. studied mice with muscle-specific depletion of nicotinamide phosphoribosyltransferase (Nampt), an enzyme needed to restore concentrations of NAD in working muscle.

Abstract 3006: Arsenic trioxide inhibits overexpression of NAMPT in oral cancer

Abstract Purpose: Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme for a rate-limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. Tumor cells are reliant on the NAMPT salvage pathway for NAD regeneration, thus making this enzyme an attractive therapeutic target for cancer treatment. Overexpression of NAMPT has been observed across a broad range of solid tumors including ovarian, breast, and prostate cancers. However, it is unknown regarding the expression and function of NAMPT in oral cancer. Arsenic trioxide (ATO) has been approved for the treatment of acute promyelocytic leukemia, and accumulating evidence indicate that it is a promising therapeutic drug for certain solid malignant tumors. Up to now, the effect of ATO on NAMPT in oral cancer remains unclear. In the present study, we investigated the expression of NAMPT in different differentiated degree oral cancer and examined the effects of ATO on NAMPT in oral cancer cell line. Materials and methods: Tissue microarray (TMA) was performed by immunohistochemistry to examine the NAMPT expression in well differentiated (n = 39), moderately differentiated (n = 6), and poorly differentiated (n = 5) tissue samples collected from surgical specimens of the oral cancer patients. Human oral squamous cell carcinoma Tca-8113 cells were treated with ATO for 48 hrs. PrestoBlue activity assay was used to detect cell activity and NAMPT mRNA expression was measured by RT-PCR. Results: In paracancer tissue, NAMPT was mainly distributed in cytoplasma and lightly located in nuclear. Intensely increased staining of NAMPT was found in all differentiated grade oral cancer by immunohistochemistry. Moreover, while increased NAMPT are mainly located in cytoplasma in well-differentiated cancer, the marked increase of NAMPT occurred both in cytoplasma and nuclear in moderate- and poor-differentiated cancer. TMA results showed that comparing with normal oral tissue, the protein expression of NAMPT increased by 2.20, 2.66, and 3.03 fold in well-, moderate-, and poor- differentiated oral cancer, respectively. ATO (1, 2.5, 5, and 10 μmol/L) pretreatment of Tca8113 cells significantly reduced the cell activity in an ATO concentration-dependent manner. More importantly, NAMPT mRNA expression was markedly decreased 8.60%, 26.92%, 71.01%, and 78.26%, respectively, compared with control cells. Conclusion: Our novel findings suggest that increased NAMPT expression were highly associated with cellular differentiation in oral cancer, indicating that NAMPT may participate in the development of oral cancer. ATO can inhibit cell activity by depressing NAMPT transcription in oral cancer. These findings may have implications for exploring the NAMPT pathway for oral cancer treatment. Citation Format: Jin Zhi Wang, Yan Liu, Xin Yue Wang, Li Chi Han, Fu Yin Zhang, Ke Liu, Bin Xiang. Arsenic trioxide inhibits overexpression of NAMPT in oral cancer. [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 3006.

DEVELOPMENT OF A PHOTOELECTROCHEMICAL LACTIC DEHYDROGENASE BIOSENSOR USING MULTI-WALL CARBON NANOTUBE -TIO2 NANOPARTICLE COMPOSITE AS COENZYME REGENERATION TOOL

A novel photoelectrochemical (PEC) lactic dehydrogenase (LDH) biosensor was developed based on a multi-wall carbon nanotube (MWCNT)-TiO2 nanoparticle (TNP) composite platform. This composite platform can not only aid in regeneration of nicotinamide adenine dinucleotide (NAD+) in the enzymatic cycle, but also immobilize enzymes on electrode surface. TNPs were grown on MWCNT surface through a hydrothermal method and the composite was characterized by various spectroscopic techniques. The electrochemical performance of the LDH biosensors has demonstrated that the composite is a feasible immobilization matrix for LDH. The PEC experiments have confirmed that NAD+ can be regenerated by the holes produced by irradiating MWCNT-TNP composite to fulfill the enzyme catalytic cycle. The analytical performance of the PEC LDH biosensor was studied by measuring its photocurrents. The dynamic range, sensitivity and limit of detection of the biosensor were estimated to be 0.5 to 120μM, 0.0242μAμM−1 and 0.1μM respectively, which are superior to those of electrochemical LDH biosensors.

Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways.

Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer's glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD+) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD+ and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD+. This effect was specific for p53+/+ cancer cells and correlated with (i) reduced activity of NAD+-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53+/+ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53+/+ cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD+ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.