Decrease In NAD Levels Research Articles

Apoptosis induced by NAD depletion is inhibited by KN-93 in a CaMKII-independent manner

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme that catalyzes the synthesis of nicotinamide mononucleotide from nicotinamide (Nam) in the salvage pathway of mammalian NAD biosynthesis. Several potent NAMPT inhibitors have been identified and used to investigate the role of intracellular NAD and to develop therapeutics. NAD depletion induced by NAMPT inhibitors depolarizes mitochondrial membrane potential and causes apoptosis in a range of cell types. However, the mechanisms behind this depolarization have not been precisely elucidated. We observed that apoptosis of THP-1 cells in response to NAMPT inhibitors was reduced by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 via an unknown mechanism. The inactive analog of KN-93, KN-92, exhibited the same activity, but the CaMKII-inhibiting cell-permeable autocamtide-2-related inhibitory peptide II did not, indicating that the inhibition of THP-1 cell apoptosis was not dependent on CaMKII. In evaluating the mechanism of action, we confirmed that KN-93 did not inhibit decreases in NAD levels but did inhibit decreases in mitochondrial membrane potential, indicating that KN-93 exerts inhibition upstream of the mitochondrial pathway of apoptosis. Further, qPCR analysis of the Bcl-2 family of proteins showed that Bim is efficiently expressed following NAMPT inhibition and that KN-92 did not inhibit this expression. The L-type Ca2+ channel blockers verapamil and nimodipine partially inhibited apoptosis, indicating that part of this effect is dependent on Ca2+ channel inhibition, as both KN-93 and KN-92 are reported to inhibit L-type Ca2+ channels. On the other hand, KN-93 and KN-92 did not markedly inhibit apoptosis induced by anti-cancer agents such as etoposide, actinomycin D, ABT-737, or TW-37, indicating that the mechanism of inhibition is specific to apoptosis induced by NAD depletion. These results demonstrate that NAD depletion induces a specific type of apoptosis that is effectively inhibited by the KN-93 series of compounds.

Thrombin or Ca++-Ionophore-Mediated Fall in Endothelial ATP Levels Independent of Poly(ADP-Ribose) Polymerase Activity and NAD Levels - Comparison with the Effects of Hydrogen Peroxide

To test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca++-ionophore A23187, or hydrogen peroxide (H2O2), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H2O2 induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca++-ionophore-mediated lowering of ATP. H2O2 caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD.

Abstract 1896: Bioluminescent assays for investigating cell metabolism.

Abstract Cell metabolism is an important and expanding area of research in the field of cancer biology. Simple, rapid assays for studying cellular metabolic pathways, their enzymes, metabolites and cofactors can facilitate these studies. We have developed two novel bioluminescent technologies, one for monitoring cell viability, and the other for monitoring changes in cellular nicotinamide adenine dinucleotides, that can facilitate the study of cell metabolism and its regulation. The bioluminescent cell viability assay is based on the metabolic activity of live cells. Metabolically active cells reduce a proluminogenic substrate in vivo which is then detected by a luciferase enzyme in the media. The luminescence output is correlated with the number of viable cells. The assay is non-toxic, sensitive (detects less than 10 cells/well) and has a large dynamic range (S/B>100). The key, differentiating feature of this assay is the incorporation of the recently developed stable and very bright NanoLuc™ luciferase and its profurimazine substrate, which allows changes in viability to be monitored in real-time and continuously, over extended periods of time, from the same sample. Three homogeneous, one-step, bioluminescent assays have been developed for the detection of nicotinamide adenine dinucleotides, specific for: (1) reduced forms NADH and NADPH, (2) non-phosphorylated forms NAD and NADH, and (3) phosphorylated forms NADP and NADPH. The assays are based on the reaction of the enzyme diaphorase with a proluciferin substrate, which in the presence of NADH or NADPH is converted to luciferin, a substrate for luciferase. These assays are sensitive (LOD ≤ 50 nM) and have large maximum signal windows (S/B > 100), two advantages over currently available colorimetric and fluorescent detection methods. They are also robust, and amenable to automated and high-throughput protocols (Z’ > 0.8). The high sensitivity, one-step reagent addition, and no requirement for sample processing, permit an in-well protocol for the assay of cells directly in culture plate wells. Changes in cellular NAD levels can be rapidly monitored, as demonstrated using FK866, an inhibitor of NAD biosynthesis. FK866 caused a decrease in NAD levels in multiple cell types with good signal windows (S/B > 10) and expected pharmacology (IC50 values = 1-10 nM). Citation Format: Donna Leippe, Mary Sobol, Sarah Duellman, Jolanta Vidugiriene, Wenhui Zhou, Gediminas Vidugiris, Troy Good, Laurent Bernad, Poncho Meisenheimer, James Cali. Bioluminescent assays for investigating cell metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1896. doi:10.1158/1538-7445.AM2013-1896

Molecular mechanisms underlying selective cytotoxic activity of BZL101, an extract of Scutellaria barbata, towards breast cancer cells

We studied the mechanism of the cytotoxic activity of BZL101, an aqueous extract from the herb Scutellaria barbata D. Don, which is currently in phase II clinical trial in patients with advanced breast cancer. The phase I trial showed favorable toxicity profile and promising efficacy. We report here that BZL101 induces cell death in breast cancer cells but not in non-transformed mammary epithelial cells. This selective cytotoxicity is based on strong induction by BZL101 of reactive oxygen species (ROS) in tumor cells. As a consequence, BZL101 treated cancer cells develop extensive oxidative DNA damage and succumb to necrotic death. Data from the expression profiling of cells treated with BZL101 are strongly supportive of a death pathway that involves oxidative stress, DNA damage and activation of death-promoting genes. In breast cancer cells oxidative damage induced by BZL101 leads to the hyperactivation of poly (ADP-ribose) polymerase (PARP), followed by a sustained decrease in levels of NAD and depletion of ATP, neither of which are observed in non-transformed cells. The hyperactivation of PARP is instrumental in the necrotic death program induced by BZL101, because inhibition of PARP results in suppression of necrosis and activation of the apoptotic death program. BZL101 treatment leads to the inhibition of glycolysis selectively in tumor cells, evident from the decrease in the enzymatic activities within the glycolytic pathway and the inhibition of lactate production. Because tumor cells frequently rely on glycolysis for energy production, the observed inhibition of glycolysis is likely a key factor in the energetic collapse and necrotic death that occurs selectively in breast cancer cells. The promising selectivity of BZL101 towards cancer cells is based on metabolic differences between highly glycolytic tumor cells and normal cells.

A role for MAP kinase in the antitumor activity of a nucleoside analog.

Nucleoside analogs (NAs) have been used extensively in both antitumor and antiviral therapies. Their general mechanism of action has been postulated to result from incorporation into DNA, leading to disruption of DNA synthesis and DNA polymerase inhibition. To further explore the antitumor mechanisms of NAs we have evaluated ganciclovir (GCV), an NA antiviral agent, in herpes simplex virus thymidine kinase (HSV-TK) gene-modified tumor cells. This system allows specific evaluation of the antitumor effects of NAs because the antitumor effect is directly related to the phosphorylation of the prodrug GCV by the HSV-TK enzyme in the gene-modified tumor cells. We demonstrated that GCV incorporates into DNA and inhibits DNA polymerase, as has been observed in HSV-infected cells and with other antitumor NAs in tumor cells. A novel observation is that GCV activates MAP kinase within 1 hour of GCV exposure. This activation directly correlates with cytotoxicity, because inhibition of the MAP kinase extracellular regulated kinase (Erk) by PD98059, reversed GCV-mediated cytotoxicity. This effect appears to be specific to the Erk pathway, because inhibition of the p38 kinase with SB203580 had no effect on cytotoxicity. Further, GCV does not act as a DNA-damaging agent or activate general DNA-repair mechanisms, but does produce a number of metabolic disruptions, including a reversible decrease in NAD levels. These effects appear to be downstream of the earlier activation of Erk in this system, which may be a novel mechanism of action for GCV cytotoxicity in HSV-TK gene-modified tumor cells, and thus, needs to be further evaluated as the mechanism of tumor cell killing by other antitumor NAs.

A comparative study on the effect of tumor promoters on poly ADP‐ribosylation in A431 cells

Poly ADP-ribosylation is a post-translational modification of chromatin proteins catalyzed by the enzyme poly-ADPR transferase (poly-ADPRT) and affects the structure as well as the functional properties of chromatin. It is of particular relevance in carcinogenesis, as it represents an epigenetic mechanism for modulation of gene expression. In the present study, A431 cells were exposed to tumor promoters phorbol-12-myristate-13-acetate (PMA), benzoyl peroxide (BP), mezerein and 6-keto-lithocholic acid (KA), and their effect on poly-ADP-ribosylation was studied. All these tumor promoters increased the activity of poly-ADPRT in these cells--PMA 2.3-fold, BP and mezerein 2.2-fold each and KA 1.3-fold. The enzyme inhibitor 3-amino benzamide (3AB) partially prevented the stimulation of poly-ADPRT by these promoters. There was a concomitant decrease in NAD levels, the substrate for poly-ADPRT. The decrease was 44% for PMA, 46% for BP, 21% for KA and 34% for mezerein. The induction of poly-ADP-ribose synthesis by PMA and BP appears to be mediated at least in part by active oxygen species, as they induced an increase in superoxide anions and anti-oxidants prevented the increase of poly-ADPRT activity to varying extents.

Stimulation of poly(ADP-ribose) synthesis by free radicals in C3H10T1/2 cells: relationship with NAD metabolism and DNA breakage

We have studied the effect of H2O2 and O2- produced by xanthine and xanthine oxidase on NAD catabolism, poly(ADP-ribose) synthesis, and production of DNA single-strand breaks in C3H10T1/2 cells. The results show a correlation between the induction of DNA single-strand breaks, the decrease of NAD pool, and the accumulation of polymer. New techniques, based on affinity chromatography and reversed-phase high pressure liquid chromatography, have allowed an accurate determination of polymer contents and showed a 20-fold stimulation of polymer biosynthesis induced by active oxygen species. Inhibition experiments performed with 3-aminobenzamide have shown that the decrease in NAD levels after exposure of cells to active oxygen species was caused by stimulation of poly(ADP-ribosyl)ation and of another cellular process.

Poly(ADP-ribose) levels in carcinogen-treated cells

Several reports indicate that intracellular levels of NAD are rapidly decreased by DNA-damaging agents such as strep-tozotocin1–7, neocarzinostatin8,9 and ionising radiation8–11. Similarly, evidence obtained from studies in isolated nuclei and permeabilised cells indicates that DNA damage caused by several alkylating chemicals12–15, X rays16,17 and DNA-hydrolysing enzymes18,19 elicit a considerable increase in the activity of poly(ADP-ribose) polymerase, a chromosomal enzyme which uses NAD as a substrate in the formation of poly(ADP-ribose) in histones and other nuclear proteins20,21. Thus, it has been suggested that DNA-damaging agents lower NAD levels by causing an increase in poly(ADP-ribose) synthesis9,12,13. As DNA damage initiates DNA repair mechanisms, it has also been argued that poly(ADP-ribose) is involved in DNA repair8,12,14,17,22,23. Such possibilities would be strongly supported if an actual increase in the intracellular levels of poly(ADP-ribose) could be shown to occur after induction of DNA damage. We now present data obtained using a new technique which has enabled us to measure the levels of poly(ADP-ribose) in vivo in SV40 virus-transformed 3T3 cells (SVT2). We show that treatment of these cells with the powerful mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) produces, concomitantly with a decrease in NAD levels, a dramatic increase in the intracellular levels of poly (ADP-ribose).

Intrinsic ADP-Ribose Transferase Activity versus Levels of Mono(ADP-Ribose)Protein Conjugates in Proliferating Ehrlich Ascites Tumor Cells

Transition of proliferating Ehrlich ascites tumor cells (3 days after transplantation) to the non-proliferating status (8--14 days after transplantation) was associated with an increase in total mono (ADP-ribose) protein conjugates. This increase was largely confined to the NH2OH-resistant subfraction. When the amounts of mono-(ADP-ribose) conjugates from 20% trichloroacetic acid precipitates were compared with those from 5% perchloric acid precipitates, no significant differences were seen. This fact excludes histone H1 as a major mono (ADP-ribose) acceptor in vivo in these cells. Transition to the resting state was also associated with a small decrease in NAD levels, and with no significant changes of total ADP-ribose transferase activity. However intrinsic ADP-ribose transferase activity as expressed in permeabilized cells was increased, being correlated with the changes in the level of the NH2OH-resistant mono (ADP-ribose) protein conjugates. This shows that alterations in intrinsic transferase activity may, in general, indicate similar alterations in major subfractions of ADP-ribose conjugates. Intrinsic ADP-ribose transferase activity exhibited an inverse relationship to ornithine decarboxylase activity.

Chicken embryo nad levels lowered by teratogenic organophosphorus and methylcarbamate insecticidesag

Many organophosphorus (OP) and methylcarbamate (MC ) insecticides and related compounds. when injected into the yolk sac on day 4 of incubation, markedly lower chicken embryo NAD levels at day 12 of incubation. The same compounds also produce drastic developmental defects. suggesting that the lowered level of NAD may be the cause of teratogenesis. The most potent OP compounds of those tested are diazinon and dicrotophos acting at 0.1 and 1 mg/kg. respectively, while the MC compounds, escrine sulfate and carbaryl. are effective at 1 and 4 mg/kg respectively. The injection of nicotinamide and other NAD precursors prevents the decrease in NAD levels and alleviates most of the teratogenic signs. Previous hypotheses, including that relating the inhibition of yolk sac membrane esterases to embryonic abnormalities, are less satisfactory or are inadequate to explain this type of chemically induced teratogenesis. Areas remaining to be defined are the biochemical lesion leading to decreased chicken embryo NAD levels and the relevance of these findings to mammals.

Effects of estradiol on the levels of pyridine nucleotide coenzymes in the rat uterus.

NADP has been shown to induce the synthesis of a uterine enzyme, glucose-6- phosphate dehydrogenase (G6PDH), a response which is also seen after administration of 17β- estradiol to the ovariectomized rat. The relative kinetics of cofactor and hormone induction suggest that estradiol might act first by induction of NADP synthesis which in turn might induce the synthesis of the enzyme. An enzymatic cycling technique with sufficient sensitivity to measure pmole amounts of NAD, NADH, NADP and NADPH was used to determine the levels of these coenzymes in the rat uterus at various times after administration of 17β-estradiol. The results of this study indicate that the levels of all 4 pyridine nucleotide coenzymes increase during the first 24 hr of the uterine response to estradiol. Control levels are 14,601, 1506, 620 and 1494 pmoles/uterus for NAD, NADH, NADP and NADPH, respectively. An initial rise is seen in the levels of NAD by 2 hr. NADP levels are constant for 2 hr and increase to 160 % of control levels by the sixth hr. Total NADP coenzyme levels (NADP+NADPH) remain relatively constant for 4 hr and increase rapidly during the interval from 4 to 6 hr. This coincides with a decrease in NAD levels during this period, suggesting that NADP levels increase via de novo synthesis. The ratio of oxidized/reduced NAD reaches a maximum (16.72) at 4 hr, while a similar ratio for NADP reaches a maximum (0.64) at 6 hr. The ratios for both cofactors approach control values by the 24th hr after hormone treatment. The time sequence for these events lends strength to the hypothesis that the estrogen induced increases in G6PDH levels are, in part, mediated through an estrogen induced increase in uterine NADP levels. (Endocrinology86: 1118, 1970)