Nitric Oxide-donating Aspirin Research Articles

Nitric oxide-donating aspirin (NO-Aspirin) suppresses lung tumorigenesis in vitro and in vivo and these effects are associated with modulation of the EGFR signaling pathway.

Although regular aspirin use has been shown to lower the risk of colorectal cancer, its efficacy against lung cancer is weak or inconsistent. Moreover, aspirin use increases the risk of ulcers and stomach bleeding. In this study, we determined the efficacy of nitric oxide-donating aspirin (NO-Aspirin), a safer form of aspirin in which the parent drug is linked to a nitric oxide-releasing moiety through a spacer, to suppress lung tumorigenesis. Under in vitro conditions, NO-Aspirin significantly reduced the proliferation and survival of tumorigenic bronchial cell line (1170) and non-small cell lung cancer (NSCLC) cell lines (A549, H1650, H1975 and HCC827) and colony formation by NSCLC cells at sub- or low micromolar concentrations (≤1 µM for 1170 cells and ≤6 µM for NSCLC cells) in a COX-2 independent manner. These effects were paralleled by suppression of phospho-epidermal growth factor receptor (EGFR), -STAT3, -Akt and -ERK and enhanced caspase 3 and PARP cleavage. Among NSCLC cells, EGFR mutant cells (H1650, H1975 and HCC827) were more sensitive than cells expressing wild-type EGFR (A549) and H1650 cells were the most sensitive. Moreover, NO-Aspirin sensitized H1650 and H1975 cells to the antiproliferative effects of erlotinib, a tyrosine kinase inhibitor. In in vivo studies using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) + lipopolysaccharide (LPS)-induced model of lung tumorigenesis, NO-Aspirin significantly reduced the number and size of lung tumors, expression of phospho-EGFR and -Akt as well as the pro-inflammatory molecules TNF-α and interferon-gamma. Overall, these results indicate the potential of NO-Aspirin for the chemoprevention of lung cancer in high risk populations.

NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

In this study, the effects and underlying mechanisms of NCX 4040, a nitric oxide (NO)-donating aspirin derivative, on the production of proinflammatory mediators were examined using murine macrophages exposed to lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in the etiology of periodontal disease. NCX 4040 significantly reduced P. intermedia LPS-induced production of inducible NO synthase (iNOS)-derived NO, IL-1β and IL-6 as well as their mRNA expression in RAW264.7 cells. Notably, NCX 4040 was much more effective than the parental compound aspirin in reducing LPS-induced production of inflammatory mediators. NCX 4040 induced the expression of heme oxygenase-1 (HO-1) in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced NO production was significantly reversed by SnPP, a competitive HO-1 inhibitor. NCX 4040 did not influence LPS-induced phosphorylation of JNK and p38. IκB-α degradation as well as nuclear translocation and DNA-binding activities of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. Besides, LPS-induced phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040. Further, NCX 4040 elevated the SOCS1 mRNA in cells stimulated with LPS. This study indicates that NCX 4040 inhibits P. intermedia LPS-induced production of NO, IL-1β and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NF-κB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. NCX 4040 could potentially be a promising tool in the treatment of periodontal disease, although further studies are required to verify this.

Nitric oxide-donating aspirin induces G2/M phase cell cycle arrest in human cancer cells by regulating phase transition proteins

NO-aspirin (NO-ASA), consisting of aspirin and a nitric oxide-releasing group, is safer than aspirin and effective in colon cancer prevention. Here, we examined the mechanism of action of NO-ASA by focusing primarily on its effects on the cell cycle. NO-ASA reduced the growth of several cell lines from colon, pancreas, skin, cervix and breast cancer much more potently than aspirin, with 24-h IC(50) values of 133-268 µM, while those of ASA were >1,000 µM. NO-ASA elevated the intracellular levels of reactive oxygen species, generating a state of oxidative stress. In all cell lines examined, NO-ASA induced cell cycle arrest in the G(2)/M phase transition accompanied by altered expression of G(2)/M transition-related proteins. In SW480 colon cancer cells NO-ASA modulated proteins controlling this transition. Thus, it markedly increased the levels of cyclin B1, decreased the expression of cyclin D1 and Cdc25C, and increased the Thr14/Tyr15-phosphorylation of Cdk1 while leaving unchanged its protein levels. These changes, including the G2/M arrest, were prevented by pretreating the cells with the anti-oxidant N-acetyl-cysteine, indicating that redox signaling is likely responsible for the cell cycle changes, a conclusion consistent with the known redox regulation of these proteins. Collectively, these results confirm the profound cytokinetic effect of NO-ASA and provide strong evidence that it regulates cell cycle transitions through its ability to induce oxidative stress, which activates redox signaling in the target cell.

Abstract 3817: Nitric oxide-donating aspirin induces G2/M phase cell cycle arrest in human cancer cells by regulating phase transition proteins

Abstract NO-aspirin (NO-ASA), consisting of aspirin, a non-steroidal anti-inflammatory drug (NSAID), and a nitric oxide (NO)-releasing group, is reportedly safer than aspirin and effective in colon cancer chemoprevention. NO-ASA reduced the growth of several cancer cell lines much more potently than aspirin, with IC50 values at 24-h as follows: (a) colon (HT-29, SW480, HCT-15, and LoVo) 174 to 257 µM; (b) pancreas (BxPC-3), 175 µM; (c) skin (A431), 133 µM; (d) cervix (Hela), 240 µM; and (e) breast (MCF-7), 268 µM. In all instances, the IC50 values for the parent compound were >1,000 µM. To assess NO-ASA's mechanism of action, we studied in vitro its effect on colonocyte kinetics. In all cell lines examined, the growth inhibitory effect of NO-ASA was accompanied by cell cycle arrest in the G2/M phase transition. This inhibition directly correlated to altered expression of G2/M transition related proteins. Specifically, NO-ASA had a time- and concentration-dependent effect on the expression of cyclin D1. Here we propose that NO-ASA-mediated G2/M arrest is probably caused by ROS generation. This conclusion is based on (a) treatment with NO-ASA generates ROS in SW480 and (b) the G2/M arrest induced by NO-ASA is abolished on pretreatment with the antioxidant NAC. Thus, NO-ASA's superior effectiveness compared to traditional aspirin is due in part to its ability to induce G2/M phase cell cycle arrest in human cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3817. doi:1538-7445.AM2012-3817

Aspirin and low-dose nitric oxide-donating aspirin increase life span in a Lynch syndrome mouse model.

Nonsteroidal anti-inflammatory drugs (NSAID) appear to be effective cancer chemopreventives. Previous cellular studies showed that aspirin (acetylsalicylic acid: ASA) and nitric oxide-donating ASA (NO-ASA) suppressed microsatellite instability (MSI) in mismatch repair (MMR)-deficient cells linked to the common cancer predisposition syndrome hereditary nonpolyposis colorectal cancer or Lynch syndrome (LS/HNPCC), at doses 300- to 3,000-fold less than ASA. Using a mouse model that develops MMR-deficient intestinal tumors that appear pathologically identical to LS/HNPCC, we show that ASA (400 mg/kg) and low-dose NO-ASA (72 mg/kg) increased life span by 18% to 21%. We also note a trend where ASA treatment resulted in intestinal tumors with reduced high MSI (H-MSI) and increased low MSI (L-MSI) as defined by the Bethesda Criteria. Low-dose NO-ASA had a minimal effect on MSI status. In contrast to previous studies, high-dose NO-ASA (720/1,500 mg/kg) treatments increased tumor burden, decreased life span, and exacerbated MSI uniquely in the LS/HNPCC mouse model. These results suggest that MMR-deficient tissues/mice may be specifically sensitive to intrinsic pharmacokinetic features of this drug. It is likely that long-term treatment with ASA may represent a chemopreventive option for LS/HNPCC patients. Moreover, as low-dose NO-ASA shows equivalent life span increase at 10-fold lower doses than ASA, it may have the potential to significantly reduce the gastropathy associated with long-term ASA treatment.

Protein nitration and nitrosylation by NO-donating aspirin in colon cancer cells: Relevance to its mechanism of action

Nitric oxide-donating aspirin (NO-ASA) is a promising agent for cancer prevention. Although studied extensively, its molecular targets and mechanism of action are still unclear. S-nitrosylation of signaling proteins is emerging as an important regulatory mechanism by NO. Here, we examined whether S-nitrosylation of the NF-κB, p53, and Wnt signaling proteins by NO-ASA might explain, in part, its mechanism of action in colon cancer. NO-ASA releases significant amounts of NO detected intracellularly in HCT116 and HT-29 colon cells. Using a modified biotin switch assay we demonstrated that NO-ASA S-nitrosylates the signaling proteins p53, β-catenin, and NF-κB, in colon cancer cells in a time- and concentration-dependent manner. NO-ASA suppresses NF-κB binding to its cognate DNA oligonucleotide, which occurs without changes in the nuclear levels of the NF-κB subunits p65 and p50 and is reversed by dithiothreitol that reduces ―S―NO to ―SH. In addition to S-nitrosylation, we documented both in vitro and in vivo widespread nitration of tyrosine residues of cellular proteins in response to NO-ASA. Our results suggest that the increased intracellular NO levels following treatment with NO-ASA modulate cell signaling by chemically modifying key protein members of signaling cascades. We speculate that S-nitrosylation and tyrosine nitration are responsible, at least in part, for the inhibitory growth effect of NO-ASA on cancer cell growth and that this may represent a general mechanism of action of NO-releasing agents.

Inhibitory effect of nitric oxide-donating aspirin against prostatic cancer angiogenesis in tumor-bearing mice

Objective To study the inhibitory effect of nitric oxide-donating aspirin(NO-ASA) on prostatic cancer growth and angiogenesis in subcutaneously-injected tumor in mice.Methods The tumor-bearing mice were subcutaneously injected with NO-ASA,and the tumor growth was observed.ELISA was used to assay the expression of VEGF in mouse sera;real-time PCR was used to examine the expression of VEGF mRNA in tumor tissues;immunohistochemistry was used to detect CD34 expression and to quantify the number of microvessels in the tumor;and Hen's egg test chorioallantoic membrane (HET-CAM) was employed to detect the in vitro angiogenesis.Results NO-ASA inhibited the prostate cancer growth in mice.The expression of VEGF was greatly decreased in the serum and tumor tissues,and CD34 expression was significantly decreased in the tumor tissues compared with the control group (P 0.05,P 0.01).NO-ASA also greatly inhibited the angiogenesis in HET-CAM.Conclusion NO-ASA can inhibit prostate cancer growth and angiogenesis.

NCX 4040, a Nitric Oxide-Donating Aspirin, Exerts Anti-Inflammatory Effects through Inhibition of IκB-α Degradation in Human Monocytes

NO-donating aspirins consist of aspirin to which a NO-donating group is covalently linked via a spacer molecule. NCX 4040 and NCX 4016 are positional isomers with respect to the -CH(2)ONO(2) group (para and meta, respectively) on the benzene ring of the spacer. Because positional isomerism is critical for antitumor properties of NO-donating aspirins, we aimed to compare their anti-inflammatory effects with those of aspirin in vitro. Thus, we assessed their impacts on cyclooxygenase-2 activity (by measuring PGE(2) levels), protein expression, and cytokine generation(IL-1beta, IL-18, TNF-alpha, and IL-10) in human whole blood and isolated human monocytes stimulated with LPS. Interestingly, we found that micromolar concentrations of NCX 4040, but not NCX 4016 or aspirin, affected cyclooxygenase-2 expression and cytokine generation. We compared the effects of NCX 4040 with those of NCX 4016 or aspirin on IkappaB-alpha stabilization and proteasome activity in the LPS-stimulated human monocytic cell line THP1. Differently from aspirin and NCX 4016, NCX 4040, at a micromolar concentration range, inhibited IkappaB-alpha degradation. In fact, NCX 4040 caused concentration-dependent accumulation of IkappaB-alpha and its phosphorylated form. This effect was not reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase, thus excluding the contribution of NO-dependent cGMP generation. In contrast, IkappaB-alpha accumulation by NCX 4040 may involve an inhibitory effect on proteasome functions. Indeed, NCX 4040 inhibited 20S proteasome activity when incubated with intact cells but not in the presence of cell lysate supernatants, thus suggesting an indirect inhibitory effect. In conclusion, NCX 4040 is an inhibitor of IkappaB-alpha degradation and proteasome function, and it should be taken into consideration for the development of novel anti-inflammatory and chemopreventive agents.

The differential cell signaling effects of two positional isomers of the anticancer NO-donating aspirin

We studied the mechanism by which the para and meta positional isomers of nitric oxide-donating aspirin (NO-ASA) inhibit human colon cancer cell growth. These compounds are promising chemopreventive agents and represent a broader class of novel drugs. The two isomers differ drastically in their 24-h IC50s for cell growth, which are 12 microM for p-NO-ASA and 230 microM for m-NO-ASA. We examined their effects on cell signaling cascades, including predominantly the mitogen activated protein kinases (MAPKs). The principal differences between the two isomers were: a) p-NO-ASA exerts its effect earlier than m-NO-ASA; b) the predominant effect of m-NO-ASA is on ERK1/2 and Akt; whereas that of p-NO-ASA is on JNK1/2, while both activate p38, with p-NO-ASA showing a stronger and earlier effect; c) ATF-2 is more responsive to m-NO-ASA and c-Jun to p-NO-ASA; d) both isomers seem to have similar effects on AP-1 binding, the main difference between them being the timing of the effect; p-NO-ASA's effect is early and m-NO-ASA's is late; e) p-NO-ASA has an earlier and stronger effect on p21, while m-NO-ASA's effect occurs later and is weaker; and f) cell cycle changes follow the effect on p21 expression. Our findings underscore the role of positional isomerism in modulating the pharmacological effects of drugs and have potentially important implications for the further development of these chemoprevention agents.

Suppression of Wnt/β-catenin signaling inhibits prostate cancer cell proliferation

Although mounting evidence has demonstrated an important role of Wnt/β-catenin signaling in the development and progression of cancer, the therapeutic potential of small molecules that target this pathway for prostate cancer remains largely unknown. We reported herein that the highly invasive androgen-independent PC-3 and DU145 human prostate cancer cells exhibited higher levels of Wnt/β-catenin signaling than the androgen-dependent LNCaP prostate cancer cells and non-cancerous PZ-HPV-7 and PWR-1E prostate cells, and that exogenous Wnt3A treatment exaggerated the difference of the Wnt/β-catenin signaling levels among these prostate cells. Furthermore, we demonstrated that the non-steroidal anti-inflammatory drug, sulindac sulfide, the cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, and the nitric oxide-donating aspirin derivative, NO-ASA, blocked Wnt/β-catenin signaling in PC-3 and DU145 cells. These effects occurred at concentrations comparable to those required to inhibit cell proliferation, indicating that the inhibitory effect of these drugs on prostate cancer cell proliferation may involve the suppression of Wnt/β-catenin signaling. Finally, we showed that a novel small molecule inhibitor of Wnt/β-catenin signaling, PKF118- 310, inhibited Wnt/β-catenin signaling and proliferation in prostate cancer cells within the same concentration range. Together, these results suggest that small molecules that inhibit Wnt/β-catenin signaling have therapeutic potential for the prevention or treatment of prostate cancer.

Chemopreventive agents induce oxidative stress in cancer cells leading to COX-2 overexpression and COX-2-independent cell death

Chemopreventive agents generate oxidative stress, which culminates in cell death and may be part of a general mechanism of chemoprevention. The redox-responsive cyclooxygenase (COX)-2, overexpressed during carcinogenesis, has been a target for cancer prevention. To assess the potential link between chemopreventive agents, oxidative stress and COX-2, we studied the chemopreventive sulindac and nitric oxide-donating aspirin (NO-ASA). Both generated oxidative stress and induced COX-2 in various cell lines, more prominently in dying cells. Two antioxidants and an inhibitor of NADPH oxidase abrogated the induction of COX-2 and cell death. Exogenous xanthine/xanthine oxidase, which produce O(2)(-)., had the same effect. Inhibition of caspases and cox-2 knockdown showed that COX-2 did not participate in reactive oxygen species (ROS) generation or cell death induction in response to NO-ASA. Our results support three potentially useful ideas: (i) the concept that ROS are a critical component of the action of chemopreventive agents; (ii) the notion that COX-2 may not be an ideal target for chemoprevention and (iii) the possibility that COX-2 may be overexpressed in cancer cells due to their state of oxidative stress. It is conceivable that, if further substantiated, these findings may inform the rational design of chemotherapeutic strategies, in particular the choice of agents in combination approaches.

Nitric oxide-donating aspirin inhibits the growth of pancreatic cancer cells through redox-dependent signaling

The novel chemopreventive nitric oxide-donating aspirin (NO-ASA) prevents nearly 90% of ductal adenocarcinomas in a animal tumor model. To decipher the mechanism of this effect, we studied in BxPC-3 human pancreatic cancer cells the sequence of signaling events leading from NO-ASA treatment to cell growth inhibition. NO-ASA inhibited the growth of BxPC-3 cells (IC 50 = 13 μM), by inhibiting proliferation modestly and inducing apoptosis, necrosis and G 1/S cell cycle block. At 15 min of treatment with NO-ASA, the intracellular levels of reactive oxygen species (ROS) began increasing (peak at 8 h, baseline levels by 24 h). ROS activated almost immediately in a time- and concentration-dependent manner the MAPK pathways p38, ERK and JNK (their activation was abrogated by the antioxidant N-acetylcysteine). MAPK activation induced p21 cip-1, which suppressed the levels of cyclin D1 that controls the G 1/S cell cycle transition. NO-ASA induced COX-2 expression starting 90 min after p21 cip-1 was induced. When COX-2 expression was knocked down using siRNA against cox-2, the expression of p21 cip-1 was induced by NO-ASA, regardless of the level of expression of COX-2, suggesting a marginal, if any, role for COX-2 in the growth inhibitory effect of NO-ASA. These findings along with the temporal sequence of individual changes indicate a signaling sequence that involves ROS → MAPKs → p21 cip-1 → cyclin D1 → cell death. Our findings establish the critical role of ROS as proximal signaling molecules in the action of anticancer compounds and may be useful in designing mechanism-driven approaches to cancer control.

Induction of oxidative stress as a mechanism of action of chemopreventive agents against cancer

Prevention is a promising option for the control of cancer. Cellular redox changes have emerged as a pivotal and proximal event in cancer. In this review, we provide a brief background on redox biochemistry, discuss the important distinction between redox signalling and oxidative stress, and outline the ‘multiple biological personalities’ of reactive oxygen and nitrogen species: at low concentrations they protect the cell; at higher concentrations they can damage many biological molecules, such as DNA, proteins, and lipids; and, as we argue here, they may also prevent cancer by initiating the death of the transformed cell. Nitric oxide-donating aspirin is discussed as an instructive example: it generates a state of oxidative stress through which it affects several redox-sensitive signalling pathways, leading ultimately to the elimination of the neoplastic cell via apoptosis or necrosis. As additional examples, we discuss the chemopreventive n–3 polyunsaturated fatty acids and conventional nonsteroidal anti-inflammatory drugs, which induce cell death through redox changes. We conclude that modulation of redox biochemistry represents a fruitful approach to cancer prevention.

NO-donating aspirin inhibits the activation of NF-kappaB in human cancer cell lines and Min mice.

Nitric oxide-donating aspirin (NO-ASA) is a promising agent for the control of cancer, whose mechanism of action remains unclear. NF-kappaB is an important signaling molecule in the pathogenesis of cancer. We studied in several human colon (HT-29, HCT-15, LoVo, HCT116 and SW-480), pancreatic (BxPC-3, MIA PaCa-2) and breast (MDA-MB-231 and MCF-7) cancer cell lines, the effect of NO-ASA on NF-kappaB activation, determined by electrophoretic mobility shift assays, immunofluorescence and western blot analyses of nuclear proteins. NO-ASA inhibited NF-kappaB activation, as early as 30 min and with IC(50)s ranging between 0.83 and 64 microM. Such inhibition was also observed at NO-ASA concentrations that had an insignificant or marginal effect on cell growth. The effect of NO-ASA on NF-kappaB binding to DNA was significantly correlated with its effect on cell growth (P < 0.05) indicating that the growth inhibitory effect of NO-ASA may be mediated by its effect on NF-kappaB. Compared with control, NO-ASA decreased NF-kappaB activation in intestinal epithelial cells of APC(min+/-) mice by 38.4% (P < 0.01). Western blot and immunofluorescence analyses revealed that the nuclear levels of the p50 and p65 NF-kappaB subunits were virtually unaffected, suggesting an inhibitory mechanism different from suppressed subunit translocation into the nucleus. Inhibition of NF-kappaB activation by NO-ASA may account, at least in part, for its chemopreventive efficacy.

Nitric oxide–donating aspirin (NCX 4016) inhibits neointimal thickening in a pig model of saphenous vein-carotid artery interposition grafting: A comparison with aspirin and morpholinosydnonimine (SIN-1)

Despite its proven value in reducing thrombotic complications in patients undergoing coronary artery bypass graft surgery, aspirin does not reduce the incidence of late vein graft failure. It was suggested, therefore, that co-administration of nitric oxide with aspirin may compensate for these limitations. A drug class that fulfills this pharmacologic criterion is nitric oxide-donating aspirin (NCX 4016). The effect of administration of the aspirin-nitric oxide adduct, NCX 4016, compared with those of aspirin alone and the nitric oxide donor, morpholinosydnonimine, alone (once daily for 1 month) on thickening of saphenous vein-carotid artery interposition grafts was investigated. NCX 4016, at 10 mg, 30 mg, and 60 mg x kg(-1) x d(-1), inhibited neointimal thickness and area in porcine vein grafts. Aspirin alone (60 mg x kg(-1) x d(-1)) and morpholinosydnonimine alone (1 mg x kg(-1) x d(-1)), also inhibited neointimal thickness and neointimal area, although they were less potent than NCX 4016. At 30 mg x kg(-1) x d(-1), aspirin had no effect. Compared with untreated controls, NCX 4016 had little effect on medial thickness or area at 10 mg/kg or 30 mg x kg(-1) x d(-1) but had a significant effect at 60 mg x kg(-1) x d(-1). Aspirin alone and morpholinosydnonimine alone also inhibited medial thickness and area. NCX 4016 at 60 mg x kg(-1) x d(-1) and aspirin at 60 mg x kg(-1) x d(-1) increased luminal area. The range of properties displayed by NCX 4016 (inhibition of neointima formation, gastroprotection, antithrombotic and antiatherogenic effects) renders them potentially useful in treating both early and late vein graft failure and indicates that a clinical study on this novel drug class in patients undergoing coronary bypass grafting is warranted.

The mechanism of action of nitric oxide-donating aspirin

NO-donating aspirin (NO-ASA) is a promising anticancer drug. We studied the contribution of NO-ASA’s components ( ASA, NO-releasing moiety, and spacer linking them) to its effect. The ASA and NO-releasing moieties play no biological role: ASA inhibits the growth of colon cancer cells >100-fold less potently that NO-ASA; and denitrated NO-ASA plus the NO-donor SNAP releasing the same amount of NO as NO-ASA, inhibit the growth of cancer cells >50-fold less potently than NO-ASA. The biologically active moiety of NO-ASA is the spacer: it is chemically reactive (studies with NO-ASA radiolabeled at the spacer demonstrated that it binds to proteins); and compounds in which the ASA or the NO-releasing groups are replaced inhibit cell growth similar to NO-ASA. We propose a mechanism of action of NO-ASA involving formation of quinone methide from its para and ortho isomers and of a carbocation from the meta, with the NO-releasing group functioning as a leaving group.

Chemical Insights in the Concept of Hybrid Drugs: The Antitumor Effect of Nitric Oxide-Donating Aspirin Involves A Quinone Methide but Not Nitric Oxide nor Aspirin

Hybrid drug 1 (NO-ASA) continues to attract intense research from chemists and biologists alike. It consists of ASA and a -ONO2 group connected through a spacer and is in preclinical development as an antitumor drug. We report that, contrary to current beliefs, neither ASA nor NO contributes to this antitumor effect. Rather, an unsubstituted QM was identified as the sole cytotoxic agent. QM forms from 1 after carboxylic ester hydrolysis and, in accordance with the HSAB theory, selectively reacts with cellular GSH, which in turn triggers cell death. Remarkably, a derivative lacking ASA and the -ONO2 group is 10 times more effective than 1. Thus, our data provide a conclusive molecular mechanism for the antitumor activity of 1. Equally importantly, we show for the first time that a "presumed invisible" linker in a hybrid drug is not so invisible after all and is in fact solely responsible for the biological effect.

Prevention by NCX 4016, a nitric oxide-donating aspirin, but not by aspirin, of the acute endothelial dysfunction induced by exercise in patients with intermittent claudication

Ischemia/reperfusion damage evokes systemic inflammation and endothelial dysfunction in patients with intermittent claudication. We compared the effects of aspirin with those of a nitric oxide-donating aspirin in preventing the acute, systemic endothelial dysfunction provoked by exercise-induced ischemia of the lower limbs in patients with intermittent claudication. In a prospective, randomized, single-blind, parallel-groups trial among 44 patients with intermittent claudication we compared four weeks of aspirin (100 mg o.d.) with NCX 4016 (800 mg b.i.d.). Primary end point was the exercise-induced changes in brachial flow-mediated vasodilation (FMD) at day 28; secondary end points were effort-induced changes of markers of neutrophil (plasma elastase) and endothelial (soluble VCAM-1) activation. Baseline FMD was comparable in the two groups, both on day 1 (pre-treatment: aspirin = 3.1 +/- 0.5%, nitroaspirin = 3.9 +/- 0.7%, p = NS), and on day 28 (aspirin = 3.4 +/- 0.7%, NCX 4016 = 3.2 +/- 0.6%, p = NS). Maximal treadmill exercise induced an acute worsening of FMD in both groups at baseline (aspirin = -1.15%, nitroaspirin = -1.76%); after four weeks treatment, the impairment of FMD induced by exercise was still present in the aspirintreated group (-1.46%) while it was abolished in the NCX 4016-treated group (+0.79%, p = 0.038 vs. aspirin). Similarly, exercise induced an increase of plasma elastase and of sVCAM-1 which were not affected by aspirin while they were suppressed by NCX 4016. Maximal treadmill exercise induces a systemic arterial endothelial dysfunction in patients with intermittent claudication. A nitric oxide-donating aspirin, but not aspirin, prevents effort-induced endothelial dysfunction.

The use of nitric oxide-donating nonsteroidal anti-inflammatory drugs in the chemoprevention of colorectal neoplasia

Nitric oxide-donating nonsteroidal anti-inflammatory drugs are emerging as a promising class of compounds for the chemoprevention of colon cancer. Recent progress in their preclinical and mechanistic evaluation is reviewed. Compared to their parent compounds, nitric oxide-donating nonsteroidal anti-inflammatory drugs are up to several hundred times more potent in inhibiting the growth of colon cancer cell lines and also quite effective in preventing colon cancer in various tumor animal models. Their chemopreventive effect is brought about through a strong cell kinetic effect, including inhibition of proliferation, induction of cell death and inhibition of cell cycle phase transitions. The induction of oxidative stress appears mechanistically crucial. Pleiotropic effects on cell signaling have been identified including Wnt, NOS2, mitogen-activated protein kinase and Nrf2 signaling. Nitric oxide-donating nonsteroidal anti-inflammatory drugs, particularly nitric oxide-donating aspirin, appear to be very safe compounds, as evidenced from many animal and early human studies. Nitric oxide-donating nonsteroidal anti-inflammatory drugs hold the promise of being safe and effective chemopreventive agents against colon cancer. Clinical trials are needed to determine whether these drugs can be applied clinically.

Nitric oxide-donating aspirin (NCX 4016): an overview of its pharmacological properties and clinical perspectives

In more than a century of use, the benefits of aspirin have been exploited in different therapeutic areas, from inflammation to cardiovascular disease for which it is an approved treatment for acute coronary heart disease and a recommended treatment for secondary cardiovascular prevention in patients at risk. Nitric oxide (NO), a small gaseous free radical molecule, is an important messenger of cellular and biological function that demonstrates an interesting anti-inflammatory profile and vascular protective activity. In addition, NO exerts a protective action on the gastrointestinal mucosa. An elegant approach for exploiting the properties of both aspirin and NO, and for eventually increasing the overall activity of aspirin, has been to create a new molecule in which aspirin and a NO-donating group are covalently linked (NCX 4016). This molecule requires enzymatic metabolism to release both components in order to provide simultaneous actions. NCX 4016 has been shown to retain the anti-inflammatory properties of aspirin and therefore possesses the same efficacy as aspirin in animal models of pain and inflammation. In addition, the release of NO accounts for the inhibition of the release of some cytokines. In cardiovascular models, NCX 4016 shows a better efficacy than aspirin in counteracting thromboembolism, myocardial infarction and vascular reactivity and remodelling (restenosis). This review describes the pharmacological profile of the NO-donating aspirin, NCX 4016, by addressing principally the pharmacological activity in inflammation and cardiovascular models. Gastrointestinal safety is specifically addressed. The potential clinical use of NCX 4016 in vascular pathologies is also discussed.