Nitric Oxide-releasing Aspirin Research Articles

Retracted: NCX-4016, a nitric oxide-releasing aspirin, protects endothelial cells against apoptosis by modulating mitochondrial function.

S. Fiorucci, A. Mencarelli, R. Mannucci, E. Distrutti, A. Morelli, P. del Soldato, S. Moncada, "NCX-4016, a nitric oxide-releasing aspirin, protects endothelial cells against apoptosis by modulating mitochondrial function," TheFASEB Journal 16, no. 12 (2002): 1645-1647. https://doi.org/10.1096/fj.02-0297fje. The above article, published online on August 07, 2002 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Dr. Loren E. Wold; the Federation of American Societies for Experimental Biology; and Wiley Periodicals LLC. The retraction has been agreed due to concerns raised by a third party regarding the similarity of blots in Figure7. The authors did not respond to inquiries by either the society or the publisher. In addition, the authors' institution did not respond to our request to begin an investigation. An investigation by the society and the publisher determined that there is overwhelming evidence that images in Figure7 were manipulated. Because the images in question were imperative to the conclusions presented in the article, all parties agreed the article must be retracted.

Abstract 6400: Synergistic combination of NCX 4040 and napabucasin induces immunogenic activation via upregulation of mitochondrial oxidative stress and STAT3 inhibition in cadmium transformed and metastatic prostate cancer cells

Abstract Despite recent diagnostic and therapeutic advances, prostate cancer (PCa) poses a significant burden on men’s health and quality of life, retaining the second highest incidence rate after lung cancer. The combinatorial therapy approach has emerged as a useful treatment option for various types of cancer to target tumor heterogeneity and overcome the limitations of existing treatment modalities. Here, we explored the combination of NCX 4040, a nitric oxide-releasing aspirin derivative and Napabucasin, a recently developed STAT3 and cancer stemness inhibitor as a potential treatment for PCa. Two cellular models were used namely BPH-Cd, a cadmium-transformed carcinogenic cell line and DU145, a brain metastatic PCa cell line. Cell viability assay indicated reduced cell viability in both cell lines in a dose-dependent manner and more so in combination as opposed to individual treatments, with an IC50 of 5 µM for NCX 4040 and 250 nM for Napabucasin. Notably, treatment with catalase prevented the synergistic activity of NCX 4040 and Napabucasin in reducing the viability of these cells. This suggested that NCX 4040 and Napabucasin might be inducing oxidative stress related cellular death. In addition, the combination of NCX 4040 and Napabucasin robustly impeded the in-vitro tumorigenic potential in both cell lines in colony formation assay and decreased the size of spheroids with increasing doses in in-vitro 3D spheroid assay in BPH-Cd. Western blot analysis revealed that in addition to the downregulation of STAT3, NCX 4040 and Napabucasin treatment showed upregulation in cGAS-STING immunogenic activation pathway related markers (pSTING, cGAS, pTBK1 in DU145 and STING, TBK1 in BPH-Cd). This data demonstrated that combination drug treatment might be inducing cell death via immunogenic activation. Additional markers related to this pathway are being tested in both cellular models. Interestingly, thioredoxin (TRX)-interacting protein was highly upregulated in both cell lines following combination treatment indicating that the synergistic effects are mediated via targeting TRX-regulated cellular redox signaling. Subsequently, TRX and thioredoxin reductase 1 were also downregulated in both cell lines. Furthermore, confocal imaging analysis indicated that NCX 4040 and Napabucasin together might be causing DNA damage and mitochondrial stress as DNA release was clearly evident. Flow cytometric analysis demonstrated that the drug combination shows a synergistic effect in producing excess mitochondrial and cellular ROS, causing apoptosis and in inducing cell cycle arrest in both BPH-Cd and DU145 cells. Taken together, our study demonstrates that NCX 4040 and Napabucasin in combination could be a potential anti-PCa therapy which warrants further evaluation for its clinical application. Citation Format: Nishtha Pathak, Gnanasekar Munirathinam. Synergistic combination of NCX 4040 and napabucasin induces immunogenic activation via upregulation of mitochondrial oxidative stress and STAT3 inhibition in cadmium transformed and metastatic prostate cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6400.

Characterization of a novel antibiofilm effect of nitric oxide-releasing aspirin (NCX-4040) on Candida albicans isolates from denture stomatitis patients.

Candida albicans biofilms play a key role in denture stomatitis, one of the most common oral pathologies in elderly people. Because biofilms are highly resistant to antifungals, new pharmacological strategies are needed. Aspirin and nitric oxide-donor molecules have both shown antibiofilm effects on C. albicans, making them promising candidates for treatment. In this study, we evaluated the antifungal/antibiofilm effect of a nitric-oxide releasing aspirin (NO-ASA) on C. albicans isolates from denture stomatitis patients in vitro. Disk diffusion assays showed that while NO-ASA had no antifungal effect, the drug potentiated fluconazole inhibition zone diameters, increasing the effect of fluconazole by 20–30% (p<0.05). The effect of NO-ASA on the morphogenesis of C. albicans was evaluated using light microscopy after inducing hyphae formation. For all clinical strains assayed, 125 μM NO-ASA significantly decreased the number of filamentous cells present (p<0.01). Adhesion to abiotic surfaces, a critical event for biofilm formation, was evaluated in 96-well polystyrene plates using crystal violet assay; 125 μM NO-ASA significantly inhibited adhesion. Biofilms were observed with scanning electron microscopy (SEM) and quantified using XTT reduction assay. NO-ASA decreased biofilm formation (IC50 ranging from 300 μM to 700 μM), consistent with SEM findings of altered biofilm microarchitecture. PGE2 and carboxy-PTIO (an NO scavenger) both blocked the antibiofilm effects of NO-ASA, suggesting that the efficacy of NO-ASA may be associated with both inhibition of PGE2 synthesis and release of NO. NO-ASA is a promising novel antibiofilm agent for treating fluconazole-resistant strains of C. albicans.

Nitric Oxide-Releasing Aspirin Suppresses NF-κB Signaling in Estrogen Receptor Negative Breast Cancer Cells in Vitro and in Vivo.

Estrogen receptor negative (ER(−)) breast cancer is aggressive, responds poorly to current treatments and has a poor prognosis. The NF-κB signaling pathway is implicated in ER(−) tumorigenesis. Aspirin (ASA) is chemopreventive against ER(+) but not for ER(−) breast cancers. Nitric oxide-releasing aspirin (NO-ASA) is a safer ASA where ASA is linked to an NO-releasing moiety through a spacer. In vitro, we investigated anti-proliferation effects of NO-ASA (para- and meta-isomers) against ER(−) breast cancer cells MDA-MB-231 and SK-BR-23, effects on NF-κB signaling, and reactive oxygen species by standard techniques. In vivo, effects of NO-ASA were evaluated in a mouse xenograft model using MDA-MB-231 cells. p-NO-ASA inhibited the growth of MDA-MB-231 and SK-BR-3 cells at 24 h, the respective IC50s were 13 ± 2 and 17 ± 2 μM; ASA had an IC50 of >3000 μM in both cell lines. The IC50s for m-NO-ASA in MDA-MB-231 and SK-BR-3 were 173 ± 15 and 185 ± 12 μM, respectively, therefore, implying p-NO-ASA as a stronger inhibitor of growth p-NO-ASA reduced cell growth by inhibiting proliferation, inducing apoptosis and causing G0/G1 cell cycle block. Activation of NF-κB was inhibited by both isomers as demonstrated by decreases in NF-κB-DNA binding and luciferase activity at 24 h, However, m-NO-ASA produced transient effects at 3 h such as increased NF-κB-DNA-binding, increased levels of nuclear p50, even though both isomers inhibited IκB degradation. Increase in nuclear p50 by m-NO-ASA was associated with translocation of p50 in to the nucleus as observed by immunoflouresence at 3 h. NO-ASA induced reactive oxygen species (ROS) as evidenced by overall increases in both H2DCFDA (2′,7′-dichlorodihydrofluorescein) and DHE (dihydroethidium)-derived fluorescence. Inhibition of ROS by N-acetyl-cysteine reversed the m-NO-ASA-mediated translocation of p50 in to the nucleus. In xenografts, p-NO-ASA inhibited tumor growth by inhibiting proliferation (PCNA and tumor volume), inducing apoptosis (TUNEL positive cells) and reducing NF-κB expression. Both isomers inhibit cancer cells, inhibit NF-κB pathway and induce ROS, and have potential as anticancer compounds.

Inhibition of Pancreatic Intraepithelial Neoplasia Progression to Carcinoma by Nitric Oxide-Releasing Aspirin in p48Cre/+-LSL-KrasG12D/+ Mice

Nitric oxide-releasing aspirin (NO-aspirin) represents a novel class of promising chemopreventive agents. Unlike conventional nonsteroidal anti-inflammatory drugs, NO-aspirin seems to be free of adverse effects while retaining the beneficial activities of its parent compound. The effect of NO-aspirin on pancreatic carcinogenesis was investigated by assessing the development of precursor pancreatic lesions and adenocarcinomas in KrasG12D/+ transgenic mice that recapitulate human pancreatic cancer progression. Six-week-old male p48Cre/+-LSL-KrasG12D/+ transgenic mice (20 per group) were fed diets containing 0, 1000, or 2000 ppm NO-aspirin. The development of pancreatic tumors was monitored by positron emission tomography imaging. All mice were killed at the age of 41 weeks and assessed for pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) and for molecular changes in the tumors. Our results reveal that NO-aspirin at 1000 and 2000 ppm significantly suppressed pancreatic tumor weights, PDAC incidence, and carcinoma in situ (PanIN-3 lesions). The degree of inhibition of PanIN-3 and carcinoma was more pronounced with NO-aspirin at 1000 ppm (58.8% and 48%, respectively) than with 2000 ppm (47% and 20%, respectively). NO-aspirin at 1000 ppm significantly inhibited the spread of carcinoma in the pancreas (∼97%; P < .0001). Decreased expression of cyclooxygenase (COX; with ∼42% inhibition of total COX activity), inducible nitric oxide synthase, proliferating cell nuclear antigen, Bcl-2, cyclin D1, and β-catenin was observed, with induction of p21, p38, and p53 in the pancreas of NO-aspirin-treated mice. These results suggest that low-dose NO-aspirin possesses inhibitory activity against pancreatic carcinogenesis by modulating multiple molecular targets.

Nitric oxide-releasing aspirin but not conventional aspirin improves healing of experimental colitis

To determine the effect of non-selective cyclooxygenase (COX) inhibitors, selective COX-2 inhibitors and nitric oxide (NO)-releasing aspirin in the healing of ulcerative colitis. Rats with 2,4,6 trinitrobenzenesulfon-ic acid (TNBS)-induced colitis received intragastric (ig) treatment with vehicle, aspirin (ASA) (a non-selective COX inhibitor), celecoxib (a selective COX-2 inhibitor) or NO-releasing ASA for a period of ten days. The area of colonic lesions, colonic blood flow (CBF), myeloperoxidase (MPO) activity and expression of proinflammatory markers COX-2, inducible form of nitric oxide synthase (iNOS), IL-1β and tumor necrosis factor (TNF)-α were assessed. The effects of glyceryl trinitrate (GTN), a NO donor, and 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-​tetramethyl-1H-imidazolyl-1-oxy-3-oxide, onopotassium salt (carboxy-PTIO), a NO scavenger, administered without and with ASA or NO-ASA, and the involvement of capsaicin-sensitive afferent nerves in the mechanism of healing the experimental colitis was also determined. Rats with colitis developed macroscopic and microscopic colonic lesions accompanied by a significant decrease in the CBF, a significant rise in colonic weight, MPO activity and plasma IL-1β and TNF-α levels. These effects were aggravated by ASA and 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560), but not celecoxib and counteracted by concurrent treatment with a synthetic prostaglandin E₂ (PGE₂) analog. Treatment with NO-ASA dose-dependently accelerated colonic healing followed by a rise in plasma NO(x) content and CBF, suppression of MPO and downregulation of COX-2, iNOS, IL-1β and TNF-α mRNAs. Treatment with GTN, the NO donor, significantly inhibited the ASA-induced colonic lesions and increased CBF, while carboxy-PTIO or capsaicin-denervation counteracted the NO-ASA-induced improvement of colonic healing and the accompanying increase in the CBF. These effects were restored by co-treatment with calcitonin gene related peptide (CGRP) and NO-ASA in capsaicin-denervated animals. NO-releasing ASA, in contrast to ASA, COX-1 inhibitors, and SC-560, accelerated the healing of colitis via a mechanism involving NO mediated improvement of microcirculation and activation of sensory nerves releasing CGRP.

Emerging antiplatelet agents, differential pharmacology, and clinical utility

The aspirin-clopidogrel combination is the current gold standard antiplatelet regimen following percutaneous coronary intervention and for the treatment of acute coronary syndrome. Despite the clinical benefit of this combination, patients continue to have vascular events. Another purinergic (P2Y(12)) receptor antagonist, prasugrel, became available last year. Although prasugrel is superior to clopidogrel in reducing clinical endpoints, a higher bleeding rate has been identified particularly in high-risk patients. Ticagrelor, a reversible P2Y(12) receptor antagonist currently being evaluated for approval, is also more potent than clopidogrel but has a similar bleeding risk. Two additional P2Y(12) antagonists are being investigated that will be available as an intravenous formulation. Apart from the P2Y(12) receptor antagonists, multiple other agents are being developed with unique mechanisms of platelet inhibition. These agents are being studied as an alternative to or in combination with clopidogrel. The antiplatelet agents currently under development include: thrombin receptor antagonists, phosphodiesterase inhibitors, a thromboxane-prostaglandin receptor antagonist, a serotonin receptor blocker, a platelet adhesion antagonist, nitric oxide-releasing aspirin, a glycoprotein VI antagonist, and a cyclooxygenase inhibitor. The purpose of this review is to describe the efficacy and safety profiles of the emerging antiplatelet agents and their role in the treatment of atherosclerotic cardiovascular diseases.

Modulation of stress genes expression profile by nitric oxide-releasing aspirin in Jurkat T leukemia cells

NO-donating aspirin (NO-ASA, para isomer) has been reported to exhibit strong growth inhibitory effect in Jurkat T-acute lymphoblastic leukemia (T-ALL) cells mediated in part by β-catenin degradation and caspase activation, but the mechanism(s) still remains unclear. In this study, DNA oligoarrays with 263 genes were used to examine the gene expression profiles relating to stress and drug metabolism, and characterize the stress responses at IC 50 and subIC 50 concentrations of p-NO-ASA (20 and 10 μM, respectively) in Jurkat T cells. A total of 22 genes related to heat shock response, apoptosis signaling, detoxifiers and Phase II enzymes, and regulators of cell growth were altered in expression by array analysis based on the expression fold change criteria of ≥1.5-fold or ≤0.65-fold. Real time quantitative RT-PCR confirmed that 20 μM p-NO-ASA strongly upregulated the mRNA levels of two heat shock genes HSPA1A (41.5 ± 7.01-fold) and HSPA6 (100.4 ± 8.11-fold), and FOS (16.2 ± 3.2-fold), moderately upregulated HSPH1 (1.71 ± 0.43-fold) , FMO4 (4.5 ± 1.67-fold), CASP9 (1.77 ± 0.03-fold), DDIT3 (5.6 ± 0.51-fold), and downregulated NF- κB1 (0.54 ± 0.01-fold) and CCND1 (0.69 ± 0.06-fold). Protein levels of Hsp70, the product of HSPA1A, and fos were increased in p-NO-ASA-treated Jurkat T and HT-29 colon cancer cells in a dose-dependent manner. Silencing of Hsp70 enhanced the growth inhibitory effect of p-NO-ASA at low concentrations. The altered gene expression patterns by NO-ASA in Jurkat T cells suggest mechanisms for carcinogen metabolism, anti-proliferative activity and possible chemoprotective activity in T-ALL.

Nitro-aspirin inhibits MCF-7 breast cancer cell growth: Effects on COX-2 expression and Wnt/β-catenin/TCF-4 signaling

There is current evidence implicating the Wnt/β-catenin/TCF pathway in breast cancer. We investigated the effect of para- and meta-positional isomers of nitric oxide-releasing aspirin (NO-ASA), and aspirin (ASA) on MCF-7 human breast cancer cell growth and β-catenin/TCF signaling. The p- and m-NO-ASA isomers strongly inhibited cell growth and β-catenin/TCF transcriptional activity compared to ASA; the IC 50s for growth inhibition were 57 ± 4, 193 ± 10 and >5000 μM, and for transcriptional inhibition they were 12 ± 1.8, 75 ± 6.5 and >5000 μM for p-, m-NO-ASA and ASA, respectively. p-NO-ASA reduced the expression of Wnt/β-catenin downstream target gene cyclin D1, and total cellular β-catenin levels. COX-2 expression was induced by p-NO-ASA, protein kinase C inhibitors reversed this induction. p-NO-ASA blocked the cell cycle transition at S to G 2/M phase. These studies suggest a targeted chemopreventive/chemotherapeutic potential for NO-ASA against breast cancer.

T1902 Effect of Selective and Non-Selective Cyclooxygenase (COX)-1 and COX-2 Inhibitors and Nitric Oxide Releasing Aspirin (No-ASA) On the Chronic Esophagitis in the Rat Model of Barrett's Esophagus

T1902 Effect of Selective and Non-Selective Cyclooxygenase (COX)-1 and COX-2 Inhibitors and Nitric Oxide Releasing Aspirin (No-ASA) On the Chronic Esophagitis in the Rat Model of Barrett's Esophagus

Abstract A23: Expression profile of genes altered by ester-protected hydroxybenzyl phosphates (EHBP) in T-leukemia cells

Abstract A23 Introduction Acute lymphoblastic leukemia (ALL) is the primary cause of cancer-related mortality in children. Novel compounds and different therapeutic approaches are required for more effective treatments. Nitric oxide-releasing aspirin (NO-ASA), comprised of an ASA molecule covalently bound to an NO liberating moiety through an aromatic spacer was reported to inhibit Jurkat T cell growth and other cancer cell lines of colon, pancreatic and breast origin, including experimentally-induced tumors in various animal models. Recently, we and others showed that the mechanism of action of NO-ASA did not involve released NO or aspirin but that the spacer molecule joining these two entities was the biologically active component. The role of the NO-releasing moiety was simply that of a leaving group in the production of a reactive quinone methide (QM) intermediate. The ASA component apparently had a very limited or no biological contribution. On this basis, we synthesized two compounds as models in which the NO-releasing group was replaced with a substituted phosphate (Agent 1: No NO, NO-ASA); and one in which the aspirin component was replaced with an acetyl group and the -ONO2 leaving group was replaced by a substituted phosphate group (Agent 2: No NO, No-aspirin, NO-ASA). In this study we investigated whether these three compounds may alter the gene expression of biological signaling pathways that are potential targets in carcinogenesis, and to elucidate the role of the NO-releasing moiety and the aspirin component of NO-ASA in the differential expression of these genes. Methods Agents: NO-ASA (2-(acetyloxy)-4-[(nitrooxy)methyl] phenyl ester, Agent 2: (2-(acetyloxy)-4-[(diethylphosphate)methyl] phenyl ester, Agent 3: (4-acetyloxybenzyl diethylphosphate) were synthesized, purified and NMR verified by us. Cell line: Jurkat T-acute lymphoblastic leukemia; the IC50 for cell growth inhibition for the three compounds was determined by an MTT assay. Following treatment of the Jurkat cells with NO-ASA, agent 2 or agent 3, at their respective IC50s, mRNA was isolated and labeled according to standard protocols and kits. The oligo arrays set from SuperArray were used. The blots were hybridized and the ratios of the expression levels quantified and calculated. Potential candidate genes that were differentially expressed were validated by real time RT-PCR. Results All three drugs were potent growth inhibitors of human leukemia Jurkat T cells. The genes that were prominently induced by all three drugs included FMO4, HSPA1A, HSPA6, HSPH1, FOS, CYP24 A1, DDIT3; this induction occurred in a concentration dependent manner. These may provide mechanistic insights into the growth inhibition observed with these agents. Other moderately altered genes included HYOU1, SOD1, SOD2. Interestingly, all 3 agents had the same effect in terms of fold-changes in gene expression for many of these candidate genes. This strongly suggests that the NO-releasing group and the aspirin component of NO-ASA have a negligible role in modulating these gene expressions. We propose that the growth inhibitory properties of all three agents are associated with the spacer component of the molecules which generates a quinone methide intermediate. The significance of the genes induced or repressed and the relationship of their gene expression with the cell growth inhibition will be discussed. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A23.

NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols

BackgroundOvarian carcinoma is the leading cause of mortality among gynecological cancers in the world. The high mortality rate is associated with lack of early diagnosis and development of drug resistance. The antitumor efficacy and mechanism of NCX-4040, a nitric oxide-releasing aspirin derivative, against ovarian cancer is studied.MethodsNCX-4040, alone or in combination with cisplatin (cis-diamminedichloroplatinum, cDDP), was studied in cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) cell lines as well as xenograft tumors grown in nude mice. Electron paramagnetic resonance (EPR) was used for measurements of nitric oxide and redox state. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice was used for mechanistic studies.ResultsCells treated with NCX-4040 (25 μM) showed a significant reduction of cell viability (A2780 WT, 34.9 ± 8.7%; A2780 cDDP, 41.7 ± 7.6%; p < 0.05). Further, NCX-4040 significantly enhanced the sensitivity of A2780 cDDP cells (cisplatin alone, 80.6 ± 11.8% versus NCX-4040+cisplatin, 26.4 ± 7.6%; p < 0.01) and xenograft tumors (cisplatin alone, 74.0 ± 4.4% versus NCX-4040+cisplatin, 56.4 ± 7.8%; p < 0.05), to cisplatin treatment. EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice treated with NCX-4040 and cisplatin revealed significant downregulation of pEGFR (Tyr845 and Tyr992) and pSTAT3 (Tyr705 and Ser727) expression.ConclusionThe results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols. Thus NCX-4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies.

Study of molecular mechanisms of pro-apoptotic activity of NCX 4040, a novel nitric oxide-releasing aspirin, in colon cancer cell lines

BackgroundDespite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, in in vitro human colon cancer models.MethodsThe effect on tumor growth was evaluated in four human colon cancer cell lines (LoVo, LRWZ, WiDr and LoVo Dx) by sulforhodamine B assay, oxidative stress by immunohistochemistry, apoptosis by laddering assay, mitochondrial membrane potential (ΔΨm) by flow cytometry, and apoptosis- and chemoresistance-related markers by western-blot and real-time method, respectively. Prostaglandin E2 levels were determined by ELISA.ResultsNCX 4040 produced a higher cytotoxic effect in all the cell lines than that produced by other NO donors tested. In particular, in LoVo and LRWZ cells, NCX 4040 induced a cytocidal effect and apoptosis through p53 and NAG-1 expression, an early ΔΨm collapse, and a sequential release of cytoplasmatic cytochrome c and caspase -9 and -3 active forms. 8-hydroxyguanine lesions, indicative of oxidative stress, were also observed. Conversely, in WiDr line, the drug caused a cytocidal effect, albeit not through apoptosis, and a concomitant increase in COX-2 activity. In LoVo Dx line, characterized by high levels drug resistance and DNA repair-related markers, only a cytostatic effect was observed, again in concomitance with the increase in COX-2 enzyme activity.ConclusionThis study highlights the multiplicity of mechanisms involved in sensitivity or resistance to NCX 4040 and could provide useful indications for tailored therapy by identifying potentially drug-responsive tumors.

Roles of platelet and endothelial cell COX-1 in hypercholesterolemia-induced microvascular dysfunction

Aspirin is a common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, nitric oxide-releasing aspirin (NCX-4016), a selective cyclooxygenase (COX)-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last week of a 2-wk HC diet. COX-1-deficient (COX-1(-/-)) and wild-type (WT) mice were transplanted with WT (WT/COX-1(-/-)) or COX-1(-/-) (COX-1(-/-)/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, whereas adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1(-/-)/WT chimeras was comparable to that in SC560-treated mice, whereas the largest reductions in blood cell adhesion were in WT/COX-1(-/-) chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet nitric oxide-releasing aspirin directly inhibits platelets independent of COX-1.

Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents

This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis.

Functional effects of nitric oxide-releasing aspirin on vein conduits of diabetic patients undergoing CABG

Background Type 2 diabetes mellitus (DM) is known to negatively affect biological properties of venous vasculature, and, particularly, to reduce endothelium-derived nitric oxide release. This condition might influence venous graft function following coronary artery bypass surgery (CABG). The aim of this study was to evaluate the functional effects of a NO-releasing aspirin (NORA) on vein grafts (VG) of diabetics and control patients undergoing elective CABG. Methods In 40 consecutive ischemic heart disease patients, the effects of NORA were tested on segments of saphenous vein conduits harvested during elective CABG. Twenty patients had type-2 DM (mean age 69 ± 2), whereas 20 patients had no DM (NDM) and represented the control group (mean age 67 ± 4). Functional responses were tested by exposing VGs to NORA and to standard vasoactive agents in an organ-bath preparation. Histological features of VGs were also assessed by light and electronic microscopy. Results Significant impairment of endothelial-dependent vasodilation (acetylcholine induced) was documented in VGs of DM subjects. NORA induced a significant and comparable vascular relaxation in all venous segments of NDM and DM patients (56 ± 12% of maximal relaxation vs 61 ± 11% in the control group, respectively). Histology showed variable extent of vascular layer and cellular abnormalities in VGs of diabetics (intimal hyperplasia, calcific deposition, endothelial cell degeneration) likely responsible of the endothelial functional impairment, whereas control group VG showed preserved structures. Conclusions This preliminary study confirms the impairment of endothelium-dependent vasodilative property of VGs in DM patients. It also indicates that NORA effectively induces vasodilation of VGs which was effective also in DM patients thereby representing a promising therapy for diabetics undergoing CABG with the use of VGs, although further studies are mandatory to conclusively assess the safety and benefits of this pharmacological agent.

The Effect of NCX4016 [2-Acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl Ester] on the Consequences of Ischemia and Reperfusion in the Streptozotocin Diabetic Rat

The aim of this study was to assess the effect of chronic administration of NCX4016 [2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester], a nitric oxide-releasing aspirin derivative on the consequences of coronary artery occlusion in streptozotocin-diabetic rats. Rats were made diabetic by injection of streptozotocin (60 mg kg(-1)) and received insulin (2.5 U kg(-1) s.c.) daily for 4 weeks. Animals received vehicle (1 ml kg(-1) polyethylene glycol), aspirin (65.2 mg kg(-1)), NCX4016 (60 mg kg(-1)), or (iv) NCX4016 (120 mg kg(-1)) orally, once daily for the last 5 days before coronary artery occlusion (CAO). One hour after the last dose, pentobarbital-anesthetized rats were subjected to CAO for 30 min followed by 120-min reperfusion. Neither drug significantly modified initial hemodynamics or plasma glucose levels compared with vehicle treatment in either nondiabetic or diabetic rats. Neither drug modified the total ventricular premature beat (VPB) count in normal animals, although NCX4016, but not aspirin, reduced the total VPB count and the incidence of ventricular tachycardia in diabetic rats. In nondiabetic animals, both aspirin and NCX4016 reduced infarct size. However, in diabetic rats, infarct size was reduced only by the larger dose of NCX4016 (120 mg kg(-1)) but not by aspirin or the lower dose of NCX4016. These results demonstrate that the cardioprotective effects of NCX4016 are reduced in the presence of diabetes compared with the effects seen in nondiabetic animals. In summary, the present study confirms the protective effect of NCX4016 against ischemia-reperfusion injury in the normal rat heart and demonstrates for the first time its protective effect in the heart of streptozotocin-diabetic rats.

NO-donating aspirin inhibits both the expression and catalytic activity of inducible nitric oxide synthase in HT-29 human colon cancer cells

Nitric oxide-releasing aspirin (NO-ASA) is emerging as a potentially important chemopreventive agent against colon cancer. We examined in HT-29 human colon adenocarcinoma cells the effect of NO-ASA on the inducible form of nitric oxide synthase (NOS2), an enzyme implicated in colon carcinogenesis. NO-ASA inhibited in a time- and concentration-dependent manner the expression of NOS2 up to 70% compared to control (IC 50 for this effect = 46 μM). NO-ASA also decreased the corresponding steady-state mRNA levels and this reduction preceded the reduction of protein levels by at least 6 h. NO-ASA also reduced the enzymatic activity of NOS2, as determined by a direct enzyme assay (maximal reduction = 80%) and by determining the accumulation of NO in the culture medium (IC 50 for this effect = 36 μM). These effects of NO-ASA on NOS2 were paralleled by inhibition in cell growth (IC 50 = 8.5 μM). These findings indicate that NO-ASA profoundly inhibits both the expression and enzymatic activity of NOS2 and suggest that these effects may represent an important mechanism for the colon cancer chemopreventive effect of NO-ASA.

Nitroaspirin plus clopidogrel versus aspirin plus clopidogrel against platelet thromboembolism and intimal thickening in mice

Clopidogrel plus aspirin is the treatment of choice for patients undergoing percutaneous, coronary interventions with stenting, but it does not prevent restenosis. NCX-4016, a nitric oxide-releasing aspirin (nitroaspirin), exerts a wider range of antiplatelet actions compared to aspirin, superior antithrombotic activity and reduces restenosis after arterial injury in animals. The aim of the present study was to compare the combination of nitroaspirin plus clopidogrel with aspirin plus clopidogrel in a model of platelet pulmonary thromboembolism, bleeding and intimal thickening in mice. Drugs were administered orally for 5 days; the antithrombotic effects were evaluated against collagen plus epinephrine-induced pulmonary thromboembolism, the haemorrhagic effects by tail transection bleeding time and the effects on neointima proliferation by histomorphology of photochemically injured femoral arteries. Lung platelet emboli were reduced significantly and more effectively by nitroaspirin plus clopidogrel (-56%, p<0.05 vs control) than by aspirin plus clopidogrel (-26%, p<0.05 vs control). Ex vivo platelet aggregation was inhibited maximally by nitroaspirin plus clopidogrel. Aspirin plus clopidogrel strikingly prolonged the bleeding time while nitroaspirin plus clopidogrel induced a lesser prolongation. Nitroaspirin plus clopidogrel significantly reduced intimal thickening of the femoral artery while aspirin plus clopidogrel was ineffective. Nitroaspirin plus clopidogrel is more effective and less prohaemorrhagic than aspirin plus clopidogrel in mice; provided these data are confirmed in other animal models, nitroaspirin plus clopidogrel may represent a new regimen to be tested in patients undergoing coronary revascularization procedures.

222 P53-mediated apoptosis induced by NCX 4040, a nitric oxide-releasing aspirin derivative, in human colon cancer cell lines

222 P53-mediated apoptosis induced by NCX 4040, a nitric oxide-releasing aspirin derivative, in human colon cancer cell lines