Loretta Lazzarato

Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells

Blocking CCL2 nitration in tumors promoted CD8+ influx and reduced tumor growth and prolonged survival in mice when combined with adoptive cell therapy.

Searching for new NO-donor Aspirin-like molecules: a new class of nitrooxy-acyl derivatives of salicylic acid

A new class of products in which the phenol group of salicylic acid is linked to alkanoyl moieties bearing nitrooxy functions has been synthesized and studied for their polyvalent actions. The products were stable in acid and neutral media, while they were hydrolyzed in human serum. Their half-lives were dependent upon the structure of alkanoyl moieties. The products showed anti-inflammatory activities similar to aspirin when tested in the carrageenan-induced paw edema assay in the rat. Interestingly, unlike aspirin, they showed reduced or no gastrotoxicity in a lesion model in rats at equimolar doses. A number of them were able to inhibit platelet aggregation induced by collagen in human platelet-rich plasma. All of the products were capable of relaxing rat aortic strips precontracted with phenylephrine in a concentration-dependent manner. Selected members of this new class of nonsteroidal anti-inflammatory drugs might represent possible safer alternatives to aspirin in different clinical settings.

Nitric oxide donor doxorubicins accumulate into Doxorubicin-resistant human colon cancer cells inducing cytotoxicity.

Products 4 and 5, obtained by conjugation of doxorubicin with nitric oxide (NO) donor nitrooxy and phenylsulfonyl furoxan moieties, respectively, accumulate in doxorubicin-resistant human colon cancer cells (HT29-dx), inducing high cytotoxicity. This behavior parallels the ability of the compounds to generate NO, detected as nitrite, in these cells. Preliminary immunoblotting studies suggest that the mechanism that underlies the cytotoxic effect could involve inhibition of cellular drug efflux due to nitration of tyrosine residues of the MRP3 protein pump.

Mitochondrial-Targeting Nitrooxy-doxorubicin: A New Approach To Overcome Drug Resistance

In previous studies, we showed that nitric oxide (NO) donors and synthetic doxorubicins (DOXs) modified with moieties containing NO-releasing groups--such as nitrooxy-DOX (NitDOX) or 3-phenylsulfonylfuroxan-DOX (FurDOX)--overcome drug resistance by decreasing the activity of ATP-binding cassette (ABC) transporters that can extrude the drug. Here, we have investigated the biochemical mechanisms by which NitDOX and FurDOX exert antitumor effects. Both NitDOX and FurDOX were more cytotoxic than DOX against drug-resistant cells. Interestingly, NitDOX exhibited a faster uptake and an extranuclear distribution. NitDOX was preferentially localized in the mitochondria, where it nitrated and inhibited the mitochondria-associated ABC transporters, decreased the flux through the tricarboxylic acid cycle, slowed down the activity of complex I, lowered the synthesis of ATP, induced oxidative and nitrosative stress, and elicited the release of cytochrome c and the activation of caspase-9 and -3 in DOX-resistant cells. We suggest that NitDOX may represent the prototype of a new class of multifunctional anthracyclines, which have cellular targets different from conventional anthracyclines and greater efficacy against drug-resistant tumors.

A novel hybrid aspirin-NO-releasing compound inhibits TNFalpha release from LPS-activated human monocytes and macrophages

BackgroundThe cytoprotective nature of nitric oxide (NO) led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNFα release from lipopolysaccharide (LPS)-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-κB) activation.MethodsPeripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 μM), their respective furazan NO-free counterparts (B16, B15; 10 μM), aspirin (10 μM), existing nitroaspirin (NCX4016; 10 μM), an NO donor (DEA/NO; 10 μM) or dexamethasone (1 μM), in the presence and absence of LPS (10 ng/ml; 4 h). Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNFα release and by lactate dehydrogenase (LDH) assay for cell necrosis. To assess NF-κB activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-κB, IκBα (assessed by western blotting) and nuclear localisation (assessed by immunofluorescence) of the p65 subunit of NF-κB were determined.ResultsB8 significantly reduced TNFα release from LPS-treated macrophages to 36 ± 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNFα release to 28 ± 5, and 49 ± 9% of control, respectively. The B8 effect was equivalent in magnitude to that of dexamethasone, but was not shared by 10 μM DEA/NO, B7, the furazans, aspirin or NCX4016. LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic IκBα and nuclear translocation of the p65 NF-κB subunit was inhibited by the active NO-furoxans.ConclusionHere we show that furoxan-aspirin, B8, significantly reduces TNFα release from both monocytes and macrophages and suggest that inhibition of NF-κB activation is a likely mechanism for the effect. This anti-inflammatory action highlights a further therapeutic potential of drugs of this class.

Synthesis and Biological Evaluation of the First Example of NO- Donor Histone Deacetylase Inhibitor

The NO-donor histone deacetylase inhibitor 2, formally obtained by joining Entinostat 1, a moderately selective Class I histone deacetylases (HDACs) inhibitor, to a 4-(methylaminomethyl)furoxan-3-carbonitrile scaffold, is described and its preliminary biological profile discussed. This hybrid regulates Classes I and II HDACs. Nitric oxide (NO) released by the compound activates soluble guanylate cyclase (sGC), causing Class II nuclear shuttling and chromatin modifications, with consequences on gene expression. The hybrid affects a number of micro-RNAs not modulated by its individual components; it promotes myogenic differentiation, inducing the formation of larger myotubes with significantly more nuclei per fiber, in a more efficient manner than the 1:1 mixture of its two components. The hybrid is an example of a new class of NO-donor HDACs now being developed, which should be of interest for treating a number of diseases.

New Nitric Oxide or Hydrogen Sulfide Releasing Aspirins

A new series of (((R-oxy)carbonyl)oxy)methyl esters of aspirin (ASA), bearing nitric oxide (NO) or hydrogen sulfide (H(2)S) releasing groups, was synthesized, and the compounds were evaluated as new ASA co-drugs. All the products were quite stable in buffered solution at pH 1 and 7.4. Conversely, they were all rapidly metabolized, producing ASA and the NO/H(2)S releasing moiety used for their preparation. Consequent on ASA release, the compounds were capable of inhibiting collagen-induced platelet aggregation of human platelet-rich plasma (PRP). The simple NO/H(2)S donor substructures were able to relax contracted rat aorta strips, with a NO- and H(2)S-dependent mechanism, respectively, but they either did not trigger antiaggregatory activity or displayed antiplatelet potency markedly below that of the related co-drug. The new products might provide a safer and improved alternative to the use of ASA principally in its anti-inflammatory and antithrombotic applications.

Nitrooxyacyloxy)methyl Esters of Aspirin as Novel Nitric Oxide Releasing Aspirins

A series of (nitrooxyacyloxy)methyl esters of aspirin were synthesized and evaluated as new NO-donor aspirins. Different amounts of aspirin were released in serum from these products according to the nature of nitrooxyacyloxy moiety present. In the aromatic series, there is a rather good linear correlation between the amount of aspirin released and the potencies of the products in inhibiting platelet aggregation induced by collagen. Both the native compounds and the related nitrooxy-substituted acid metabolites were able to relax rat aorta strips precontracted with phenylephrine, in keeping with a NO-induced activation of the sGC as a mechanism that underlies the vasodilator effect. The products here described are new improved examples of NO-donor aspirins containing nitrooxy groups. They could represent an alternative to the use of aspirin in a variety of clinical applications.

Mechanistic insights into cyclooxygenase irreversible inactivation by aspirin.

A mechanistic hypothesis for the acetylation of cyclooxygenase (COX) by aspirin is proposed on the basis of a QM/MM study. This mechanism is consistent with previous experimental findings by other investigators. Ser 530 appears to be acetylated under intramolecular general base catalysis provided by the carboxylate moiety of aspirin, while Tyr 385 plays a crucial role in orienting and polarizing the acetyl group.

Searching for new NO-donor aspirin-like molecules: Furoxanylacyl derivatives of salicylic acid and related furazans.

A new group of derivatives of salicylic acid containing NO-donor furoxans, and the related des-NO-furazans, were synthesized and evaluated as new aspirin-like molecules. Their stability was assessed in acid (pH 1) and physiological solutions (pH 7.4), and in human serum. No compound exhibited COX-inhibitory activity against COX-1 and COX-2 isoforms, when tested up to 100μM, respectively, on isolated platelets and on monocytes. Phenylsulfonyl- and cyano-substituted furoxans inhibited platelet aggregation induced by collagen in human platelet-rich plasma, through a cGMP dependent mechanism. Furoxan derivatives displayed cGMP-dependent vasodilator activities, tested on rat aorta strips precontracted with phenylephrine. All products showed anti-inflammatory activity similar to that of ASA, tested on rats by the carrageenan-induced paw edema assay. Unlike ASA, all products showed markedly reduced gastrotoxicity in a rat lesion model.