Studio della regolazione genetica della via di trasduzione del segnale iNOS dipendente nell'epatocarcinoma sperimentale e umano

Mounting evidence underlines the role of inducible nitric oxide synthase (iNOS) in hepatocellular carcinoma (HCC) development,but its functional interactions with pathways involved in HCC progression remain uninvestigated. We analyzed in preneoplastic and neoplastic livers from Fisher 344 and Brown Norway rats, possessing different genetic predisposition to HCC and in human HCC iNOS function and interactions with nuclear factor-kB (NF-kB) and Ha-RAS/extracellular signal-regulated kinase (ERK) during hepatocarcinogenesis; influence of genetic predisposition to liver cancer on these pathways. We found progressive iNos induction in rat at higher levels in the most aggressive rats. iNOS, inhibitor of kB kinase/NF-kB and RAS/ERK upregulation was significantly higher in HCC with poorer prognosis, positively correlated with tumor proliferation, genomic instability and microvascularization and negatively with apoptosis. Suppression of iNOS signaling led to decreased HCC growth and NF-kB and RAS/ERK expression and increased apoptosis both in vivo and in vitro. Conversely, block of NF-kB signaling or ERK signaling caused iNOS downregulation in HCC cell lines. These findings indicate that iNOS cross talk with NFkB and Ha-RAS/ERK cascades influences HCC growth and prognosis, suggesting that key component of iNOS signaling could represent important therapeutic targets for human HCC

Influence of temperature on the equilibria of oxidovanadium(IV) complexes in solution.

The equilibria at different temperatures of VIVOL2complexes were investigated in order to elucidate their interaction with proteins

A Friendly Complexing Agent for Spectrophotometric Determination of Total Iron

Iron, one of the most common metals in the environment, plays a fundamental role in many biological as well as biogeochemical processes, which determine its availability in different oxidation states. Its relevance in environmental and industrial chemistry, human physiology, and many other fields has made it necessary to develop and optimize analysis techniques for accurate determination. Spectrophotometric methods are the most frequently applied in the analytical determination of iron in real samples. Taking advantage of the fact that desferrioxamine B, a trihydroxamic acid used since the 1970s in chelation therapy for iron overload treatment, forms a single stable 1:1 complex with iron in whichever oxidation state it can be found, a smart spectrophotometric method for the analytical determination of iron concentration was developed. In particular, the full compliance with the Lambert-Beer law, the range of iron concentration, the influence of pH, and the interference of other metal ions have been taken into account. The proposed method was validated in terms of LoD, LoQ, linearity, precision, and trueness, and has been applied for total iron determination in natural water certified material and in biological reference materials such as control human urine and control serum

Interaction of Vanadium(IV) Species with Ubiquitin : A Combined Instrumental and Computational Approach

Altres ajuts: COST Action CM1306The interaction of VIVO2+ ion and five VIVOL2 compounds with potential pharmacological application, where L indicates maltolate (ma), kojate (koj), acetylacetonate (acac), 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (dhp), and l-mimosinate (mim), with ubiquitin (Ub) was studied by EPR, ESI-MS, and computational (docking and DFT) methods. The free metal ion VIVO2+ interacts with Glu, Asp, His, Thr, and Leu residues, but the most stable sites (named 1 and 2) involve the coordination of (Glu16, Glu18) and (Glu24, Asp52). In the system with VIVOL2 compounds, the type of binding depends on the vanadium concentration. When the concentration is in the mM range, the binding occurs with cis-VOL2(H2O), L = ma, koj, dhp, and mim, or with VO(acac)2: in the first case, the equatorial coordination of His68, Glu16, Glu18, or Asp21 residues yields species with formula n[VOL2]-Ub where n = 2-3, while with VO(acac)2 only noncovalent surface interactions are revealed. When the concentration of V is on the order of micromolar, themono-chelated species VOL(H2O)2+ with L = ma, koj, acac, dhp, and mim, favored by the hydrolysis, interact with Ub, and adducts with composition n[VOL]-Ub (n = 1-2) are observed with the contemporaneous coordination of (Glu18, Asp21) or (Glu16, Glu18), and (Glu24, Asp52) or (Glu51, Asp52) donors. The results of this work suggest that the combined application of spectroscopic, spectrometric, and computational techniques allow the complete characterization of the ternary systems formed by a V compound and a model protein such as ubiquitin. The same approach can be applied, eventually changing the spectroscopic/spectrometric techniques, to study the interaction of other metal species with other proteins

The Oxidative state of LDL is the major determinant of anti/prooxidant effect of coffee on Cu²⁺ catalysed peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

Thermodynamic Study of Oxidovanadium(IV) with Kojic Acid Derivatives: A Multi-Technique Approach

The good chelating properties of hydroxypyrone (HPO) derivatives towards oxidovanadium(IV) cation, VIVO2+, constitute the precondition for the development of new insulinmimetic and anticancer compounds. In the present work, we examined the VIVO2+ complex formation equilibria of two kojic acid (KA) derivatives, L4 and L9, structurally constituted by two kojic acid units linked in position 6 through methylene diamine and diethyl-ethylenediamine, respectively. These chemical systems have been characterized in solution by the combined use of various complementary techniques, as UV-vis spectrophotometry, potentiometry, NMR and EPR spectroscopy, ESI-MS spectrometry, and DFT calculations. The thermodynamic approach allowed proposing a chemical coordination model and the calculation of the complex formation constants. Both ligands L4 and L9 form 1:1 binuclear complexes at acidic and physiological pHs, with various protonation degrees in which two KA units coordinate each VIVO2+ ion. The joined use of different techniques allowed reaching a coherent vision of the complexation models of the two ligands toward oxidovanadium(IV) ion in aqueous solution. The high stability of the formed species and the binuclear structure may favor their biological action, and represent a good starting point toward the design of new pharmacologically active vanadium species

Decoding Surface Interaction of VIVO Metallodrug Candidates with Lysozyme

Altres ajuts: COST Action CM1306The interaction of metallodrugs with proteins influences their transport, uptake, and mechanism of action. In this study, we present an integrative approach based on spectroscopic (EPR) and computational (docking) tools to elucidate the noncovalent binding modes of various VIVO compounds with lysozyme, a prototypical model of protein receptor. Five VIVO-flavonoid drug candidates formed by quercetin (que), morin (mor), 7,8-dihydroxyflavone (7,8-dhf), chrysin (chr), and 5-hydroxyflavone (5-hf) - effective against several osteosarcoma cell lines - and two benchmark VIVO species of acetylacetone (acac) and catechol (cat) are evaluated. The results show a gradual variation of the EPR spectra at room temperature, which is associated with the strength of the interaction between the square pyramidal complexes [VOL2] and the surface residues of lysozyme. The qualitative strength of the interaction from EPR is [VO(que)2]2- ? [VO(mor)2] > [VO(7,8-dhf)2]2- > [VO(chr)2] ? [VO(5-hf)2] > [VO(acac)2] ? [VO(cat)2]2-. This observation is compared with protein-ligand docking calculations with GOLD software examining the GoldScore scoring function (F), for which hydrogen bond and van der Waals contact terms have been optimized to account for the surface interaction. The best predicted binding modes display an energy trend in good agreement with the EPR spectroscopy. Computation indicates that the strength of the interaction can be predicted by the Fmax value and depends on the number of OH or CO groups of the ligands that can interact with different sites on the protein surface and, more particularly, with those in the vicinity of the active site of the enzyme. The interaction strength determines the type of signal revealed (rigid limit, slow tumbling, or isotropic) in the EPR spectra. Spectroscopic and computational results also suggest that there are several sites with comparable binding energy, with the V complexes distributing among them in a bound state and in aqueous solution in an unbound state. This kind of study and analysis could be generalized to determine the noncovalent binding modes of a generic metal species with a generic protein

Biospeciation of Potential Vanadium Drugs of Acetylacetonate in the Presence of Proteins

Among vanadium compounds with potential medicinal applications, [V IV O(acac)] is one of the most promising for its antidiabetic and anticancer activity. In the organism, however, interconversion of the oxidation state to +III and +V and binding to proteins are possible. In this report, the transformation of V III (acac), V IV O(acac), and V V O(acac) after the interaction with two model proteins, lysozyme (Lyz) and ubiquitin (Ub), was studied with ESI-MS (ElectroSpray Ionization-Mass Spectroscopy), EPR (Electron Paramagnetic Resonance), and computational (docking) techniques. It was shown that, in the metal concentration range close to that found in the organism (15-250 μM), V III (acac) is oxidized to V IV O(acac) + and V IV O(acac), which-in their turn-interact with proteins to give n [V IV O(acac)]-Protein and n [V IV O(acac)]-Protein adducts. Similarly, the complex in the +IV oxidation state, V IV O(acac), dissociates to the mono-chelated species V IV O(acac) + which binds to Lyz and Ub. Finally, V V O(acac) undergoes complete dissociation to give the 'bare' V V O ion that forms adducts n [V V O]-Protein with n = 1-3. Docking calculations allowed the prediction of the residues involved in the metal binding. The results suggest that only the V IV O complex of acetylacetonate survives in the presence of proteins and that its adducts could be the species responsible of the observed pharmacological activity, suggesting that in these systems V IV O 2+ ion should be used in the design of potential vanadium drugs. If V III or V V O potential active complexes had to be designed, the features of the organic ligand must be adequately modulated to obtain species with high redox and thermodynamic stability to prevent oxidation and dissociation

Chelating Agents in Soil Remediation: A New Method for a Pragmatic Choice of the Right Chelator

Soil pollution by metal ions constitutes one of the most significant environmental problems in the world, being the ecosystems of extended areas wholly compromised. The remediation of soils is an impelling necessity, and different methodologies are used and studied for reaching this goal. Among them, the application of chelating agents is one of the most promising since it could allow the removal of metal ions while preserving the most meaningful properties of the original soils. The research in this field requires the joined contribute of different expertise spanning from biology to chemistry. In this work, we propose a parsimonious and pragmatic approach for screening among a range of potential chelating agents. This methodology, the Nurchi's method, is based on an extension of the Reilley procedure for EDTA titrations. This allows forecasting the binding ability of chelating agents toward the target polluting metal ions and those typically found in soils, based on the knowledge of the related protonation and complex formation constants. The method is thoroughly developed, and then tested by application to some representative cases. Its use and relevance in biomedical and industrial applications is also discussed