Maria Pia Rigobello

Biochemical and toxicological properties of the oxidation products of catecholamines

The normal catabolism of catecholamines proceeds through enzymatic pathways (monoaminooxidase, catechol-o-methyltranserase, and phenolsulphotransferase). In addition, nonenzymatic oxidative pathways might take place since catechols are readily oxidized. In this review article, the pathways of formation of the oxidation products of catecholamines and their reactions are described. The interactions of these products with different biological systems and their toxicity are examined. Among the reactions known to occur is that with sulfhydryls, which results in either a covalently linked adduct or disulfide production. Another interesting pathway to toxicity involves the oxidation of these catecholamine products by oxygen, with the formation of damaging oxygen-derived species. The action of the oxidation products of catecholamines is outlined, with special attention to the nervous and cardiac systems.

Emerging Protein Targets for Anticancer Metallodrugs: Inhibition of Thioredoxin Reductase and Cathepsin B by Antitumor Ruthenium(II)−Arene Compounds

A series of ruthenium(II)-arene (RAPTA) compounds were evaluated for their ability to inhibit thioredoxin reductase (either cytosolic or mitochondrial) and cathepsin B, two possible targets for anticancer metallodrugs. In general, inhibition of the thioredoxin reductases was lower than that of cathepsin B, although selected compounds were excellent inhibitors of both classes of enzymes in comparison to other metal-based drugs. Some initial structure-activity relationships could be established. On the basis of the obtained data, different mechanisms of binding/inhibition appear to be operative; remarkably the selectivity of the ruthenium compounds toward solid metastatic tumors also correlates to the observed trends. Notably, docking studies of the interactions of representative RAPTA compounds with cathepsin B were performed that provided realistic structures for the resulting protein-metallodrug adducts. Good agreement was generally found between the inhibiting potency of the RAPTA compounds and the computed stability of the corresponding cat B/RAPTA adducts.

Gold(I) Carbene Complexes Causing Thioredoxin 1 and Thioredoxin 2 Oxidation as Potential Anticancer Agents

Gold(I) complexes with 1,3-substituted imidazole-2-ylidene and benzimidazole-2-ylidene ligands of the type NHC-Au-L (NHC = N-heterocyclic carbene L = Cl or 2-mercapto-pyrimidine) have been synthesized and structurally characterized. The compounds were evaluated for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), as well in the nontumorigenic human embryonic kidney cell line (HEK-293T), showing in some cases important cytotoxic effects. Some of the complexes were comparatively tested as thioredoxin reductase (TrxR) and glutathione reductase (GR) inhibitors, directly against the purified proteins or in cell extracts. The compounds showed potent and selective TrxR inhibition properties in particular in cancer cell lines. Remarkably, the most effective TrxR inhibitors induced extensive oxidation of thioredoxins (Trxs), which was more relevant in the cancerous cells than in HEK-293T cells. Additional biochemical assays on glutathione systems and reactive oxygen species formation evidenced important differences with respect to the classical cytotoxic Au(I)-phosphine compound auranofin.

Induction of mitochondrial permeability transition by auranofin, a Gold(I)-phosphine derivative

Gold(I)‐thiolate drugs are compounds that specifically interact with thiol and/or selenol groups and are essentially utilized in the treatment of rheumatoid arthritis. Considering the importance of thiol groups in regulating mitochondrial membrane permeability, the effects of auranofin (S‐triethylphosphinegold(I)‐2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐β‐D‐glucopyranoside), a second‐generation gold drug, were studied on mitochondria isolated from rat liver. Auranofin, at submicromolar concentrations, was able to induce the mitochondrial membrane permeability transition observed as swelling and loss of membrane potential. Both events are completely inhibited by cyclosporin A, the specific inhibitor of mitochondrial permeability transition. Calcium ions and energization by succinate are required for the occurrence of permeability transition. By interacting with the active site selenol group, auranofin results as an extremely potent inhibitor of mitochondrial thioredoxin reductase, both isolated and in its mitochondrial environment. It is concluded that auranofin, in the presence of calcium ions, is a highly efficient inducer of mitochondrial membrane permeability transition, potentially referable to its inhibition of mitochondrial thioredoxin reductase.

Anticancer therapeutics that target selenoenzymes: synthesis, characterization, in vitro cytotoxicity, and thioredoxin reductase inhibition of a series of gold(I) complexes containing hydrophilic phosphine ligands.

Gold(I) complexes bearing water‐soluble phosphine ligands, including 1,3,5‐triaza‐7‐phosphaadamantane (PTA), 3,7‐diacetyl‐1,3,7‐triaza‐5‐phosphabicyclo[3.3.1]nonane (DAPTA), and sodium triphenylphosphine trisulfonate (TPPTS), in combination with thionate ligands, were screened for their antiproliferative activities against human ovarian cancer cell lines A2780 either sensitive or resistant to cisplatin. In addition, the compounds were screened for their inhibition of mammalian thioredoxin reductases (TrxR), enzymes that are overexpressed in many tumor cells and contribute to drug resistance. The gold(I)–phosphine complexes efficiently inhibited cytosolic and mitochondrial TrxRs at concentrations that did not affect the related oxidoreductase glutathione reductase (GR). Additional complementary information on the enzyme metallation process and potential gold binding sites was obtained through the application of a specific biochemical assay using a thiol‐tagging reagent, BIAM (biotin‐conjugated iodoacetamide).

Principles in Redox Signaling: From Chemistry to Functional Significance

Reactive oxygen and nitrogen species are currently considered not only harmful byproducts of aerobic respiration but also critical mediators of redox signaling. The molecules and the chemical principles sustaining the network of cellular redox regulated processes are described. Special emphasis is placed on hydrogen peroxide (H(2)O(2)), now considered as acting as a second messenger, and on sulfhydryl groups, which are the direct targets of the oxidant signal. Cysteine residues of some proteins, therefore, act as sensors of redox conditions and are oxidized in a reversible reaction. In particular, the formation of sulfenic acid and disulfide, the initial steps of thiol oxidation, are described in detail. The many cell pathways involved in reactive oxygen species formation are reported. Central to redox signaling processes are the glutathione and thioredoxin systems controlling H(2)O(2) levels and, hence, the thiol/disulfide balance. Lastly, some of the most important redox-regulated processes involving specific enzymes and organelles are described. The redox signaling area of research is rapidly expanding, and future work will examine new pathways and clarify their importance in cellular pathophysiology.

Fluorescent silver(I) and gold(I)-N-heterocyclic carbene complexes with cytotoxic properties

Silver(I) and gold(I)-N-heterocyclic carbene (NHC) complexes bearing a fluorescent anthracenyl ligand were examined for cytotoxicity in normal and tumor cells. The silver(I) complex exhibits greater cytotoxicity in tumor cells compared with normal cells. Notably, in cell extracts, this complex determines a more pronounced inhibition of thioredoxin reductase (TrxR), but it is ineffective towards glutathione reductase (GR). Both gold and silver complexes lead to oxidation of the thioredoxin system, the silver(I) derivative being particularly effective. In addition, the dimerization of peroxiredoxin 3 (Prx3) was also observed, demonstrating the ability of these compounds to reach the mitochondrial target. The fluorescence microscopy visualization of the subcellular distribution of the complexes shows a larger diffusion of these molecules in tumor cells with respect to normal cells.

Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress

The reversible and irreversible conversion of xanthine dehydrogenase to xanthine oxidase during ischemia/reperfusion and oxidative stress induced by hydrogen peroxide or diamide and its relationship with glutathione and protein SH groups were studied. The direct spectrophotometric measurement of the various forms of the xanthine-converting enzyme indicates that, in the fresh rat heart or after normoxic perfusion, there always is a basal level of 80% xanthine dehydrogenase and 20% of xanthine oxidase (15% irreversible and 5% reversible) that could contribute to the background production of free radicals. There is no significant increase of irreversible xanthine oxidase during ischemia nor during reperfusion. After global ischemia the reversible oxidase shows almost no increase while, when ischemia is followed by reperfusion, there is a limited increase (less then 9%) of the reversible xanthine oxidase. In the latter conditions there is a decrease of glutathione and of SH groups of about 70% and 25%, respectively. Perfusion for 1 h with oxidizing agents like hydrogen peroxide (60 microM) or diamide (100 microM) determines a marked conversion of xanthine dehydrogenase to reversible xanthine oxidase of about 40% and 60%, respectively; this oxidase activity partially reconverts to the dehydrogenase after withdrawing the oxidizing agents from the perfusion medium. The level of irreversible xanthine oxidase remains unchanged in all the conditions tested. Both hydrogen peroxide and diamide induce a strong decrease in SH groups and depletion of glutathione. The xanthine dehydrogenase----xanthine oxidase conversion thus appears to be sensitive to the redox state of thiol groups.

In Vitro Antitumour Activity of Water Soluble Cu(I), Ag(I) and Au(I) Complexes Supported by Hydrophilic Alkyl Phosphine Ligands

Hydrophilic, monocationic [M(L)(4)]PF(6) complexes (M = Cu or Ag; L: thp = tris(hydroxymethyl)phosphine, L: PTA = 1,3,5-triaza-7-phosphaadamantane, L: thpp = tris(hydroxypropyl)phosphine) were synthesized by ligand exchange reaction starting from [Cu(CH(3)CN)(4)]PF(6) or AgPF(6) precursors at room temperature in the presence of an excess of the relevant phosphine. The related [Au(L)(4)]PF(6) complexes (L = thp, PTA or thpp) were synthesized by metathesis reactions starting from [Au(L)(4)]Cl at room temperature in the presence of equimolar quantity of TlPF(6). The three series of complexes [M(L)(4)]PF(6) were tested as cytotoxic agents against a panel of several human tumour cell lines also including a defined cisplatin resistant cell line. These investigations have been carried out in comparison with the clinically used metallodrug cisplatin and preliminary structure-activity relationships are presented. The best results in terms of in vitro antitumour activity were achieved with metal-thp species and, among the coinage metal complexes, copper derivatives were found to be the most efficient drugs. Preliminary studies concerning the mechanism of action of these [M(L)(4)]PF(6) species pointed to thioredoxin reductase as one of the putative cellular targets of gold and silver complexes and provided evidence that copper derivatives mediated their cytotoxic effect through proteasome inhibition.

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase.

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.