Gabriele D'Andrea

Resveratrol: A Natural Polyphenol with Multiple Chemopreventive Properties (Review)

Resveratrol, a naturally occurring polyphenol, shows pleiotropic health beneficial effects, including anti-oxidant, anti-inflammatory, anti-aging, cardioprotective and neuroprotective activities. Due to the several protective effects and since this compound is widely distributed in the plant kingdom, resveratrol can be envisaged as a chemo-preventive/curative agent introduced almost daily with the diet. Currently, a number of preclinical findings suggest resveratrol as a promising natures weapon for cancer prevention and treatment. A remarkable progress in elucidating the molecular mechanisms underlying anti-cancer properties of resveratrol has been achieved in the last years. Concerning the resveratrol mechanism of action as a protective (vs. normal cells and tissues) and toxic (vs. cancer cells) compound, many studies focus on its antioxidant capacity as well as on its ability to trigger and favor the apoptotic cascade in malignant cells. However, a generalized mechanism of action able to explain this dual effect of resveratrol has not yet been clearly established. In addition to these important functions, resveratrol is reported to exhibit several other biological/biochemical protective effects on heart, circulation, brain and age-related diseases which are summarized in this Review.

Quercetin: A flavonol with multifaceted therapeutic applications?

Great interest is currently centered on the biologic activities of quercetin a polyphenol belonging to the class of flavonoids, natural products well known for their beneficial effects on health, long before their biochemical characterization. In particular, quercetin is categorized as a flavonol, one of the five subclasses of flavonoid compounds. Although flavonoids occur as either glycosides (with attached glycosyl groups) or as aglycones, most altogether of the dietary intake concerning quercetin is in the glycoside form. Following chewing, digestion, and absorption sugar moieties can be released from quercetin glycosides. Several organs contribute to quercetin metabolism, including the small intestine, the kidneys, the large intestine, and the liver, giving rise to glucuronidated, methylated, and sulfated forms of quercetin; moreover, free quercetin (such as aglycone) is also found in plasma. Quercetin is now largely utilized as a nutritional supplement and as a phytochemical remedy for a variety of diseases like diabetes/obesity and circulatory dysfunction, including inflammation as well as mood disorders. Owing to its basic chemical structure themost obvious feature of quercetin is its strong antioxidant activity which potentially enables it to quench free radicals from forming resonance-stabilized phenoxyl radicals. In this review the molecular, cellular, and functional bases of therapy will be emphasized taking strictly into account data appearing in the peer-reviewed literature and summarizing the main therapeutic applications of quercetin; furthermore, the drug metabolism and the main drug interaction as well as the potential toxicity will be also spotlighted.

Pycnogenol: A blend of procyanidins with multifaceted therapeutic applications?

Great interest is currently centred on the biologic activities of pycnogenol a standardized plant extract obtained from the bark of the French maritime pine Pinus pinaster (formerly known as Pinus maritima), Aiton, subspecies Atlantica des Villar (Pycnogenol, Horphag Research Ltd., UK, Geneve, Switzerland), which grows in the coastal southwest France. The quality of this extract is specified in the United States Pharmacopeia (USP 28). Between 65% and 75% of Pycnogenol are procyanidins comprising of catechin and epicatechin subunits with varying chain lengths. Other constituents are polyphenolic monomers, phenolic or cinnamic acids and their glycosides. As many studies indicate, pycnogenol components are highly bioavailable. Uniquely, pycnogenol displays greater biologic effects as a mixture than its purified components do individually indicating that the components interact synergistically. Pycnogenol is now utilized throughout the world as a nutritional supplement and as a phytochemical remedy for various diseases ranging from chronic inflammation to circulatory dysfunction, including several impaired psycho-physiological functions. Owing to the basic chemical structure of its components, the most obvious feature of pycnogenol is its strong antioxidant activity. In fact, phenolic acids, polyphenols, and in particular flavonoids, are composed of one (or more) aromatic rings bearing one or more hydroxyl groups and are therefore potentially able to quench free radicals by forming resonance-stabilized phenoxyl radicals. In this review, emphasizing the molecular, cellular, and functional bases of therapy, data appearing in the peer-reviewed literature and focussing the main therapeutic applications of pycnogenol will be summarized and critically evaluated.

Temperature, pH and UV irradiation effects on ascorbate oxidase

The activity of ascorbate oxidase (AO) purified from Cucurbita pepo medullosa was assayed after incubation at different temperatures. The optimum temperature was 35°; activity was completely lost after 4 min at 65°. The half-time of denaturation at 55° was 21.5 min. The activation energy for the AO reaction was 10 kJmol−1. A bell-shaped pH profile of the enzymatic activity was obtained with a pH optimum of 6. The enzyme was fairly stable in the pH range 4–10. A decrease of enzymatic activity was observed upon exposure of AO to UV irradiation (t12 of inactivation, 8.5 min). The presence of 0.1 M mannitol made the enzyme more prone to UV damage (t12 of inactivation, 6.5 min). Exposure of both native and copper-free AO to UV light resulted in a decrease of intrinsic fluorescence which was larger in the presence of 0.1 M mannitol. In both cases copper-free AO was more sensitive to photoinduced damage. Under anaerobic conditions the loss of intrinsic flu orescence was lower.

AZT: An Old Drug With New Perspectives

The science of antiviral research was well advanced when HIV/AIDS appeared as a major new virus disease in the early 1980s. The first effective antiviral compound (AZT, azidothymidine, zidovudine) was already among the library of compounds screened and was promptly reported to be a specific inhibitor of retroviruses, including HIV. Due to the pivotal role of AZT in HIV treatment, this review summarizes the most known effects -some of which are toxic side effects- induced by AZT a drug which is still used in the combined therapy of HIV-infected patients. Among the toxic side effects, a severe bone marrow toxicity manifested as anemia, neutropenia and siderosis, and caused by inhibition of heme and globin synthesis together with a general derangement of iron supply, have been reported. In this regard, we proved that while AZT and its monophosphorylated derivative AZTMP were unable to chelate iron, the triphosphate form AZTTP displayed a significant capacity to remove iron from transferrin. Moreover, we have previously demonstrated that AZT-exposed K562 cells showed an increase of transferrin receptors located on the cell membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. Interestingly, literature data report the impairement of glycosylation reactions by AZT. Indeed, we have shown that AZT-treated K562 cells exhibited a reduced sialylation of proteins and lipids, and a strong inhibition of alpha,(2-->8) sialyltransferase activity while beta,(1-->4)galactosyltransferase and beta-galactosidase activities were significantly increased. These latter observations could be of clinical relevance since alterations of intracellular and cell surface carbohydrate expression and composition, often are associated with several diseases. However, contrarily to previous reports by other authors on AZT as an inhibitor of plant and bacterial toxins activity, we have demonstrated that AZT not only did not inhibit saporin toxicity, but even increased the cytotoxic activity of this plant toxin on K562 cells. Furthermore, the review enlightens the potential utilization of AZT as a tool in proteomics since in the recent years several genes responding to this drug have been identified in different cell lines. We have shown, for the first time, an over-expression of two proteins (PDI-A3 and sthatmin), and a full repression of two others (HSP-60 and SOD1) in AZT-exposed K562 cells. At present, we are investigating if the above reported alterations are a general feature of AZT-treatment of cultured cells, or they represent a peculiar characteristic of a specific cell line. Finally, the paper reviews a number of novel methodologies aimed at enhancing the AZT plasma levels and its bioavailability in all human organs in order to improve its therapeutic efficacy against HIV infection. These new possibilities, namely the AZT prodrug strategy, the AZT transdermal delivery and the targeted brain delivery, are yet not in use for humans but they are under experimental studies.

Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium.

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 microM) or riboflavin (500 microM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.

Peroxidase-labelling of human serum transferrin by conjugation to oligosaccharide moieties

The generation of reactive aldehydes on the carbohydrate moieties of the human serum transferrin was performed by a derivatization procedure based on the mild oxidation with sodium periodate and subsequent reaction with peroxidase hydrazide. The synthesized conjugate was compared to that obtained by modification of the amino acid side chains of transferrin. The conjugate reaction mixture assayed by SDS-PAGE consisted, besides unreacted compounds, of three main bands, corresponding to a molar ratio transferrin:peroxidase of 1:1, 1:2, 1:3. After blotting, these bands were identified by either anti-peroxidase and anti-transferrin antibodies on nitrocellulose membrane. ELISA detection method showed that the conjugate via oligosaccharide moieties (glycans) was still recognized not only by the anti-transferrin antibodies but also by the specific cellular receptor, while the conjugate via amino acids failed to display this latter ability. The different behaviour can be probably due to a significant damage of the protein structure or, possibly, to the peroxidase binding at sites recognized by the receptor. The results reported here indicate that the conjugation procedure through glycans leads to stable and selected transferrin-conjugates fully exhibiting their biological activity.

Effects of AZT on cellular iron homeostasis

Abstract3′-azido-3′-deoxythymidine (AZT), the first chemotherapeutic drug approved by FDA for treatment of HIV-infected patients and still used in combination therapy, has been shown to induce, upon prolonged exposure, severe bone marrow toxicity manifested as anemia, neutropenia and siderosis. These toxic effects are caused by inhibition of heme synthesis and, as a consequence, transferrin receptor (TfR) number appears increased and so iron taken up by cells. Since iron overload can promote the frequency and severity of many infections, siderosis is viewed as a further burden for AIDS patients. We have previously demonstrated that AZT-treated K562 cells showed an increase of the number of TfRs located on the surface of the plasma membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. In spite of the higher number of receptors on the plasma-membrane of AZT-treated cells, intracellular accumulation of iron showed a similar level in control and in drug-exposed cells. The chelating ability of AZT and of its phosphorylated derivatives, both in an acellular system and in K562 cells, was also checked. The results demonstrated that AZT and AZTMP were uneffective as iron chelators, while AZTTP displayed a significant capacity to remove iron from transferrin (Tf). Our results suggest that AZT may be not directly involved in the iron overloading observed upon its prolonged use in AIDS therapy. The iron accumulation found in these patients is instead caused by other unknown mechanisms that need further studies to be clarified.

Protein glycans alteration and a different distribution of some enzymatic activities involved in the glycan processing are found in AZT-treated K562 cells

AbstractIn this paper we report that 3