Dietary polyphenols: focus on resveratrol, a promising agent in the prevention of cardiovascular diseases and control of glucose homeostasis.

Plants continuously produce an extraordinary variety of biologically active low-molecular-mass compounds. Among them, resveratrol (3,5,4'-trihydroxystilbene) is endowed with significant positive activities by protecting against cardiovascular diseases and preventing the development and progression of atherosclerosis. Furthermore, the molecule significantly ameliorates glucose homeostasis in obese mice. These beneficial effects have driven considerable interest towards resveratrol molecular activities, and intensive efforts for the identification of the stilbene targets have been made. The molecule shows a pleiotropic mode of action. Particularly, its cellular targets are crucial for cell proliferation and differentiation, apoptosis, antioxidant defence and mitochondrial energy production. The complexity of resveratrol activities might account for its effectiveness in ameliorating multifactorial processes, including the onset and/or progression of several degenerative diseases such as myocardial infarction, atherosclerosis and type 2 diabetes. This article reports the actions of resveratrol on cardiovascular diseases and the molecular bases of its activity. We also discuss recent data on the effect of resveratrol on glucose homeostasis and obesity. Finally, the relevance of the stilbene use in the development of new pharmacological strategies is evaluated.

A novel Leu153Ser mutation of the Fanconi anemia FANCD2 gene is associated with severe chemotherapy toxicity in a pediatric T-cell acute lymphoblastic leukemia.

Fanconi anemia (FA) is an autosomal recessive disease characterized by pancitopenia, congenital malformations, predisposition to cancers and chromosomal instability. We report the clinical and molecular features of a patient initially identified as a potential FA case only because of chemotherapy toxicity during the treatment of a T-lineage acute lymphoblastic leukemia (ALL). Cells from this patient showed a moderate chromosomal instability, increasing sensitivity to DNA crosslinking agents but normal response to ionizing radiation. The analysis of FA proteins demonstrated a marked reduction of FANCD2 (>95%), but normal levels of FANCA or FANCG. Interestingly, this defect was associated with a homozygous missense mutation of FANCD2, resulting in a novel amino-acid substitution (Leu153Ser) at residue Leu153, which is highly conserved through evolution. The FANCD2(L153S) protein, whose reduced expression was not due to impaired transcription, was detected also in its monoubiquitinated form in the nucleus, suggesting that the mutation does not affect post-translation modifications or subcellular localization but rather the stability of FANCD2. Therefore, the hypomorphic Leu153Ser mutation represents the first example of a FANCD2 defect that might promote clonal progression of tumors, such as T-ALL, and severe chemotherapy toxicity in patients without any clinical manifestations typical of FA.

Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis.

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells.

3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated.

Pyrrolidine dithiocarbamate induces apoptosis by a cytochrome c-dependent mechanism.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 microM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. Programmed cell death was demonstrated by biochemical analyses, including the activation of procaspase 3 and the cleavage of poly(ADP-ribose) polymerase (PARP). PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21(CIP1) level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.

Olive oil hydroxytyrosol protects human erythrocytes against oxidative damages.

Hydroxytyrosol, the major representative phenolic compound of virgin olive oil, is a dietary component. Its possible protective effect on hydrogen peroxide (H(2)O(2))-induced oxidative alterations was investigated in human erythrocytes. Cells were pretreated with micromolar hydroxytyrosol concentrations and then exposed to H(2)O(2) over different time intervals. Subsequently, erythrocytes were analyzed for oxidative hemolysis and lipid peroxidation. Our data demonstrate that hydroxytyrosol prevents both oxidative alterations, therefore, providing protection against peroxide-induced cytotoxicity in erythrocytes. The effect of oxidative stress on erythrocyte membrane transport systems, as well as the protective role of hydroxytyrosol, also were investigated in conditions of nonhemolytic mild H(2)O(2) treatment. Under these experimental conditions, a marked decrease in the energy-dependent methionine and leucine transport is observable; this alteration is quantitatively prevented by hydroxytyrosol pretreatment. On the other hand, the energy-independent glucose transport is not affected by the oxidative treatment. The reported data give new experimental support to the hypothesis of a protective role played by nonvitamin antioxidant components of virgin olive oil on oxidative stress in human systems.

Resveratrol arrests the cell division cycle at S/G2 phase transition.

Resveratrol (3,5,4'-trihydroxystilbene) is a naturally occurring phytoalexin, found in grapes and wine, which has been reported to exert a variety of important pharmacological effects. We have investigated the activity of resveratrol on proliferation and differentiation of the promyelocitic cell line HL-60. A concentration as low as 30 microM causes a complete arrest of proliferation and a rapid induction of differentiation towards a myelo-monocytic phenotype. Analyses by flow cytometry showed the absence of the G2/M peak and the accumulation of cells in G1 and S phases. Moreover, at the concentrations employed, a very low amount of apoptotic cells was evidenced. A detailed biochemical analysis demonstrated that the G1 phase of the cell division cycle engine was completely unmodified by resveratrol addition, thus indicating that the G1 --> S transition occurs normally. Conversely, after only 24 h treatment, a significant increase of cyclins A and E could be observed along with the accumulation of cdc2 in the inactive phosphorylated form. These data demonstrate that resveratrol causes a complete and reversible cell cycle arrest at the S phase checkpoint.

The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells.

We investigated the injurious effects of reactive oxygen metabolites on the intestinal epithelium and the possible protective role played by two olive oil phenolic compounds, (3,4-dihydroxyphenyl)ethanol and (p-hydroxyphenyl)ethanol, using the Caco-2 human cell line. We induced oxidative stress in the apical compartment, either by the addition of 10 mmol/L H2O2 or by the action of 10 U/L xanthine oxidase in the presence of xanthine (250 micromol/L); after the incubation, we evaluated the cellular and molecular alterations. Both treatments produced significant decreases in Caco-2 viability as assessed by the neutral red assay. Furthermore, we observed a significant increase in malondialdehyde intracellular concentration and paracellular inulin transport, indicating the occurrence of lipid peroxidation and monolayer permeability changes, respectively. The H2O2-induced alterations were completely prevented by preincubating Caco-2 cells with (3,4-dihydroxyphenyl)ethanol (250 micromol/L); when the oxidative stress was induced by xanthine oxidase, complete protection was obtained at a concentration of polyphenol as small as 100 micromol/L. In contrast, (p-hydroxyphenyl)ethanol was ineffective up to a concentration of 500 micromol/L. Our data demonstrate that (3,4-dihydroxyphenyl)ethanol can act as a biological antioxidant in a cell culture experimental model and that the ortho-dihydroxy moiety of the molecule is essential for antioxidant activity. This study suggests that dietary intake of olive oil polyphenols may lower the risk of reactive oxygen metabolite-mediated diseases such as some gastrointestinal diseases and atherosclerosis.

Effect of an oxidative stress on methionine and S-adenosylmethionine metabolism in cultured bovine eye lens.

The sulphonium compound [Formula: see text] (AdoMet) plays a central role in many metabolic reactions of cellular metabolism, acting both as a propylamine donor in the biosynthesis of polyamines as well as a methyl donor in the transmethylation reactions. Moreover, AdoMet is a key intermediate of the transsulphuration pathway by which methionine is converted into cysteine, a precursor of glutathione. The aim of this study was to investigate the methionine and AdoMet metabolism in bovine lenses cultured in the presence of labelled methionine, upon treatment with H(2)O(2), as the experimental model for studying the molecular mechanisms responsible for the onset of senile cataract. The results reveal that one of the earliest changes following an oxidative stress is a severe impairment of protein synthesis. As far as the synthesis of AdoMet is concerned, a small but significant decrease in the conversion of labelled methionine into AdoMet occurs in treated lenses compared to the controls. In order to verify if the decreased AdoMet synthesis would lead in turn to alterations of methyl transfer reactions, we examined changes in the levels of various macromolecular methylations, such as protein methyl esterification and phospholipid methylation. The data clearly indicate that both the synthesis of AdoMet and the methyl transfer reactions could be significantly affected in eye lens upon an oxidative stress, suggesting that these alterations could be one of the biochemical events related to the ethiology of senile cataract. Finally, the question of whether or not H(2)O(2)-induced alterations of methionine and AdoMet metabolism could, in turn, affect some closely related metabolism, such as glutathione-associated reactions, is also discussed.