To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.

High intake of natural antioxidants (NA) from plant-derived foods and beverages is thought to provide cardiovascular benefits. The endothelium plays a pivotal role in cardiovascular homeostasis, and for this reason, the molecular events resulting from NA actions on endothelial cells (ECs) are actively investigated. Here, we show the direct impact of two NA, coumaric acid and resveratrol, on intracellular reactive oxygen species levels, protein carbonylation, and cell physiology in human ECs. While at lower doses, both NA promoted antioxidant effects, at moderately high doses, NA elicited a dose-dependent pro-oxidant effect, which was followed by apoptosis, cell damage, and phospho-Akt downregulation. NA-induced pro-oxidant effects were counteracted by N-acetyl cysteine and diphenyleneiodonium (DPI), suggesting a role for flavin oxidases in NA-induced toxicity. DPI also prevented NA-induced phospho-Akt downregulation indicating that Akt can work downstream of flavin oxidases in mediating cellular responses to NA. Stimulation of phospho-Akt by insulin dramatically counteracted NA-induced cell death, an effect abolished by Akt inhibition further suggesting that mechanistically Akt regulates cell survival in response to NA-induced stress. Although further studies are required to better characterize the molecular mechanism of NA-induced cell toxicity, our study is the first to show in a human vascular model that moderately high doses of NA can induce cell damage mediated by flavoproteins and the Akt pathway.

Polyamines appear to inhibit peroxidation of vesicles containing acidic phospholipids. A correlation exists between polyamine binding to phospholipid vesicles and its protective effect. However, phosphatidylinositol-containing vesicles which bind spermine are not protected by the polyamine [Tadolini, Cabrini, Landi, Varani & Pasquali (1985) Biogenic Amines 3, 97-106]. In the present paper I tested the hypothesis that polyamines, in particular spermine, by forming a ternary complex with iron and the phospholipid polar head may change the susceptibility of Fe2+ to autoxidation and thus its ability to generate free oxygen radicals. Different compounds mimicking phospholipid polar heads were studied, namely AMP, mimicking phosphatidic acid, CDP-choline, mimicking phosphatidylcholine, and glycerophosphoinositol, mimicking phosphatidylinositol. The results support the proposed hypothesis. In the presence of CDP-choline or of glycerophosphoinositol, spermine poorly affects Fe2+ autoxidation, whereas a considerable inhibition is observed in the presence of AMP. The ability of other phosphorus-containing compounds (ATP, ADP, cyclic AMP, sodium phosphate) to affect Fe2+ autoxidation in the presence of polyamines was also evaluated to understand the molecular mechanism of this phenomenon. It is proposed that polyamines may be part of the passive cellular defence mechanism against the oxidative damage caused by Fe2+.

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

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