Resveratrol (3,4,5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antioxidant and antiproliferative activities. In this study we have investigated whether these properties are dependent on similar or different structural determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each single hydroxylic function were selectively substituted with methyl groups, were synthesized. Analogues with the stilbenic double bond reduced or with the stereoisomery modified were also investigated. The antioxidant activity of these compounds was evaluated by measuring the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. In addition, the protection against lipid peroxidation was determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and DNA synthesis. The results showed that the hydroxyl group in 4 position is not the sole determinant for antioxidant activity. In contrast, the presence of 4-OH together with stereoisomery in the trans-conformation (4-hydroxystyryl moiety) was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases ␣ and ␦.

Key words: inositol 1,4,5-tris-phosphate kinase, low phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP 6 ), myo-inositol-phosphate monophosphatase (IMP), myo-inositol-3-phosphate synthase (MIPS), multidrug resistance protein (MRP) type ATP-binding cassette (ABC) transporter, Phaseolus vulgaris (common bean). Summary• We previously identified the lpa1 (low phytic acid) 280-10 line that carries a mutation conferring a 90% reduction in phytic acid (InsP 6 ) content. In contrast to other lpa mutants, lpa1(280-10) does not display negative pleiotropic effects. In the present paper, we have identified the mutated gene and analysed its impact on the phytic acid pathway.• Here, we mapped the lpa1(280-10) mutation by bulk analysis on a segregating F 2 population, an then, by comparison with the soybean genome, we identified and sequenced a candidate gene. The InsP 6 pathway was analysed by gene expression and quantification of metabolites.• The mutated Pvmrp1(280-10) cosegregates with the lpa1(280-10) mutation, and the expression level of several genes of the InsP 6 pathway are reduced in the lpa1(280-10) mutant as well as the inositol and raffinosaccharide content. PvMrp2, a very similar paralogue of PvMrp1 was also mapped and sequenced.• The lpa1 mutation in beans is likely the result of a defective Mrp1 gene (orthologous to the lpa genes AtMRP5 and ZmMRP4), while its Mrp2 paralog is not able to complement the mutant phenotype in the seed. This mutation appears to down-regulate the InsP 6 pathway at the transcriptional level, as well as altering inositol-related metabolism and affecting ABA sensitivity.Abbreviations: ABC, ATP-binding cassette; MRP, multidrug resistance-associated protein; InsP 6, myo-inositol-1,2,3,4,5,6-hexakisphosphate; IMP, myo-inositolphosphate monophosphatase; MIPS, myo-inositol-3-phosphate synthase; MIK, myo-inositol kinase; IPK2, inositol 1,4,5-tris-phosphate kinase; ITPK, inositol 1,3,4-triphosphate 5 ⁄ 6-kinase; IPK1, inositol 1,3,4,5,6 pentakisphosphate 2-kinase.

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Resveratrol (3,4 ,5-trihydroxy-trans-stilbene) is a phytoalexin found in grapes that has anti-inflammatory, cardiovascular protective, and cancer chemopreventive properties. It has been shown to target prostaglandin H 2 synthase (COX)-1 and COX-2, which catalyze the first committed step in the synthesis of prostaglandins via sequential cyclooxygenase and peroxidase reactions. Resveratrol discriminates between both COX isoforms. It is a potent inhibitor of both catalytic activities of COX-1, the desired drug target for the prevention of cardiovascular disease, but only a weak inhibitor of the peroxidase activity of COX-2, the isoform target for nonsteroidal anti-inflammatory drugs. We have investigated the unique inhibitory properties of resveratrol. We find that it is a potent peroxidase-mediated mechanism-based inactivator of COX-1 only (k inact ‫؍‬ 0.069 ؎ 0.004 s ؊1 , K i(inact) ‫؍‬ 1.52 ؎ 0.15 M), with a calculated partition ratio of 22. Inactivation of COX-1 was time-and concentration-dependent, it had an absolute requirement for a peroxide substrate, and it was accompanied by a concomitant oxidation of resveratrol. Resveratrolinactivated COX-1 was devoid of both the cyclooxygenase and peroxidase activities, neither of which could be restored upon gel-filtration chromatography. Inactivation of COX-1 by [ 3 H]resveratrol was not accompanied by stable covalent modification as evident by both SDS-PAGE and reverse phase-high performance liquid chromatography analysis. Structure activity relationships on methoxy-resveratrol analogs showed that the m-hydroquinone moiety was essential for irreversible inactivation of COX-1. We propose that resveratrol inactivates COX-1 by a "hit-and-run" mechanism, and offers a basis for the design of selective COX-1 inactivators that work through a mechanism-based event at the peroxidase active site.

Neovascularization plays an important role in neoplasia and angioproliferative diseases. Two major modalities have been developed so far to affect neovascularization: its prevention by antiangiogenic compounds, and immature vessel disruption by vascular-targeting agents. trans-Resveratrol, found in grapes and wine, exerts antioxidant, antineoplastic, and antiangiogenic activities. Here, among various synthetic trans-resveratrol derivatives tested, 3,5,4Ј-trimethoxystilbene was an antiangiogenic agent 30 to 100 times more potent than parent compound in inhibiting endothelial cell proliferation, sprouting, collagen gel invasion, and morphogenesis (ID 50 ϭ 0.3-3.0 M). In addition, 3,5,4Ј-trimethoxystilbene acts as a vascular-targeting agent by causing microtubule disassembling and tubulin depolymerization and by impairing the repositioning of the microtubule organization center and the formation of membrane ruffles in migrating endothelial cells. In keeping with a vascular-targeting ability, 3,5,4Ј-trimethoxystilbene induced apoptosis only in subconfluent endothelial cells and apoptotic regression of immature vessels in the ex vivo rat aorta ring assay. In vivo, 3,5,4Ј-trimethoxystilbene caused the rapid stasis of blood flow and regression of intersegmental vessels in the trunk of zebrafish embryos. In addition, it inhibited blood vessel growth and caused the disappearance of pre-existing blood vessels in the area vasculosa of the chick embryo. In conclusion, 3,5,4Ј-trimethoxystilbene associates an antiangiogenic profile to a significant vascular-targeting activity.