Stéphanie Krisa

Distribution of [14C]-trans-resveratrol, a cancer chemopreventive polyphenol, in mouse tissues after oral administration

Trans-resveratrol, a phenolic compound present in wine, has been reported to be a potential cancer chemopreventive agent. However, although it has numerous biological activities in vitro, there are few data about its bioavailability and tissue distribution in vivo. The objectives of this study were to investigate the absorption and tissue distribution of 14C-trans-resveratrol following oral administration to mice. Male Balb/c mice were given a single oral dose of 14C-trans-resveratrol and were sacrificed at 1.5, 3 or 6 h postdose. The distribution of radioactivity in tissues was evaluated using whole-body autoradiography, quantitative organ-level determination and microautoradiography. In addition, identification of radioactive compounds in kidney and liver was done with high-performance liquid chromatography. Autoradiographic survey of mice sections as well as radioactivity quantification in various organs revealed a preferential fixation of 14C-trans-resveratrol in the organs and biological liquids of absorption and elimination (stomach, liver, kidney, intestine, bile, urine). Moreover, we show that 14C-trans-resveratrol derived radioactivity is able to penetrate the tissues of liver and kidney, a finding supported by microautoradiography. The presence of intact 14C-trans-resveratrol together with glucurono- and/or sulfoconjugates in these tissues was also shown. This study demonstrates that trans-resveratrol is bioavailable following oral administration and remains mostly in intact form. The results also suggest a wide range of target organs for cancer chemoprevention by wine polyphenols in humans.

Integrin αVβ3 contains a receptor site for resveratrol

Resveratrol is a naturally occurring polyphenol, which causes apoptosis in cultured cancer cells. We describe a cell surface resveratrol receptor on the extracellular domain of hetero‐dimeric αVβ3 integrin in MCF‐7 human breast cancer cells. This receptor is linked to induction by resveratrol of extracellular‐regulated kinases 1 and 2 (ERK1/2)‐ and serine‐15‐p53‐dependent phosphorylation leading to apoptosis. The integrin receptor is near the Arg‐Gly‐Asp (RGD) recognition site on the integrin; an integrin‐binding RGD peptide inhibits induction by resveratrol of ERK1/2‐and p53‐dependent apoptosis. Antibody (Ab) to integrin αVβ3, but not to αVβ5, inhibits activation by resveratrol of ERK1/2 and p53 and consequent apoptosis in estrogen receptor‐α (ERα) positive MCF‐7, and ERα‐negative MDA‐MB231 cells. Resveratrol is displaced from the purified integrin by an RGD, but not RGE, peptide, and by αVβ3 integrin‐specific Ab. Resveratrol action is blocked by siRNAβ3, but not by siRNAαV. [14C]‐Resveratrol binds to commercially purified integrin αVβ3 and to αVβ3 prepared from MCF‐7 cells;binding of [14C]‐resveratrol to the β3, but not to the αV monomer, is displaced by unlabeled resveratrol. In conclusion, binding of resveratrol to integrin αVβ3, principally to the β3 monomer, is essential for transduction of the stilbene signal into p53‐dependent apoptosis of breast cancer cells.—Lin, H.‐Y., Lansing, L., Merillon, J.‐M., Davis, F. B., Tang, H.‐Y., Shih, A., Vitrac, X., Krisa, S., Keating, T., Cao, H. J., Bergh, J., Quackenbush, S., Davis, P. J. Integrin αVβ3 contains a receptor site for resveratrol. FASEB J. 20, E1133–E1138 (2006)

Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars

Abstract Sucrose was found to modulate polyphenol accumulation in Vitis vinifera cell cultures. The production of anthocyanins increased 12-fold after addition of 0.15 m sucrose, while that of stilbenes was only slightly affected. Sucrose did not play a physical role because metabolic sugars were required for the induction of polyphenol accumulation. Indeed, the polyols, mannitol and sorbitol, had no effect on this accumulation. We established a model system to investigate the mechanism of sucrose regulation of polyphenol production without inhibition of grape cell growth. After addition of sucrose to the culture medium, the major sugars accumulated in grape cells were glucose and fructose, reaching 40% of the dry weight. The increase in the level of these hexoses closely coincided with the increase in anthocyanin accumulation in grape cells.

Production of 13C-labelled anthocyanins by Vitis vinifera cell suspension cultures

The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported. Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium. Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside. Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e. at the beginning of the exponential growth phase. After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated. Incorporation of [1-13C]-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum. The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65%. The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.

Protective effect of ε-viniferin on β-amyloid peptide aggregation investigated by electrospray ionization mass spectrometry

Abnormal β-amyloid peptide accumulation and aggregation is considered to be responsible for the formation and cerebral deposition of senile plaques in the brains of patients with Alzheimers disease (AD). Inhibition of the formation of β-amyloid (Aβ) fibrils would be an attractive therapeutic target for the treatment of AD. Resveratrol and its derivatives exhibit a broad range of pharmacological properties such as protection against cardiovascular diseases and cancers, as well as promoting antiaging effects. We reported previously that ε-viniferin glucoside (VG), a resveratrol-derived dimer, strongly inhibits Aβ (25-35) fibril formation in vitro. In this study, we investigated the effects of VG on the aggregation of the full-length peptides (Aβ (1-40) and Aβ (1-42)) and on the β-amyloid-induced toxicity in PC12 cells. VG inhibited Aβ cytotoxicity and the non-covalent complex between VG and Aβ was observed by electrospray ionization mass spectrometry.

Radiolabelled cyanidin 3-O-glucoside is poorly absorbed in the mouse.

Anthocyanins are natural pigments abundant in various fruits and berries that are involved in the prevention of various chronic diseases. Their low concentrations in plasma and urine are explained in part by their complex chemistry and the formation of still uncharacterised metabolites. The aim of the present study was to follow the distribution of anthocyanins in the body using 14C-labelled cyanidin 3-O-glucoside (Cy3G) fed by gavage to mice. After the administration of 22.2 kBq 14C-Cy3G (0.93 mg), radioactivity was detected in most organs tested over the following 24 h with a peak observed in inner tissues at 3 h. The major fraction of the radioactivity (44.5 %) was found in the faeces collected 24 h after ingestion. At 3 h after oral administration of 141 kBq 14C-Cy3G (4.76 mg), most of the radioactivity (87.9 % of intake) was recovered in the gastrointestinal (GI) tract, especially in the small intestine (50.7 %) and the caecum (23 %). At this time, 3.3 % of the radioactivity was detected in urine. There was minimal accumulation (0.76 %) of radioactivity in tissues outside the GI tract. Distribution of radioactivity varied among organs, with liver, gallbladder and kidneys showing the highest radioactivity. Taken as a whole, these results show that Cy3G is poorly absorbed in the mouse.

Carbon-14 biolabelling of wine polyphenols in Vitis vinifera cell suspension cultures

14C-L-phenylalanine is incorporated into a range of polyphenolic compounds when fed to grape cell cultures. Optimisation of several parameters such as the quantity of precursor applied and the duration of metabolism led to incorporation yields of 15% and to specific activities of 875 mu Ci g(-1) in stilbenes. Purification of the products by several chromatographic steps is reported. Both trans- and cis-resveratrols were easily obtained by enzymatic hydrolysis of their corresponding glucosides, with specific activity of 1200-1400 mu Ci g(-1). The specific radioactivity obtained for all the compounds is suitable for in vivo feeding trials to trace their metabolic fate when consumed by animals and for in vitro activity mechanism studies. Indeed, these polyphenols seem to be implicated in the health benefits associated with regular and moderate wine consumption but little is known about their pharmacokinetics and cellular uptake.

Relaxation induced by red wine polyphenolic compounds in rat pulmonary arteries: lack of inhibition by NO‐synthase inhibitor

Some red wine polyphenols exert nitric oxide (NO)‐dependent relaxation in systemic arteries, following activation of endothelial NO synthase (eNOS). In this study, the effect of red wine polyphenols was determined in rat intrapulmonary arteries, and the effect of some of these compounds was compared with the responses obtained in rat aorta. In pulmonary arteries, red wine polyphenolic extract (> 300 μg/mL) exerted relaxation that was not inhibited by the NOS inhibitor Nω‐nitro‐l‐arginine methylester (l‐NAME) or endothelium removal. Among the several fractions obtained from the extract, the one enriched with anthocyanins was less active than fractions containing non‐anthocyanins. Among the latter, the most active for relaxing pulmonary arteries was the one enriched in the stilbene derivative trans‐resveratrol (relaxation for concentration >10 μg/mL). Trans‐piceid, the glucoside derivative of trans‐resveratrol, was almost inactive. Trans‐resveratrol‐induced relaxation, as well as relaxation to the anthocyanin delphinidin, was l‐NAME‐insensitive in pulmonary arteries. In aorta, trans‐resveratrol and trans‐piceid exerted similar effects to those in pulmonary arteries that were also not inhibited by l‐NAME. However, red wine polyphenolic extract and delphinidin induced relaxation of aorta at much lower concentrations (about 10 μg/mL) than in pulmonary arteries, and their effects were inhibited by l‐NAME. These data show differences between small intrapulmonary arteries and systemic conductance arteries in their responses to red wine polyphenols, the major difference being that the relaxant effect of these compounds is not blunted by NOS inhibitor in pulmonary arteries. They suggest that red wine polyphenols act directly on smooth muscle to promote pulmonary artery relaxation.

Phenolic contents and bioactive potential of peach fruit extracts.

Several cultivars of peach fruit (Prunus persica L.) were investigated. Their phenolic composition and concentration were assessed by LC-MS. Concentrations were calculated in mg per g of dry weight extract. Their antioxidant capacity (Folin-Ciocalteu, ORAC, DPPH, ABTS, PFRAP and ICA), inhibitory property against β-amyloid and α-synuclein fibril formation and protective capacity against Aβ-induced toxicity on PC12 cell lines (viability assessed by MTT assay and intracellular ROS production by DCFH-DA assay) were evaluated. Fifteen different phenolic compounds were identified and quantified. In particular, new isorhamnetin derivatives were identified. Phenolic contents were ranged between 19 and 82mg/g. Spring Belle extract had the highest content and Romea the lowest. Except for the ICA assay, a good correlation between phenolic content and the antioxidant capacities of peach fruit extracts was found, indicating that phenolic compounds are major contributors to their antioxidant capacity. Results indicate that the phenolic extract of peach cultivars inhibits Aβ and αS fibril formation and protects PC12 cell lines against Aβ-induced toxicity.

Inhibitory activity of plant stilbenoids against nitric oxide production by lipopolysaccharide-activated microglia.

Microglia-driven inflammatory processes are thought to play an important role in ageing and several neurological disorders. Since consumption of a diet rich in polyphenols has been associated with anti-inflammatory and neuroprotective effects, we studied the effects of twenty-five stilbenoids isolated from Milicia excelsa, Morus alba, Gnetum africanum, and Vitis vinifera. These compounds were tested at 5 and 10 µM on BV-2 microglial cells stimulated with bacterial lipopolysaccharide. Ten stilbenoids reduced lipopolysaccharide-induced nitric oxide production at 5 and/or 10 µM. Two tetramers, E-vitisin A and E-vitisin B, were the most effective molecules. Moreover, they attenuated the expression of the inducible NO synthase protein and gene.