Procyanidins can interact with Caco-2 cell membrane lipid rafts: Involvement of cholesterol

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol–Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex–cholesterol bondings. Procyanidin–lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.     

Flavan-3-ols and procyanidins protect liposomes against lipid oxidation and disruption of the bilayer structure

The antioxidant activity and the membrane effects of the flavanols (-)-epicatechin, (+)-catechin, and their related oligomers, the procyanidins, were evaluated in liposomes composed by phosphatidylcholine:phosphatidylserine (60:40, molar ratio). When liposomes were oxidized with a steady source of free radicals, the flavanols and procyanidins (25 microM monomer equivalents) inhibited oxidation in a manner that was related to procyanidin chain length. Flavanols and procyanidins did not influence membrane fluidity or lipid lateral phase separation. However, flavanols and procyanidins induced a decrease in the membrane surface potential and protected membranes from detergent-induced disruption. These effects were dependent on flavonoid concentration, procyanidin chain length, and membrane composition. Flavanol- and procyanidin-induced inhibition of lipid oxidation was correlated with their effect on membrane surface potential and integrity. These results indicate that the interaction of flavanols and procyanidins with phospholipid head groups, particularly with those containing hydroxyl groups, is associated with a reduced rate of membrane lipid oxidation. Thus, flavanols and procyanidins can potentially reduce oxidative modifications of membranes by restraining the access of oxidants to the bilayer and the propagation of lipid oxidation in the hydrophobic membrane matrix.     

Flavanols: digestion,absorption and bioactivity

Flavanols, or flavan-3-ols, are a family of bioactive compounds present in cocoa, red wine, green tea, red grapes, berries and apples. With a basic monomer unit of (−)-epicatechin or (+)-catechin, flavanols can be present in foods and beverages as monomers or oligomers (procyanidins). Most, but not all, procyanidins are degraded into monomer or dimer units prior to absorption. The bioavailability of flavanols can be influenced by multiple factors, including food processing, cooking, digestion, and biotransformation. Flavanols are potent antioxidants, scavenging free radicals in vitro and in vivo. While some of the actions of flavanols can be linked to antioxidant activities, other modes of action may also occur, including modulation of intracellular signaling, effects on membrane fluidity and regulation of cytokine release or action. Physiologically, flavanol-rich foods and beverages can affect platelet aggregation, vascular inflammation, endothelial nitric oxide metabolism, and may confer protective effects against neurodegeneration. Epidemiological data suggests that intake of cocoa, a rich source of flavanols, is inversely associated with 15-year cardiovascular and all-cause mortality in older males. (−)-Epicatechin and its metabolite, epicatechin-7-O-glucuronide, have been identified as independent predictors of some of the vascular effects associated with the consumption of a flavanol-rich beverage. Targeted dietary components and nutrition supplements that can influence the vascular system will be of great value in the prevention and treatment of chronic disease.     

Membrane effects of cocoa procyanidins in liposomes and Jurkat T cells

We investigated the effects of the interaction between flavanols and related procyanidins (dimer to hexamer) with both cell and synthetic membranes, on bilayer fluidity and susceptibility to oxidation. Cocoa derived dimers (0.05 to 1 microg/ml) protected Jurkat T cells from AMVN-mediated oxidation and increased plasma membrane fluidity. These effects occurred in a concentration- and chain length-dependent manner. In liposomes, procyanidins prevented the Fe2+ -induced permeabilization of the membrane. Together, these results support the hypothesis that procyanidins could interact with the polar headgroup of lipids, increasing membrane fluidity and also, preventing the access of molecules that could affect membrane integrity.     

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.     

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.     

Dietary polyphenols suppress chronic inflammation by modulation of multiple inflammation-associated cell signaling pathways

The high failure rate of the reductionist approach to discover effective and safe drugs to treat chronic inflammatory diseases has led scientists to seek alternative ways. Recently, targeting cell signaling pathways has been utilized as an innovative approach to discover drug leads from natural products. Cell signaling mechanisms have been identified playing key role in diverse diseases by inducing proliferation, cell survival and apoptosis. Phytochemicals are known to be able to modulate the cellular and molecular networks which are associated to chronic diseases including cancer-associated inflammation. In this review, the roles of dietary polyphenols (apigenin, kaempferol, quercetin, curcumin, genistein, isoliquiritigenin, resveratrol and gallic acid) in modulating multiple inflammation-associated cell signaling networks are deliberated. Scientific databases on suppressive effects of the polyphenols on chronic inflammation via modulation of the pathways especially in the recent five years are gathered and critically analyzed. The polyphenols are able to modulate several inflammation-associated cell signaling pathways, namely nuclear factor-kappa β, mitogen activated protein kinases, Wnt/β-catenin and phosphatidylinositol 3-kinase and protein kinase B via selective actions on various components of the networks. The suppressive effects of the polyphenols on the multiple cell signaling pathways reveal their potential use in prevention and treatment of chronic inflammatory disorders. Understanding the mechanistic effects involved in modulation of the signaling pathways by the polyphenols is necessary for lead identification and development of future functional foods for prevention and treatment of chronic inflammatory diseases.     

G protein-linked effector and second messenger systems involved in melatonin signal transduction

The isolation and characterization of multiple melatonin receptors in a wide range of tissues and cells signifies the functional diversity of melatonin. In different cellular environments, melatonin can regulate distinct second messengers or even positively or negatively regulate the same signal transduction pathway. The capacity by which melatonin receptors modulate the activities of various effector molecules is determined by the complement of signaling components present in any particular cell type. The specific interactions between many signaling molecules have been discerned in an increasing number of cellular systems and this information is being used to explain the physiological actions of melatonin. This review will attempt to summarize recent research by many groups that has revealed numerous subtleties of the melatonin-coupled signaling pathways.     

Cytotoxic effects of digalloyl dimer procyanidins in human cancer cell lines

Flavanols, a class of polyphenols present in certain plant-based foods, have received increasing attention for their putative anticancer activity. In vitro and in vivo studies, which have compared the effectiveness of various monomer flavanols, indicate that the presence of a galloyl residue on the 3 position on the C-ring enhances the cytotoxicity of these compounds. Procyanidins, oligomerized flavanols, have been reported to be more cytotoxic than monomer flavanols in a variety of human cancer cell lines. Given the above, we evaluated the potential anticancer properties of dimer procyanidins that contain galloyl groups. Specifically, the cytotoxicity of synthetic digalloyl dimer B1 and B2 esters {[3-O-galloyl]-(−)-epicatechin-(4β,8)-(+)-catechin-3-O-gallate (DGB1) and [3-O-galloyl]-(−)-epicatechin-(4β,8)-(+)-epicatechin-3-O-gallate (DGB2), respectively} were tested in a number of in vitro models. DGB1 produced significant cytotoxicity in a number of human cancer cell lines evaluated by three independent methods: ATP content, MTT and MTS assays. For the three most sensitive cell lines, exposure to DGB1 and DGB2 for 24, 48 or 72 h was associated with a reduction in cell number and an inhibition of cell proliferation. Digalloyl dimers exerted significantly higher cytotoxic effects than the structurally related flavanols, (−)-epicatechin, (+)-catechin, (−)-epicatechin gallate, (−)-epigallocatechin gallate, (−)-catechin gallate and dimer B1 and B2. These results support the concept that the incorporation of galloyl groups and the oligomerization of flavanols enhances the cytotoxic effects of typical monomer flavanols. The therapeutic value of these compounds and their derivative forms as anticancer agents merits further investigation in whole animal models.     

Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.     

The Interaction of Flavonoids with Membranes: Potential Determinant of Flavonoid Antioxidant Effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2′-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2′-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid–water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

The interaction of flavonoids with membranes: potential determinant of flavonoid antioxidant effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2'-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2'-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid-water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

A simple guide to biochemical approaches for analyzing protein-lipid interactions

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein-lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein-lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.     

Cocoa procyanidin chain length does not determine ability to protect LDL from oxidation when monomer units are controlled

Cocoa flavan-3-ols (catechin, epicatechin and oligomeric procyanidins) were tested for their ability to decrease LDL oxidative susceptibility and spare alpha-tocopherol (alpha-toc) in vitro. Physiologic concentration (0.10-0.50 &mgr;M) of flavanols were used. The flavanols increased LDL conjugated diene lag times dose-dependently from 23-207% and 15-143% in response to copper and AAPH oxidation, respectively, and delayed alpha-toc consumption. Sparing of LDL alpha-toc represents a possible mechanism for flavanols to enhance the resistance of plasma and LDL to oxidative stress. Procyanidins decreased LDL oxidative susceptibility with increasing chain length. However, when based on equivalent amounts of monomeric units, they inhibited LDL oxidation to a similar extent. This suggests that antioxidant activity of procyanidins with biologic substrates is not attributable to chain length or charge delocalization through polymeric linkages, but primarily to ring structures and catechol groups. Additionally, human plasma was analyzed for the presence of oligomeric procyanidins following consumption of a flavanol-rich cocoa product. Procyanidin dimers were detected in plasma concordant with the appearance of monomeric flavanols, with a peak of 0.08 +/- 0.01 &mgr;mol/L (n = 6) at two hours after consumption. Thus, this paper confirms the occurrence of procyanidins in human plasma, and extends previous structure-function observations regarding flavanoid protection of LDL.