Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Resveratrol and Health

Importance of lipid microdomains, rafts, in absorption,delivery, and biological effects of resveratrol

Dominique Delmas,1,2 Virginie Aires,1,2 Didier J. Colin,3 Emeric Limagne,1,2

Alessandra Scagliarini,1,2 Alexia K. Cotte,1,2 and Francois Ghiringhelli1,2

1University of Burgundy, Dijon, France. 2Chemotherapy, Lipid Metabolism and Antitumoral Immune Response Team, INSERMResearch Center U866, Dijon, France. 3Center for Biomedical Imaging (CIBM)–microPET Imaging Laboratory, University ofGeneva, Geneva, Switzerland

Address for correspondence: Dominique Delmas, Faculty of Medicine, INSERM Research Center UMR866 “Lipids, Nutrition,Cancers,” 7 Bd Jeanne d’Arc, 21000 Dijon, France. ddelmas@u-bourgogne.fr

The preventive effects of the phytoalexin trans-resveratrol toward cancer have been largely described at the cellularand molecular levels in both in vivo and in vitro models; however, its primary targets are still poorly identified.In this review, we show the crucial role of cell membrane microdomains, that is, lipid rafts, not solely in theinitiation of the early biochemical events triggered by resveratrol leading to cancer cell death, but also in resveratrolabsorption and distribution. Resveratrol accumulates in lipid rafts and is then taken up by cells through raft-dependent endocytosis. These events allow early activation of kinase pathways and redistribution of cell deathreceptors within lipid microdomains, events ultimately leading to apoptotic cell death.

Keywords: resveratrol; transport; cancer; rafts; apoptosis

Introduction

Resveratrol or 3,4′,5-trihydroxystilbene is a sec-ondary metabolite produced in limited plant speciesand found in many natural foods (e.g., grapes,red wine, purple grape juice, and some berries).1

In plants, resveratrol is mostly found in thetrans-resveratrol-3-O-�-d-glucoside form, which isoften referred to as piceid. Like many other plantpolyphenols (i.e., flavonoids, epicatechins), resver-atrol presents interesting properties against cancers.Resveratrol is able to delay or to prevent the stages ofcarcinogenesis.2,3 These beneficial effects have beensupported by observations at the cellular and molec-ular levels in both in vitro and in vivo models andreinforce the interest in grape products and dietarysupplements for cancer therapy.

In the field of chemoprevention, the ability ofresveratrol to prevent the occurrence of various car-cinomas is related to the inhibition of the tumor cellcycle,4,5 the activation of signaling pathways thatinvolve kinase activations, and/or the induction ofthe proteolytic cascade of caspases to trigger tumorcell apoptosis.6,7 Although the beneficial effects of

resveratrol have largely been elucidated, very little isknown about the mechanisms of resveratrol cellularuptake and how resveratrol initiates its biological ef-fects. Here we review the essential role played by par-ticular plasma membrane microdomains, also calledrafts or lipid rafts, in both resveratrol absorption anddistribution and in early resveratrol-induced signal-ing pathways leading to apoptotic cancer cell death.The last part of this review discusses the potentialrole of lipid rafts in resveratrol-mediated effects andin the potential restoration of tumor cell sensitivityto death receptor ligands.

Lipid rafts as dynamic membranestructures

The plasma membrane of mammalian cells is abilayer primarily composed of thousands of typesof lipids and proteins.8 Besides delineating intra-and extracellular compartments, the plasma mem-brane constitutes a selective barrier through whichinformation and matter pass. The plasma mem-brane structure was originally described by Singerand Nicholson in 1970 as a fluid mosaic in which

doi: 10.1111/nyas.12177

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Delmas et al. Rafts as key regulators of resveratrol activities

inserted proteins can drift laterally and randomly ina fluid phospholipid bilayer. The current model sug-gests a biological membrane organized into lipid mi-crodomains or platforms, which are distinguishedfrom the rest of the membrane by their high con-tent of sphingomyelin and cholesterol. This lipid raftconcept was introduced to explain the generation ofthe glycolipid-rich apical membrane of epithelialcells.9 In this model, two phases are distinguished.The first is the liquid-ordered L0 raft phase, whichresults from the lateral association between sphin-golipids in the outer leaflet and sterol in the outerand inner leaflet of the bilayer. The second phasecorresponds to a liquid-disordered phase, which isnonraft in nature due to the differences in the short-ranged liquid translational and conformationalorder.

As mentioned, lipid rafts are ordered plasmamembrane structures resulting from the preferen-tial packing of sphingolipids and cholesterol. Theyare believed to cover around 35% of the cell sur-face. Lipid rafts are also characterized by the pres-ence of various proteins, notably the glycosylphos-phatidylinositol (GPI)-anchored proteins, whichare specifically attached to membrane outer leafletvia their GPI anchor (Fig. 1, inset). In addition,these rafts are associated with dynamic structurescalled caveolae, which are plasma membrane in-vaginations resulting from the polymerization ofthe raft protein caveolin. Furthermore, rafts arehighly dynamic in the sense that they can spa-tiotemporally recruit and localize components re-quired for many key cellular processes, such as theintracellular transport of various molecules (e.g.,viruses, xenobiotics) and the induction or responseto various stimuli (e.g., xenobiotics, nutrients, anddrugs).

Resveratrol uptake involves lipidraft–mediated endocytosis

Cellular uptake of xenobiotics can be achievedthrough passive diffusion, carrier-mediated trans-port, or active transport. Using radiolabeled resver-atrol, we have previously shown that the polyphenolenters cells by both passive (50%) and facilitated(50%) processes based on the following observa-tions. At 4 ◦C, a temperature that blocks carrier-mediated and active processes, resveratrol uptakein hepatoblastoma,10 colon cancer, and leukemia

cells,11 is significantly reduced relative to uptakeat 37 ◦C. Results suggest that passive diffusionaccounts for ∼50% of transport. Cis-inhibitionexperiments using unlabeled resveratrol showinhibition of the total tracer uptake,10,11 thus in-dicating the contribution of a carrier-mediatedprocess.

To get insight into the nature of these carrier-mediated processes, we used pharmacological in-hibitors of endocytic pathways. Pretreatment of cellswith the pan-inhibitor of endocytosis monensinsignificantly curtailed resveratrol uptake,11 whereasspecific blockers of either clathrin- or dynamin-mediated endocytosis and macropinocytosis failedto do so. Only commonly used lipid raft disrupters(methyl-�-cyclodextrin, nystatin, and filipin) con-sistently impaired resveratrol uptake in colon can-cer cells,11 indicating that part of the polyphenolmay use lipid rafts for intracellular penetration(Fig. 1). Lipid raft–mediated endocytosis has beendescribed as part of the clathrin-independent en-docytic pathways.12,13 It is involved in the endo-cytosis of GPI-anchored proteins,14 interleukin-2linked to its receptors,15 and several ether lipids(e.g., alkyl-lysophospholipid).16 The lipophilicnature of resveratrol is consistent with its ac-cumulation in lipid microdomains before itsendocytosis (Fig. 1). Interestingly, endocytosis ofresveratrol via lipid rafts was associated with an ac-cumulation of LysoSensorTM-positive acidic vesicleswithin cytoplasm; however, lowering intracellularpH did not modify resveratrol uptake and antipro-liferative effects.11 Yang et al. also suggested the in-volvement of lipid rafts in resveratrol uptake,17 andthey identified a specific subclass of rafts that containcaveolin-1, the main component of caveolae, whichare plasma membrane invaginations implicated invarious functions including endocytosis, transcyto-sis, and potocytosis.18 In this study, they showed inboth HepG2 cells transfected with wild-type or mu-tant caveolin-1 and in a hepatocellular carcinomaanimal model that caveolin-1 expression increasedthe cytotoxic and proapoptotic activity of resvera-trol in a dose- and time-dependent manner throughan increase in its internalization and trafficking, asvisualized with a fluorescent resveratrol derivative(dansyl-chloride resveratrol).17 In addition, resvera-trol endocytosis is not mediated by estrogen receptor(ER) � and �, as suggested by a lack of competitiveinhibition by estrogen or tamoxifen.17

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Figure 1. Lipid rafts as key players in resveratrol uptake and kinase activation. Resveratrol (RSV) accumulates in lipid raftsand enters tumor cells through lipid raft–mediated endocytosis, which is independent of clathrin. Furthermore, resveratrol bindsintegrin �v�3 on the extracellular domain and promotes the formation of integrin-signaling complexes into lipid rafts, leading to thesignal transduction cascade. In parallel, resveratrol induces the redistribution of various kinase proteins and their phosphorylatedforms into lipid rafts that are essential to resveratrol action in cancer cells such as cell death. Inset: lipid raft composition.

Lipid raft–mediated endocytosis as a keyinitiator of resveratrol actions

Several studies have shown that the biological ef-fects of various drugs, xenobiotics, or nutrients arethe result of the redistribution of proteins such asreceptors or other signaling proteins at the mem-brane level. Lipid rafts are very important in theseprocesses, particularly in the induction or response

of cells to various stimuli. Moreover, the redistribu-tion of proteins inside or outside of rafts can causevarious pathologies such as cardiovascular diseases,degenerative diseases, diabetes, obesity, inflamma-tion, and cancers.

In fact, resveratrol accumulation in lipid rafts in-duces a rapid activation of various kinases, such asc-Jun NH2-terminal kinases (JNKs); extracellular

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Delmas et al. Rafts as key regulators of resveratrol activities

signal–regulated kinases (ERKs); and Akt in coloncancer, leukemia,11 prostate cancer, and melanomacells.19 These early kinase activations are impor-tant because the presence of specific kinase in-hibitors prevents resveratrol-induced apoptosis inthese cells. Resveratrol-induced kinase activation isinhibited by both the pan-inhibitor of endocytosismonensin and the lipid raft disruptors methyl-�-cyclodextrin and nystatin in colon carcinoma andleukemia cells. Subsequently, this inhibition of en-docytosis decreases resveratrol-induced apoptosisin cancer cells. A biochemical analysis of lipid raftcontent by fractionation of cell lysates on a sucrosegradient revealed that resveratrol induces the redis-tribution of JNKs, Akt, and ERKs (including theirphosphorylated forms) into rafts (Fig. 1). As ob-served for kinase activation, the use of lipid raftinhibitors suppressed the resveratrol-induced redis-tribution of these proteins into lipid rafts. In fact, theredistribution of kinases in lipid rafts facilitates theiractivation and downstream induction of apoptosis.

Integrin plays a key role in resveratroluptake and early action through lipid rafts

Rafts can be the site of interactions between re-ceptors and their ligands: this interaction takesplace first inside or outside these membrane mi-crodomains. Once the receptors are linked to theirrespective ligands, intracellular transduction cas-cades are initiated and lead to the assembly of signal-ing complexes and to actin polymerization. Thesesignaling events promote a raft migration, depen-dent on actin and myosin, to the site of signaling.As the different microdomain fields fuse to formmacrodomains, signaling proteins that were initiallyin different areas find themselves in proximity toeach other. Subsequently, the interactions betweenproteins increase, amplifying the initial signal, andtherefore recruiting more rafts and more signalingproteins into these newly formed macrodomains.This process could be triggered by transmembraneglycoproteins, such as integrins, which are able toform integrin-signaling complexes that can inducea signaling transduction cascade. Indeed, the extra-cellular ligand–binding regions of integrins senseinformation relayed by the extracellular matrix andtransduce these signals through their cytoplasmicdomain, which interacts with the cytoskeleton. In-tegrins are also known to initiate cellular signal-ing through phosphorylation cascades, as described

for the integrin �v�3, that can trigger extracellu-lar signal–related kinase 1/2 (ERK1/2) signaling.20

It has been previously shown that resveratrol caninduce ERK1/2 signaling by binding the integrin�v�3.7,11,19,21 In this context, we also demonstratedthat a prototype peptide arginine–glycine–aspartate(RGD), which is known to block integrin �v�3, caninhibit the proapoptotic effects of resveratrol to-ward cancer cells.11 Moreover, it appears that in-tegrin �v�3 is involved in resveratrol uptake, be-cause the occlusion of the RGD-binding site in theintegrin extracellular domain diminishes resvera-trol uptake and consequently decreases resveratrol-induced apoptosis.11 We analyzed the underlyingmolecular mechanisms at play, and it appears thatresveratrol promotes the recruitment of the Fak,Fyn, Ras, and Grb2 proteins to form integrin signal-ing complexes in colon carcinoma cells.11 This re-cruitment involves redistribution of these proteinsand of the integrin �v�3 into lipid rafts after ex-posure with resveratrol for 6 min (Fig. 1). Our datasupport the view that these lipid microdomains mayfunction as microcompartments for the assembly ofsignaling complexes.22 Upon integrin ligation, Fynis activated and binds to the adaptor protein SHCvia its SH3 domain. It has been previously reportedthat this sequence of events is necessary to cou-ple integrins to the Ras–ERK pathway.23 The bind-ing of resveratrol to integrin �v�3 has been furtherdemonstrated by an in silico approach showing thatit can form strong bonds with the Arg248 of the�v subunit.24 This study also showed that the 4′-OH group of resveratrol, which is implicated in itsantioxidant and antiproliferative properties, estab-lishes bonds with Glu220 of the �3 subunit.24

Lipid rafts as dynamic platforms for thelater effects of resveratrol

Lipid rafts also play an important role in the initia-tion of apoptosis in response to various molecules,such as ether lipids through the extrinsic pathway.25

Similarly, we have previously shown that lipid raftsare implicated in the clustering or aggregation ofsurface receptors and adaptor molecules into mem-brane complexes at specific sites. These redistribu-tions are shown to be essential for initiating signal-ing from a number of receptors, particularly in theinitiation of Fas-mediated apoptosis during resver-atrol treatment.26 The extrinsic pathway of apop-tosis is initiated by death receptors of the tumor

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necrosis factor (TNF) receptor superfamily. Thesereceptors have been shown to be essential in theresponses of cancer cells toward certain chemother-apeutic agents.27 The extrinsic pathway of apopto-sis primarily involves the Fas receptor (i.e., CD95,APO-1), which is the major protein for the sens-ing and transduction of extracellular death signals.It is mainly triggered by the ligation of Fas ligand(i.e., FasL, APO-1L, CD95L).28,29 The transductionof the signal by ligated Fas is mediated by subse-quent oligomerization of inactive caspases, result-ing in their proteolytic activation. The apoptoticpathway triggered by Fas has been implicated inthe anticancer response of many chemotherapeu-tic drugs, which underlies its critical role. The firstreport showing the importance of the Fas pathwayin resveratrol anticancer properties described in-creases of FasL expression and binding to Fas.30

However, a few other studies reported conflictingresults in other cell systems. They challenged theidea of resveratrol-induced Fas-L overexpressionand showed that Fas/FasL interaction is not requiredfor resveratrol-induced apoptosis by using a block-ing antibody or leukemic cells insensitive to Fas-mediated apoptosis.31–33 Other interesting reportsat this time, including our work on apoptosis, droveour subsequent studies on the mechanisms by whichresveratrol triggers its proapoptotic effect. Indeed, ithas been shown that chemotherapeutic agents caninduce apoptosis via FADD (Fas-associated deathdomain) independently of FasL.34 For instance, ac-tivation of caspase-8, which is mainly known as aconsequence of death receptor–mediated apoptosis,can also occur in the amplification loop of the intrin-sic apoptotic pathway. We thought that resveratrol-induced apoptosis might rely in part on such mech-anisms, because caspase-8 is activated in responseto the polyphenol in colon cancer cells, without rul-ing out a role for Fas signaling. Thus, we showedfor the first time that resveratrol triggers a redis-tribution of Fas into the lipid rafts of the plasmamembrane, but we did not find any effect on FasLexpression or extracellular accumulation.26 Otherstudies have confirmed the importance of the redis-tribution of Fas into lipid rafts in its capacity to trig-ger apoptosis.35 Colocalization with the raft markercaveolin and homogenous clustering of Fas seem tobe major steps in the induction of apoptosis in can-cer cells in response to resveratrol.26 Consequently,we showed that these events allow the association

of FADD and procaspase-8 in lipid rafts. Our dataobtained in colon cancer cells have been confirmedrecently by the team of Mollinedo in some nonsolidtumors (multiple myeloma and T cell leukemia), inwhich resveratrol induced clustering of Fas death re-ceptor into lipid rafts independently of its ligand.36

The mechanisms of receptor molecule trapping inmembrane rafts are yet to be characterized. It wasproposed that selective clustering of Fas involvesacid sphingomyelinase-induced release of ceramidein lymphocytes or fibroblasts.37 It could also be re-lated to hydrophobic modifications of the receptor,associations with a partner located in lipid rafts,or an initial clustering resulting in an increase ofaffinity. In all these cases, we showed that Fas redis-tribution in the lipid rafts in response to resveratrolleads to the formation of the death-inducing sig-naling complex (DISC) in colon cancer cells, andconsequently to the downstream events involved inthe apoptotic process (i.e., Bax/Bak conformationalchanges and subsequent caspase-3 activation). Wealso demonstrated that other death receptors likeDR4 and DR5 follow the same scheme of cluster-ing and signaling in response to resveratrol.38 Theseresults fit with the effects of lipid ether reported inleukemia cells,25 and highlight the role of death re-ceptor redistribution into lipid rafts in the proapop-totic effects of resveratrol toward cancer cells.

Lipid rafts to counteract chemoresistanceof tumor cells

Lipid rafts also contribute to the chemosensitizationinduced by various drugs through the transloca-tion of proteins either out of (e.g., P-glycoprotein)39

or into their structures (e.g., death receptors).Thus, we showed that in resveratrol-resistant can-cer cells, a treatment with the polyphenol can en-hance the cytotoxic effects of various agents, in-cluding TNF, anti-TNF receptor antibodies, andTNF-related apoptosis-inducing ligand (TRAIL).38

Resveratrol exhibits its sensitizing properties by fa-cilitating the formation of the DISC at the plasmamembrane. It also improves apoptosis induction in-dependently of Bax and Bak, since their knockoutdid not counteract its effects and Bcl-2 overexpress-ing cells were also affected.40 These last observationsruled out any involvement of the mitochondria-driven intrinsic pathway in these effects. Further-more, we demonstrated that inhibition of the redis-tribution of TNF-related receptors into lipid rafts

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Figure 2. Lipid rafts as key players in resveratrol-induced apoptosis. Resveratrol (RSV) induces the clustering of death receptordomains (CD95, DR4, and DR5) into lipid rafts. This redistribution leads to a functional DISC, where FADD and procaspase-8 arealso redistributed into lipid microdomains. These events contribute to the molecule’s ability to trigger apoptosis in tumor cells, andit is an essential step for its sensitizing effect when resveratrol is used in combination with death receptor agonists.

using the raft inhibitor nystatin blocked resveratrol-sensitizing effects in these drug combinationsmodels, which suggests that this clustering is a keyevent38 (Fig. 2).

These observations highlight the potential ben-efit of resveratrol as an adjuvant in chemotherapy.Indeed, it is well known that antiapoptotic proteinsof the Bcl-2 family are implicated in the resistancetoward many anticancer agents. As we showed forTRAIL- and Fas-related therapies in colon cancercells, resveratrol might be a useful and nontoxicmeans to enhance the effects of widely used cyto-toxic anticancer treatments and possibly decreasethe emergence of long-term resistance. Future stud-ies in our group will test these hypotheses in pre-clinical in vivo models.

Conclusion and perspectives

In this review, we describe the importance of themembrane dynamic microdomains, dubbed lipidrafts, in resveratrol uptake, which leads to its earlyand later effects in cancer cells. We have highlightedthe fact that lipid raft–mediated endocytosis is theearly event in the molecular signaling cascade in-duced by resveratrol. Taken together, these find-ings show that accumulation of resveratrol intolipid rafts and active endocytosis are required forresveratrol to activate kinase-dependent signaling

pathways (ERKs/JNKs/Akt) and to trigger caspase-dependent apoptosis in cancer cells. A disruptionof this process largely abolishes the biological effectof this polyphenol. Any residual activity observedwas due to resveratrol taken up by passive diffu-sion. Interestingly, although resveratrol enters nor-mal cells such as human monocytes or intestinalcells, resveratrol does not induce kinase activation,toxicity, or apoptosis.11 All these events appear tobe required for activation of downstream signalingpathways leading to cell death in tumor cells, andindicate the essential role of microdomains in thedynamics of resveratrol uptake in tumor cells andin associated biological responses.

Most of the mechanisms described in this reviewwere obtained in vitro, and future works should con-sider them as a basis for in vivo preclinical studies.Thus, the link between the chemopreventive andchemotherapeutic effects of resveratrol and its in-tegrin binding and subsequent lipid raft–mediatedendocytosis should be explored further in animalstudies. Such studies could be easily achieved bynoninvasive molecular imaging using, for exam-ple, competitive assays with RGD-based tracers.They could also provide insight into the nature ofresveratrol anticancer activity. Indeed, resveratrolmight affect various tumor components throughits binding to �v�3 integrin that is found in the

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plasma membrane of cancer and endothelial cells.The cellular responses of these cell types to resver-atrol might be mechanistically different, but theycould explain its pleiotropic effects on tumors, fromkilling cancer cells to inhibiting angiogenesis.41 Thislast effect may be comparable to promising RGD-related drug therapies.42 Metastasis is another veryimportant step in the development of cancer thatmight be targeted by resveratrol via lipid rafts. In-deed, resveratrol has been shown to increase thefluidity of the plasma membrane.43,44 Consideringthat cancer cells initiate their migration/invasion byforming lipid raft–enriched invadopodia, and thatthis phenomenon is dependent on integrins andcaveolin-1,45 it would be interesting to check ifresveratrol targets these events. Also, as the expres-sion of caveolin-1 has been strongly correlated withchemoresistance, discovering any effect of resver-atrol on the activity of this protein is important.Finally, there are still doubts about the relevance ofin vitro studies on resveratrol since its bioavailabil-ity in vivo is considered poor. Indeed, it is rapidlymetabolized by glucuronidation, sulfation, and hy-droxylation reactions. We recently showed that sul-fate derivatives of resveratrol, as well as mixtures ofsulfate and glucuronide derivatives, can exert anti-cancer effects similar to resveratrol in vitro.46 There-fore, these metabolites could account for resvera-trol’s properties in vivo. The questions posed abovewould benefit from a parallel analysis of the effects ofresveratrol metabolites and may provide importantpreclinical results.

Acknowledgments

This study was supported by the Conseil Regionalde Bourgogne and the Ligue Inter-regionale Grand-Est Contre le Cancer. A.S. received a postdoctoralfellowship from the Conseil Regional de Bourgogne,which supports D.D.’s group. We are grateful to Dr.J.A.H. Inkster, University of Geneva, for valuableEnglish corrections.

Conflicts of interest

The authors declare no conflicts of interest.

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