Endocytosis of Resveratrol via Lipid Rafts and Activation...

13
Research Article Endocytosis of Resveratrol via Lipid Rafts and Activation of Downstream Signaling Pathways in Cancer Cells Didier Colin 1,2 , Emeric Limagne 1,2 , Sylvie Jeanningros 1,2 , Arnaud Jacquel 1,3 ,G erard Lizard 1,2 , Anne Athias 1,4,6 , Philippe Gambert 1,4,6,7 , Aziz Hichami 5,7 , Norbert Latruffe 1,2 , Eric Solary 1,3 , and Dominique Delmas 1,2,7 Abstract trans-Resveratrol has been proposed to prevent tumor growth and to sensitize cancer cells to anticancer agents. Polyphenol entry into the cells has remained poorly understood. Here, we show that [ 3 H]-resveratrol enters colon cancer cells (SW480, SW620, HT29) and leukemia U937 cells through a monensin (5–20 mmol/L) -sensitive process that suggests clathrin-independent endocytosis. Uptake of the molecule can be prevented by methyl-b-cyclodextrin (2–12 mg/mL), nystatin (12 ng/mL), and filipin (1 mg/mL), which all disrupt plasma membrane lipid rafts. Accordingly, radiolabeled resveratrol accu- mulates in sphingomyelin- and cholesterol-enriched cell fractions. Interestingly, extracellular signal– regulated kinases (ERK), c-Jun NH 2 -terminal kinases (JNK), and Akt also accumulate in lipid rafts on resveratrol exposure (IC 50 at 48 h 30 mmol/L in SW480 and U937 cells). In these rafts also, resveratrol promotes the recruitment, by the integrin a V b 3 (revealed by coimmunoprecipitation with an anti-integrin a V b 3 antibody), of signaling molecules that include the FAK (focal adhesion kinase), Fyn, Grb2, Ras, and SOS proteins. Resveratrol-induced activation of downstream signaling pathways and caspase-dependent apoptosis is prevented by endocytosis inhibitors, lipid raft–disrupting molecules, and the integrin antagonist peptide arginine-glycine-aspartate (500 nmol/L). Altogether, these data show the role played by lipid rafts in resveratrol endocytosis and activation of downstream pathways leading to cell death. Cancer Prev Res; 4(7); 1095–106. Ó2011 AACR. Introduction Epidemiologic and experimental studies have suggested favorable effects of dietary polyphenols through their anti- oxidative and anticarcinogenic properties. Resveratrol (trans-3,4 0 ,5-trihydroxystilbene), a wine grape microcompo- nent, may be one of the most efficient of these polyphenols (1) in that it could prevent the occurrence of vascular diseases, neurodegenerative processes, and some malignant tumors (see for review ref. 2). These chemopreventive prop- erties are supported by observations at the cellular and molecular levels (3–5) and reinforce the interest in grape products and dietary supplements for cancer therapy. The potential ability of trans-resveratrol to prevent the occurrence of colon cancer and other carcinomas is related to its effects on the tumor cell cycle (5–7) through activation of signaling pathways that involve c-Jun NH 2 -terminal kinases (JNK) or extracellular signal–regulated kinases (ERK; refs. 8, 9) and/or its ability to trigger tumor cell death by apoptosis (4, 10–12). There is also compelling evidence that resvera- trol can sensitize tumor cells to various anticancer drugs (13, 14) and cytokines such as TRAIL (11), possibly because of the clustering of death receptors in detergent-resistant mem- branes (DRM) known as lipid rafts (4, 11, 15). An unsolved question remains the mechanisms of resver- atrol entry into cells and how resveratrol initiates its bio- logical effects in tumor cells. This study shows that resveratrol-induced apoptosis involves dependent mito- gen-activated protein kinase (MAPK) activation requiring accumulation in lipid rafts and monensin-sensitive active endocytosis. These 2 events are essential for the downstream activation of kinase-dependent signaling pathways and caspase activation leading to cell death. Interestingly, resver- atrol also promotes the redistribution of MAPKs and the integrin b 3 protein into lipid rafts, which seem as key plat- forms for anticancer effects of the anticancer effects of this polyphenol. These findings contribute new information on Authors' Affiliations: 1 Inserm U866; 2 Facult e des Sciences Gabriel, Cen- tre de Recherche-Biochimie M etabolique et Nutritionnelle (LBMN); 3 Centre de Recherche-Cancer et diff erenciation and 4 Centre de Recherche-Phy- siopathologie des dyslipid emies, Facult e de M edecine; 5 UPRES Lipides et Signalisation cellulaire, Universit e de Bourgogne; and 6 Plateau de lipido- mique and 7 Plateau Raftomique et Signalosome, IFR Sant e-STIC, Dijon, France Note: Supplementary data for this article are available at Cancer Preven- tion Research Online (http://cancerprevres.aacrjournals.org/). D. Colin and E. Limagne contributed equally to this work. Corresponding Author: Dominique Delmas, Inserm UMR866, 6 Boule- vard Gabriel, 21000 Dijon, France. Phone: 0033-380396237; Fax: 0033- 380396250. E-mail: [email protected] doi: 10.1158/1940-6207.CAPR-10-0274 Ó2011 American Association for Cancer Research. Cancer Prevention Research www.aacrjournals.org 1095 for Cancer Research. on May 4, 2018. © 2011 American Association cancerpreventionresearch.aacrjournals.org Downloaded from Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Transcript of Endocytosis of Resveratrol via Lipid Rafts and Activation...

Page 1: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

Research Article

Endocytosis of Resveratrol via Lipid Rafts and Activationof Downstream Signaling Pathways in Cancer Cells

Didier Colin1,2, Emeric Limagne1,2, Sylvie Jeanningros1,2, Arnaud Jacquel1,3, G�erard Lizard1,2,Anne Athias1,4,6, Philippe Gambert1,4,6,7, Aziz Hichami5,7, Norbert Latruffe1,2,Eric Solary1,3, and Dominique Delmas1,2,7

Abstracttrans-Resveratrol has been proposed to prevent tumor growth and to sensitize cancer cells to anticancer

agents. Polyphenol entry into the cells has remained poorly understood. Here, we show that

[3H]-resveratrol enters colon cancer cells (SW480, SW620, HT29) and leukemia U937 cells through a

monensin (5–20 mmol/L) -sensitive process that suggests clathrin-independent endocytosis. Uptake of the

molecule can be prevented by methyl-b-cyclodextrin (2–12 mg/mL), nystatin (12 ng/mL), and filipin

(1 mg/mL), which all disrupt plasma membrane lipid rafts. Accordingly, radiolabeled resveratrol accu-

mulates in sphingomyelin- and cholesterol-enriched cell fractions. Interestingly, extracellular signal–

regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), and Akt also accumulate in lipid rafts on

resveratrol exposure (IC50 at 48 h � 30 mmol/L in SW480 and U937 cells). In these rafts also, resveratrol

promotes the recruitment, by the integrin aVb3 (revealed by coimmunoprecipitation with an anti-integrin

aVb3 antibody), of signaling molecules that include the FAK (focal adhesion kinase), Fyn, Grb2, Ras, and

SOS proteins. Resveratrol-induced activation of downstream signaling pathways and caspase-dependent

apoptosis is prevented by endocytosis inhibitors, lipid raft–disrupting molecules, and the integrin

antagonist peptide arginine-glycine-aspartate (500 nmol/L). Altogether, these data show the role played

by lipid rafts in resveratrol endocytosis and activation of downstream pathways leading to cell death.

Cancer Prev Res; 4(7); 1095–106. �2011 AACR.

Introduction

Epidemiologic and experimental studies have suggestedfavorable effects of dietary polyphenols through their anti-oxidative and anticarcinogenic properties. Resveratrol(trans-3,40,5-trihydroxystilbene), awine grapemicrocompo-nent, may be one of the most efficient of these polyphenols(1) in that it could prevent the occurrence of vasculardiseases, neurodegenerative processes, and some malignanttumors (see for review ref. 2). These chemopreventive prop-erties are supported by observations at the cellular and

molecular levels (3–5) and reinforce the interest in grapeproducts and dietary supplements for cancer therapy.

The potential ability of trans-resveratrol to prevent theoccurrenceof coloncancer andother carcinomas is related toits effects on the tumor cell cycle (5–7) through activation ofsignaling pathways that involve c-Jun NH2-terminal kinases(JNK) or extracellular signal–regulated kinases (ERK; refs. 8,9) and/or its ability to trigger tumor cell death by apoptosis(4, 10–12). There is also compelling evidence that resvera-trol can sensitize tumor cells to various anticancer drugs (13,14) and cytokines such as TRAIL (11), possibly because ofthe clustering of death receptors in detergent-resistantmem-branes (DRM) known as lipid rafts (4, 11, 15).

An unsolved question remains themechanisms of resver-atrol entry into cells and how resveratrol initiates its bio-logical effects in tumor cells. This study shows thatresveratrol-induced apoptosis involves dependent mito-gen-activated protein kinase (MAPK) activation requiringaccumulation in lipid rafts and monensin-sensitive activeendocytosis. These 2 events are essential for the downstreamactivation of kinase-dependent signaling pathways andcaspase activation leading to cell death. Interestingly, resver-atrol also promotes the redistribution of MAPKs and theintegrin b3 protein into lipid rafts, which seem as key plat-forms for anticancer effects of the anticancer effects of thispolyphenol. These findings contribute new information on

Authors' Affiliations: 1Inserm U866; 2Facult�e des Sciences Gabriel, Cen-tre de Recherche-BiochimieM�etabolique et Nutritionnelle (LBMN); 3Centrede Recherche-Cancer et diff�erenciation and 4Centre de Recherche-Phy-siopathologie des dyslipid�emies, Facult�e deM�edecine; 5UPRES Lipides etSignalisation cellulaire, Universit�e de Bourgogne; and 6Plateau de lipido-mique and 7Plateau Raftomique et Signalosome, IFR Sant�e-STIC, Dijon,France

Note: Supplementary data for this article are available at Cancer Preven-tion Research Online (http://cancerprevres.aacrjournals.org/).

D. Colin and E. Limagne contributed equally to this work.

Corresponding Author: Dominique Delmas, Inserm UMR866, 6 Boule-vard Gabriel, 21000 Dijon, France. Phone: 0033-380396237; Fax: 0033-380396250. E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-10-0274

�2011 American Association for Cancer Research.

CancerPreventionResearch

www.aacrjournals.org 1095

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 2: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

resveratrol uptake–associated mechanisms and on activa-tion of downstream signaling pathways in tumor cells.

Materials and Methods

Cell linesSW480, SW620, HT29, and HCT116 human colon carci-

noma cells, human leukemic monocyte lymphoma U937cells, and rat nontransformed small intestinal IEC-18 cellswere obtained from the American Type Culture Collection.U937 cells were maintained in RPMI 1640 and SW480,SW620, HT29, HCT116, and IEC18 cells were cultured inEagle’s minimum essential medium. Both media were com-plemented with 10% (v/v) fetal calf serum (Sigma-Aldrich).Cells were also maintained at different pH values (7.4 and6.0) before 24 hours and during resveratrol treatment.Human peripheral blood monocytes were obtained fromhealthy donors with informed consent according to recom-mendations of an independent scientific review board, inaccordance with the Declaration of Helsinki. Cells wereenriched by the use of a monocyte isolation kit with auto-MACSSeparatoraccordingtothemanufacturer’s instructions(Miltenyi Biotec) and cultured as previously described (16).

Drugs, chemical reagents, and antibodies[3H]-trans-Resveratrol (specific activity: 74 GBq/mmol)

labeled in ortho and para positions of benzenic rings (Amer-sham). All chemicals were obtained from Sigma-Aldrichunless specified. We used rabbit polyclonal antibodies(Abs) against human phospho-JNKs (p-JNK), JNKs, phos-pho-ERK1/2 (p-ERK1/2), ERK1/2, phospho-Akt (p-Akt),Akt, Fyn, FAK, Ras, Grb2, PARP, procaspase-3, caspase-3active fragments, integrin b3 (Cell Signaling Technology),against caveolin-2 (Santa-Cruz Biotechnology), againstSOS1 (Abcam), andmousemonoclonal Abs against humanflotilin (BD Transduction Lab), integrin aVb3 (Santa-CruzBiotechnology), and b-actin (Sigma-Aldrich).

Fluorescence microscopyCells were grown on glass coverslips in 12-well plates,

exposed to resveratrol for indicated times, quickly rinsed 3times with cold PBS and mounted on slides in PBS. Con-ventional fluorescence microscopic analysis using a 40,6-diamidino-2-phenylindole (DAPI) filter was immediatelycarried out with an Axioskop (Zeiss) after UV excitation tovisualize resveratrol autofluorescence (17).

To detect acidic compartment, cells were grown on glasscoverslips in 12-well plates and incubated with 1 mmol/LLysoSensor probe (Invitrogen/Molecular Probes) for 30minutes with or without resveratrol during the last 6minutes. Cells were then rinsed 3 times with cold PBSand quickly mounted on slides. A 488-nm wavelength wasused to visualize LysoSensor green using a fluoresceinisothiocyanate (FITC) filter.

Resveratrol uptake measurementResveratrol uptake was examined by incubating cells

seeded in 6-well plates over different times with [3H]-

resveratrol (30 mmol/L; 0.5 mCi/mL) at various concen-trations in complete, serum-free, HBSS-enriched, bovineserum albumin (BSA)-enriched medium and at differentpH values (7.4 and 6.0). We carried out cis-inhibitionexperiments by adding unlabeled resveratrol to incuba-tion medium. Resveratrol uptake assays were carried outat 37�C and 4�C, with or without endocytosis inhibitors(10 mmol/L monensin, 10 mmol/L phenylarsine oxide,50 mmol/L chlorpromazine, 50 mmol/L monodansylca-daverine, 50 mmol/L amiloride, 80 mmol/L dynasore).Rafts mediated resveratrol uptake was also detected bytreating cells with [3H]-resveratrol after raft disruption bymethyl-b-cyclodextrin (MbCD) pretreatment (30 min-utes, 6 mg/mL). The integrin involvement in resveratroluptake was examined by incubating SW480 cells with anintegrin peptide inhibitor [500 nmol/L RGD peptide(Arg-Gly-Asp), 24 hours] and with [3H]-resveratrol(30 mmol/L). At the end of the incubation period, thelabeled media were removed, the cells were washed 3 timeswith cold PBS, and lysed in a lysis buffer (0.1 mol/L NaOH,2% Na2CO3, 0.1% SDS). Cell lysates and media radio-activity were counted in a liquid scintillation analyzer.

Resveratrol binding studiestrans-Resveratrol (5 mmol/L) was incubated for different

times at 37�C with standard cell-free culture medium.Unbound ligand was extracted by ethyl acetate and thenquantified by high performance liquid chromatographic(HPLC) analysis. The analyses were carried out on areversed phase. Nucleosil C18 columns were from TouzartandMatignon (Waters). AWaters 625 LC system, was used,together with a Waters 486 tunable absorbance detectorand a SP4400 ChromJet integrator (Spectra-Physics). TheUV detector was set at 306 nm and resveratrol was elutedfrom the column with a gradient containing water andacetonitrile. Resveratrol concentrations in culture mediawere calculated using a standard curve of resveratrol.

Cell fractionationColon carcinoma cells were seeded in 6-well plates and

were treatedwith 30 mmol/L [3H]-resveratrol for 1, 3, 6, and10 minutes. Cells were thoroughly washed with cold PBSand lysed on ice in a lysis buffer (10 mmol/L HEPES, 10mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1mmol/L dithiothreitol) in the presence of protease inhi-bitor cocktail (Roche Diagnostics). After 15 minutes, theextracts were homogenized with 0.6% NP40 during 15seconds and centrifuged for 10 minutes at 1,200 � g.The nuclear fractions were dissolved in lysis buffer contain-ing 1% NP40, and the cytoplasmic fractions were centri-fuged twice more to remove nuclear contaminations.Fractions were estimated for proteins concentrations andsubmitted to radioactivity measurement in a liquid scin-tillation analyzer.

Flow cytometric analysesCancer cells were seeded in 6-well plates andwere treated

with 30 mmol/L resveratrol for 6 minutes. Cells were

Colin et al.

Cancer Prev Res; 4(7) July 2011 Cancer Prevention Research1096

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 3: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

trypsinized, washed with PBS, centrifuged for 5 minutes at400� g, and incubated for 30 minutes with the fluorescentfluid marker Sulforhodamine-101 (25 mg/mL) with orwithout monensin (5 mmol/L). Cells were washed inPBS, fixed in 1% paraformaldehyde, and analyzed with aFACScan flow cytometer (BD Biosciences).

Rafts isolation and biochemical characterizationThis study was conducted as described (4). Briefly,

cells were starved for 6 hours and treated with MbCD; 6mg/mL) for the last 30 minutes for raft disruption. Cellswere exposed to or not exposed to resveratrol (30 mmol/L, 6 minutes) with or without MbCD, lysed in 2 mL(containing Tris, Nacl, and EDTA) TNE buffer (20mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, 1%Triton X-100, pH 7.4 containing antiproteases) for 30minutes at 4�C, and passed through an ice-cold cylindercell homogenizer. The lysates were diluted with 2 mLbuffer containing 80% sucrose (w/v), placed at thebottom of a linear sucrose gradient and centrifuged at39,000 rpm for 20 hours at 4�C before collecting eleven1-mL fractions. Sixty microliters of each fraction wassubjected to SDS-PAGE and immunoblotted. Lipidswere extracted and analyzed as described (4) in theindicated conditions. Flotilin and caveolin-2 describedas markers for rafts were analyzed in each fraction byWestern blotting.

ImmunoprecipitationSW480 cells were seeded into 80-cm2 flasks (2 � 106

cells) for 48 hours and starved for 6 hours before resveratroltreatment. After 15 minutes, cells were washed with coldPBS and proteins were cross-linked with disuccinimidylsuberate (DSS) according to the manufacturer’s instruc-tions (Pierce, Thermo Fisher Scientific). Cells were thenincubated in a lysis buffer (50 mmol/L HEPES, pH 7.5,150 mmol/L NaCl, 10 mmol/L EDTA, 10 mmol/LNa4P2O7, 100 mmol/L NaF, 2 mmol/L Na3VO4, 1% Tri-ton-X 100) complemented with antiproteases for 30 min-utes on ice. After centrifugation at 20,000 � g at 4�C for 20minutes, 1 mg of proteins from the supernatant was incu-bated in 1 mL of HNT buffer (30 mmol/L HEPES, 30mmol/L NaCl, 0.1% Triton-X 100) overnight at 4�C with2 mg of mouse monoclonal anti-aVb3 integrin Abs or withirrelevant mouse IgG1 covalently bound to magnetic beads(Dynabeads Protein G; Invitrogen) with DSS according tothemanufacturer’s instructions. Beads were washed 3 timesin HNT (containing HEPEs, Nacl, and Triton-X 100) buffer,and the precipitates were resuspended in a loading buffer,boiled for 5 minutes, and analyzed by Western blotting.

Western blottingStandard Western blot analyses were carried out as

described in Supplementary Materials and Methods.

Proliferation assaysCells were seeded in quadruplicates in 24-well plates and

treated with 30 mmol/L of resveratrol at different pH values

(7.4 and 6.0). After indicated times, cells were washed withPBS, stained with crystal violet [0.5% (w/v) crystal violet in25% (v/v) methanol] for 5 minutes, and rinsed with water3 times. Absorbance was read at 540 nm after extraction ofthe dye with 0.1 mol/L sodium citrate in 50% ethanol.

Apoptosis identificationApoptosis was identified by staining the nuclear chro-

matin of trypsinized cells with 1 mg/mL Hoechst 33342(Sigma-Aldrich) for 15 minutes at 37�C. The percentage ofapoptotic cells was determined by analyzing 300 cytos-pined cells. MAPK implication in resveratrol-inducedapoptosis was assayed by pretreating cells during 2 hoursby 10 mmol/L of U0126 [MAP/ERK kinase (MEK)inhibitor], LY294002 [phosphoinositide 3-kinase (PI3K)/Akt inhibitor], or SP600125 (JNK inhibitor) before resver-atrol exposure.

Densitometry and statistical significanceThe densitometry of blots was realized by the use of

ImageJ software (NIH). Unless indicated in the legends offigures, the reported values represent the means � SD of 1representative experiment repeated 3 times. Statistical sig-nificance was determined using the Mann–Whitney test atP < 0.05, P < 0.01, or P < 0.001.

Results

Resveratrol rapidly accumulates in colon cancer celllines

Resveratrol exhibits a spontaneous blue fluorescence onUV excitation (17), which allows the visualization of itstime- and dose-dependent accumulation in the cells. Fluor-escence was detected in SW480 colon cancer cells 1minutesafter the beginning of their treatment (Fig. 1A). Intracel-lular fluorescence was obvious at 10 minutes at resveratrolconcentrations ranging from 0.5 to 50 mmol/L with anapparent dose-dependent increase. This rapid uptake of themolecule was also observed in HCT116, HT29, and SW620colon cancer cell lines (not shown). To quantify thisuptake, cancer cells were incubated with 30 mmol/L[3H]-resveratrol, and intracellular and extracellular radio-activity was monitored. This resveratrol concentration waschosen in accordance with our results showing that itinduces a marked antiproliferative (6) and proapoptoticeffect on colon cancer cells and leukemia cells withoutinducing toxicity and apoptosis in normal human mono-cytes and in normal rat intestinal IEC18 cells (Supplemen-tary Fig. S1). After adding [3H]-trans-resveratrol at 37�C, itwas readily taken up by cancer cell lines (Fig. 1B, dia-monds) and accumulated with time to reach amaximum at6 minutes after the beginning of cell treatment. Thereafter,the radioactivity decreased gradually to reach at 48 hoursthe level obtained after 1 minutes of treatment (Fig. 3E),suggesting a prevalence of efflux over influx. When theseexperiments were conducted at 4�C, resveratrol uptake at 6minutes was lower than 50% of that measured at 37�C(Fig. 1B, triangles). This transport in the tumor cells was

Lipid Rafts Determine Resveratrol Activity

www.aacrjournals.org Cancer Prev Res; 4(7) July 2011 1097

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 4: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

specific to resveratrol, as we failed to detect any metabolitein the culture medium, up to 1 hour after the beginning ofincubation with the polyphenol (Fig. 1C).

Serum removal from the culture medium increasedresveratrol uptake at both 4�C and 37�C, indicating thatthe polyphenol was trapped by serum constituents(Fig. 1D). The molecule uptake decreased in a dose-depen-dent manner by the addition of BSA, suggesting that themajor serum protein albumin plays a role in its trapping(Fig. 1D). The kinetics of resveratrol binding to serum

proteins indicated a 50% decrease in unbound moleculeafter 2 hours of incubation in the presence of serum and avirtually complete trapping of resveratrol after 24 hours(Fig. 1D, insert).

Resveratrol is internalized by endocytosisFor many drugs, passive diffusion and carrier-mediated

transport coexist, the latter being an active or a facilitatedtransport that has a limited capacity and thus can reachsaturation. Concentration dependence of resveratrol

Time (min)

Time (min)

Concentration of resveratrol (μmol/L)

0A

C

D

B

0.5

Incubationwith medium

1-h incubationwith SW480

Resveratrol

Resveratrol

1.25

1.00

0.75

0.50

0.25

0.00

Vol

ts

1.00

0.75

0.50

0.25

0.00

4,000

10080604020

00 5 10 15 20 25

Time (h)

Time (min)

[Unb

ound

R]

(% o

f tot

al)37°C

4°C

37°C

SW4802,500

2,000

1,500

1,000

500

0

2,500

2,000

1,500

1,000

500

0

2,500

2,000

1,500

1,000

500

0

2,500

2,000

1,500

1,000

500

0

2,000

3,000

4,000

1,000

00 2 4 6

SW620

HCT116

HT29

U937

4°C

3,500

3,000

2,500

2,000

Upt

ake

of [3 H

]-re

sver

atro

l(d

pm/1

06 ce

lls)

Upt

ake

of [3 H

]-re

sver

atro

l (dp

m/1

06 ce

lls)

1,500

1,000

500

0Complete medium

Serum-free medium

HBSS

BSA mg/mL 0.5 3.5 6.0

5 10−0.13

1.25

10 50

1 10

Figure 1. Time-dependent uptakeof resveratrol in colon cancer celllines. A, time- and dose-dependent uptake analyses ofresveratrol by fluorescencemicroscopy. SW480 cells weretreated with 30 mmol/L resveratrolin culture medium for the indicatedtimes at 37�C (top) or with a gamerange of resveratrol during 10minutes (bottom). Onerepresentative experiment of atleast 3 independent experimentsis shown. B, time course ofradiolabeled resveratrol uptake incolon cancer cells and leukemiccell lines. Cells were incubatedwith [3H]-resveratrol at 37�C (^) orat 4�C (D) with complete mediumfor indicated times. C, HPLCanalysis. Resveratrolconcentrations in media weremeasured by HPLC as described,in the treating medium and 1 hourafter incubation with SW480 cells.D, decrease of resveratrol uptakeby binding to serum proteins.SW480 cells were incubated with[3H]-resveratrol (30 mmol/L) in amedium supplemented withserum, HBSS, or in serum-freemedia supplemented withincreasing concentrations of BSA.All these incubations were carriedout at 37�C (black bars) and at 4�C(white bars) for 6 minutes. Insert,kinetics of resveratrol binding toserum proteins of culture medium(B and D, means � SD of arepresentative experiment from 3independent experiments).

Colin et al.

Cancer Prev Res; 4(7) July 2011 Cancer Prevention Research1098

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 5: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

uptake reveals that intracellular radioactivity varied linearlywith the incubated concentration (Fig. 2A). When the totaluptake measured at 37�C was corrected from the passiveuptake measured at 4�C, the curve obtained, which appearsto be saturated, corresponds to a carrier-mediated transport(Fig. 2A). To confirm thismechanism, cis-inhibition experi-ments were undertaken. When unlabeled resveratrol wasadded to the radiolabeled molecule at 37�C, the intracel-lular radioactivity measured at 6 minutes decreased inSW480 and HT29 colon cancer cells (Fig. 2B), whichwas not observed at 4�C (Fig. 2B). This observation suggeststhat cancer cells could uptake resveratrol, at least in part, byan active process. Similar observations were made inSW620, HCT116, and U937 cells (not shown). To furtherexplore this active process, the 5 cell lines studiedwere pretreated for 30 minutes in a medium containing5 mmol/L monensin (18), an inhibitor of endocytosis thatdisrupts the membrane electropotential and the pH value.Monensin pretreatment provoked 50% decrease in resver-atrol uptake at 3 and 6 minutes (see 3 of the cell lines inFig. 2C). At this concentration,monensin also inhibited theaccumulation of Sulforhodamine-101, a fluorescent mar-ker for clathrin-independent fluid-phase endocytosis, inthese cells (Fig. 2D) and counteracted resveratrol-inducedapoptosis (Supplementary Fig. S1). Altogether, these resultssuggest that resveratrol taken up in cancer cells wasmediated by endocytosis.

Resveratrol endocytosis depends on lipid raftsintegrityTo better characterize the endocytic process, we used

specific inhibitors of clathrin-mediated endocytosis (phe-nylarsine oxide, chlorpromazine, monodansylcadaverine;refs. 19–21), macropinocytosis (amiloride; ref. 22), dyna-min-dependent endocytosis (dynasore; ref. 23), and lipidraft–mediated endocytosis (MbCD). Under cotreatmentwith these inhibitors, only monensin and MbCD blocked[3H]-resveratrol uptake (Fig. 2E). Two polyene antibiotics(filipin and nystatin), which are other disruptors of lipidrafts, also decreased resveratrol internalization (Fig. 2F). Atthe concentrations used, MbCD, nystatin, and filipin didnot cause any plasmamembrane leakage, as determined bytrypan blue exclusion assay (not shown). The ability ofMbCD to decrease resveratrol internalization into SW480cells was dose dependent and reached 70% at 12 mg/mL(Fig. 2G). Similar results were obtained in other cell linesstudied (not shown). These combined results argue for alipid raft–dependent endocytosis of resveratrol.

Resveratrol accumulates in lipid rafts of colon cancercellsThe lipophilic structure of resveratrol suggests that this

compound could accumulate in plasma membrane lipidrafts before endocytosis. When SW480 cells were incubatedwith 30 mmol/L [3H]-resveratrol, most of the radioactivityaccumulated within minutes in the membrane/particulatefraction (Fig. 3A). Lysates of SW480 cells exposed for6 minutes to 30 mmol/L [3H]-resveratrol were fractionated

on a sucrose gradient, and the lipid content of each fractionwas determined by HPLC-coupled mass spectrometry toidentify sphingomyelin- and cholesterol-enriched fractionscorresponding to lipid rafts (Fig. 3B) and flotilin andcaveolin-2 confirmed the DRMs enrichment in fractions3 and 4 (Fig. 3B). Radioactivity analysis showed thatresveratrol was found mainly in these 2 fractions(Fig. 3B). In the presence of MbCD (6 mg/mL), resveratrolwas dispersed in the various fractions. Furthermore, weobserved that there was no incorporation of resveratrol inmembrane phospholipids in a covalent manner (notshown). Interestingly, endocytosis of resveratrol via lipidrafts was associated with an accumulation of LysoSensor-positive acidic vesicles within cytoplasm (Fig. 3C). Theantiproliferative effect of resveratrol was not affected byan acidification of the medium (Fig. 3D). Moreover, resver-atrol was taken up by SW480 cells indifferently of pHconditions (Fig. 3E) and the lipid composition remainedunchanged (Supplementary Fig. S2).

Disruption of resveratrol endocytosis preventspolyphenol-induced signaling pathways

In agreement with the data on prostate cancer cells andmelanoma cells (24, 25), resveratrol induced a rapid acti-vation of JNK, Akt, and ERK1/2 in colon carcinoma cellsand leukemia cells but not in human normal monocytes(Fig. 4A). The combination of resveratrol with an ERK1/2inhibitor (U0126), PI3K/Akt inhibitor (LY294002), or INKinhibitor (SP600125) prevented resveratrol-induced apop-tosis in these cells (Fig. 4B). Pretreatment of cancer cellswith monensin decreased resveratrol-induced MAPK acti-vation in a dose-dependent manner (Fig. 4C; Supplemen-tary Fig. S3), without affecting their total levels in cancercells. These results underline the importance of resveratrolendocytosis in kinase activation.

Interestingly, fractionation of cell lysates on a sucrosegradient showed that JNKs, Akt, ERK1/2, and their phos-phorylated forms, which are not detected in the fractionsenriched in cholesterol and sphingomyelin of untreatedcells, accumulated in these fractions on resveratrol treat-ment (Fig. 5A and B). Moreover, resveratrol induced redis-tribution of integrin b3, which is a resveratrol receptor (25),and its associated proteins including FAK, Fyn, and Ras(Fig. 5B). It is noteworthy that MbCD suppressed theresveratrol-induced redistribution of these proteins in lipidrafts (Fig. 5B), the activation of JNKs, Akt, and ERK1/2(Fig. 5C; Supplementary Fig. S4), and caspase-dependentapoptosis (Supplementary Fig. S5).

Integrin aVb3 is involved in resveratrol uptakeConsidering the most important role played by integrin

in signal transduction, we then analyzed whether the spe-cific redistribution of FAK, Fyn, and Ras, together withintegrin b3, in the DRMs was associated with a resvera-trol-induced formation of the integrin signaling complex.Coimmunoprecipitation experiments using anti-integrinaVb3 Ab showed recruitment of FAK, Fyn, Grb2, Ras, andto a lesser extent SOS proteins to integrin aVb3 in SW480

Lipid Rafts Determine Resveratrol Activity

www.aacrjournals.org Cancer Prev Res; 4(7) July 2011 1099

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 6: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

5,000

A

B

C

E

F

G

DSW480 HT29 SW480 HT29

SW480

SW480

HT29

HT29

U937

4,000

3,000

2,000

1,000

1,000

500

0R∗

R30 R30 + monensin

R∗ + R20 R∗ R∗ + R20

1,500

2,000

1,000

500

0

1,500

2,000

2,500

1,000

1,000

00 2

Time (min)4 6

0 2Time (min)

4 6

2,000

3,000

4,000

500

0

1,500

2,000

2,500

1,000

500

0

1,500

2,000

1,000

500

0R30 2 6

MβCD (mg/mL)12

1,500

2,000

2,500

00 100 200 300

Resveratrol (μmol/L)0 100 200

100010

0E

vent

s0

100

Eve

nts

010

0E

vent

s0

100

Eve

nts

101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

Carrier-mediatedtransport

300

Ctl

R30

120

100

80

60

Intr

acel

lula

r [3

H]-

resv

erat

rol (

%)

40

20

0

Co

Chl

or-

prom

azin

e

Mon

odan

syl-

cada

verin

e

Am

ilorid

e

Dyn

asor

e

Mon

ensi

n

MβC

D

Phe

nyla

rsin

eO

xide

Sulforhodamine-101 accumulation

Upt

ake

of [3 H

]-re

sver

atro

l(d

pm/1

06 ce

lls)

Upt

ake

of [3 H

]-re

sver

atro

l(d

pm/1

06 ce

lls)

Upt

ake

of [3 H

]-re

sver

atro

l (dp

m/1

06 ce

lls)

Upt

ake

of [3 H

]-re

sver

atro

l(d

pm/1

06 ce

lls)

Upt

ake

of [3 H

]-re

sver

atro

l(d

pm/1

06 ce

lls)

37°C

4°C

Figure 2. Resveratrol uptake involves a lipid raft–dependent endocytosis. A, concentration-dependent uptake of resveratrol. SW480 and HT29 cells wereexposed to [3H]-resveratrol (0–300 mmol/L) at 37�C (^) and 4�C (*) for 6 minutes. The carrier-mediated transport obtained by subtracting the total37�C uptake from the passive 4�C uptake is also plotted (~). B, cis-inhibition experiments. Colon cancers cell lines were incubated at 37�C (black bars) andat 4�C (white bars) for 6 minutes with [3H]-resveratrol alone (R*) or with a 20-fold excess of unlabeled resveratrol (R20). C, Uptake of [3H]-resveratrol (R30:30 mmol/L; 37�C) by SW480, HT29 colon cancer cells, and U937 leukemic cells with (*) or without (^) monensin (5 mmol/L) for the indicated times. D, flowcytometric analysis of Sulforhodamine-101 endocytosis (25 mg/mL, 30 minutes) and the effect of an endocytosis inhibitor, monensin (5 mmol/L), onSulforhodamine-101 uptake. SW480 and HT29 cells were also left untreated or treated with resveratrol (R30: 30 mmol/L for 6 minutes). Red line, control Ab;black line, Sulforhodamine-101; blue line, Sulforhodamine-101 with monensin (5 mmol/L). One representative of 3 independent experiments is shown. E,SW480 cells were treated with [3H]-resveratrol (R30: 30 mmol/L; 37�C) or combinations of [3H]-resveratrol and inhibitors of endocytosis as described. F, time-dependent uptake of [3H]-resveratrol in SW480 cells treated with [3H]-resveratrol alone (30 mmol/L; 37�C) (^) or the combination of both [3H]-resveratrol andfilipin (1 mg/mL; D), nystatin (12 ng/mL; &), and MbCD (2 mg/mL; �). G, SW480 cells were treated with [3H]-resveratrol (R30: 30 mmol/L; 0.5 mCi/mL)or a combination of [3H]-resveratrol and MbCD at various concentrations (2; 6; 12 mg/mL) during 6 minutes. The uptake of tracer in A, B, C, E, and Fwas determined and expressed in dpm per 106 cells (mean � SD of a representative experiment from 3 independent experiments).

Colin et al.

Cancer Prev Res; 4(7) July 2011 Cancer Prevention Research1100

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 7: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

Upt

ake

of [3 H

]-re

sver

atro

l(d

pm/1

06 ce

lls)

Upt

ake

of [3 H

]-re

sver

atro

l (dp

m/1

06 ce

lls)

[3 H]-

Res

vera

trol

(dp

m/fr

actio

n)μg

/mg

of p

rote

ins

2,000

A C

B D

E

DAPI filter

Co

10 μμm

R30

120

Co

3,000

2,000

1,000

00 6 12

Time (min)

Time (h)0 12 24 36 48

18 24 30

3,000

2,000

1,000

0

pH 7.4 pH 6.0

pH 7.4pH 6

pH 7.4pH 6

pH 7.4 pH 6.0

R30 Co R30 Co R30 Co R30

24 h 48 h

80

40

0

Via

ble

cells

(%

)

FITC filter(Lysosensor)

Merge

1,600

1,200

800

400

600

500

400

300

200

100

0

600

500

400

300

200

100

0

1 2 3 4 5 6 7 8 9 10 11

1

Flotilin 48

kDa

42Cav-2

2 3 4 5 6 7 8 9 10 11

00 2 4

Time (min)6 8 10

Figure 3. Resveratrol accumulates into lipid rafts. A, time-dependent uptake and distribution of [3H]-resveratrol (30 mmol/L; 37�C) in SW480 cells aftersubcellular fractionation. Resveratrol content was measured in whole-cell lysates (&), cytosolic and membrane-fraction (*), and nuclear fraction (~). B, Top,lipid composition of SW480 cell lysate fractions after resveratrol treatment (30 mmol/L for 6 minutes). Black bars, cholesterol; white bars, sphingomyelin.Bottom, distribution of [3H]-resveratrol into cell fractions obtained on sucrose gradient after resveratrol treatment (30 mmol/L, for 6 minutes), in theabsence (&) or presence (*) of MbCD (6 mg/mL). Fractions 3 to 4 represent detergent-resistant rafts characterized by Western blot analysis of proteins raftsmarkers. C, distribution of resveratrol (DAPI filter set) and LysoSensor-positive acidic vesicles (FITC filter set) in SW480 cells untreated (Co) or treatedwith resveratrol (R30: 30 mmol/L for 6 minutes). D, percentages of viable SW480 cells mock treated (Co) or treated with 30 mmol/L resveratrol (R30) for 24and 48 hours in different growth medium acidity conditions (pH 7.4 or 6.0). E, time course of radiolabeled resveratrol uptake in SW480 cells (30 mmol/L)grown at 37�C in different pH conditions. Data in A, B, C, D, and E are means � SD of a representative experiment from 3 independent experiments.

Lipid Rafts Determine Resveratrol Activity

www.aacrjournals.org Cancer Prev Res; 4(7) July 2011 1101

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 8: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

cells exposed to 30 mmol/L resveratrol (Fig. 6A). Moreover,occlusionof theRGD(anarginine-glycine-aspartate peptideinhibitor) binding site in the integrin extracellular domaindiminished the resveratrol uptake (Fig. 6B) and resveratrol-induced apoptosis (Fig. 6C) in cancer cells.

Discussion

The present study shows that resveratrol, which bindsserum proteins such as albumin (26), can enter the cellsthrough both a passive diffusion and a carrier-facilitated

Time (min) 0 1 5 10 1 5 10 0 1 5 10 1 5 10 0 1 5 10 1 5 10 kDa5446

5446

60

60

4442

4442

42

kDa

5446

5446

60

60

4442

4442

SW480A

B C

TPA R30

U937

SW480 U937

SW480

U937

Co

U01

26

U01

26

SP

6001

25

SP

6001

25

LY-2

94

LY-2

9400

2

R30

R30

TPA R30

Monocytes

CSF-1 R30

p-JNK 2/31

JNK 2/31

p-Akt

p-ERK1/2

ERK1/2

β-Actin

50

40

30

20

10

0

Apo

ptot

ic c

ells

(%

)

50

40

30

20

10

0

Akt

p-JNK 2/31

Co R30

R30 + monensin(μmol/L)

R30 + monensin(μmol/L)

5 10 20 Co R30 5 10 20

JNK 2/31

p-Akt

p-ERK1/2

ERK1/2

Akt

Figure 4. Resveratrol-induced apoptosis depends on kinases activation in cancer cells. A, SW480, U937 cells lines, and human blood monocytes were leftuntreated or treated with resveratrol (R30; 30 mmol/L) or with tetradecanoyl phorbol acetate (TPA; 50 nmol/L) or CSF-1 (100 ng/mL) for the indicatedtimes and then harvested. Proteins were extracted forWestern blot analysis, using total and specific Abs to the variousMAPKs. One representative experimentof at least 3 independent experiments is shown. B, SW480 and U937 cells lines were either left untreated (Co) or treated with 30 mmol/L of resveratrol(R30) during 48 hours in the absence or presence of U0126 (U0, MEK inhibitor), SP600125 (JNK inhibitor), or LY294002 (PI3K/Akt inhibitor) before assessmentof apoptosis by Hoechst 33342 staining (means � SD of an independent experiment are shown). C, serum-starved SW480 or U937 cells werepretreated with the indicated concentrations of monensin for 30 minutes prior to resveratrol treatment (R30: 30 mmol/L) and then harvested, and the proteinwere extracted for Western blot analysis, using total and specific Abs to the various MAPKs as in A. One representative experiment of at least 3independent experiments is shown.

Colin et al.

Cancer Prev Res; 4(7) July 2011 Cancer Prevention Research1102

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 9: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

and monensin-sensitive process. Resveratrol accumulatesin DRMs known as lipid rafts and promotes the redistribu-tion of various protein kinases as well as components of theintegrin signaling complex in these membrane fractions.All these events seem to be required for activation of

downstream signaling pathways, leading to cell death intumor cells.

Resveratrol has been proposed to function as a cancerchemopreventive agent through inhibition of promutagenbioactivation and stimulation of carcinogen detoxification

Figure 5. Resveratrol redistributesMAPKs, integrin, and adhesionproteins in the rafts. A, lipidcomposition of Triton X-100lysates from untreated (Co) orresveratrol-treated (R30, 30mmol/L, 10 minutes) SW480 cellsfractionated on a sucrosegradient. Black bars, cholesterol;white bars, sphingomyelin. B,Western blot analysis of theexpression of indicated proteins inabove-defined fractions. Cellswere either left untreated (Co) ortreated with resveratrol (R30, 30mmol/L, 10 minutes) or MbCD (6mg/mL) or the combination ofboth (30 mmol/L resveratrol with6 mg/mL MbCD) beforefractionation and analysis ofindicated proteins. C, MbCDprevents resveratrol-inducedMAPK activation in colon cancercells. Serum-starved SW480 cellswere either untreated (Co) ortreated with resveratrol (R30: 30mmol/L) during 30 minutes with orwithout MbCD (2, 6, and 12 mg/mL). Western blot analysis showstotal and specific Abs to thevarious MAPKs. Onerepresentative experiment of atleast 3 independent experimentsis shown.

54kDa

kDa

R30Co

987654321987654321

987654321987654321

1062R30Cop-JNK 2/3

JNK 2/3

p-Akt

Akt

p-ERK1/2

ERK1/2Fractions

600

400

200

01 2 3 4 5 6 7 8 9

μg/m

g of

pro

tein

s

1

1

p-JNK 2/3

JNK 2/3

p-Akt

Akt

p-ERK1/2

ERK1/2

Fyn

FAK

Ras

Flotilin

Cav-2

Integrin β3

Fractions

1

1

p-JNK 2/3

JNK 2/3

p-Akt

Akt

p-ERK1/2

ERK1/2

Fyn

FAK

Ras

Flotilin

Cav-2

Integrin β3

Fractions

1

1

R30 + MβCD(mg/mL)

A

B

C

R30 + MβCD

465446

60

60

42/44

42/44

54465446

60

60

42/44

42/44

110/133

59

125

21

48

42

kDa54465446

60

60

42/44

42/44

110/133

59

125

21

48

42

MβCD

Lipid Rafts Determine Resveratrol Activity

www.aacrjournals.org Cancer Prev Res; 4(7) July 2011 1103

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 10: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

(2, 27). Resveratrol also has direct cytostatic (6, 7) orcytotoxic effects (4, 10, 12, 15). The later effect can bemediated by apoptosis through activation of both theextrinsic and the intrinsic pathways to caspase activation,oncoproteins, and kinase pathways (4, 10, 12, 15, 24).Mechanisms responsible for the resveratrol chemopreven-tive and chemosensitization properties are poorly knownin tumor cells, but evidence indicates that resveratroluptake and the cancer microenvironment plasma mem-brane play a key role in these processes.

Here, we show that resveratrol activates MAPK path-ways to induce apoptosis both in colon carcinoma celllines and in leukemic lymphoma cells but not in normalcells such as human normal monocytes or rat nontrans-formed intestinal cells in accordance with recent phase I/II trials showing the absence of resveratrol toxicity (28).The inhibition of resveratrol-induced apoptosis by mon-ensin in tumor cells suggests a clathrin-independentendocytosis of the compound. A variety of clathrin-inde-pendent endocytic pathways are responsible for takingup large particles or small solutes, together with mem-brane into cells, and some of them depend on lipid rafts(29–31), including endocytosis of glycosylphosphatidy-

linositol (GPI)-anchored proteins (32, 33), interleukin-2linked to its receptors (34), and several ether lipids (e.g.,alkyl lysophospholipid; refs. 35–37). After initial inser-tion in the outer leaflet of the plasma membrane lipidbilayer, resveratrol accumulates in lipid rafts, as describedfor epigallocatechin (38), and is taken up by lipid raft–dependent, clathrin-independent endocytosis. In addi-tion to endocytosis, disruption of lipid rafts affects resver-atrol-induced activation of downstream pathways,including kinase activation and cell death by caspase-3–mediated apoptosis. Interestingly, resveratrol seems topromote the redistribution of kinases, including ERKsand JNKs, in the lipid rafts, which may facilitate theiractivation and downstream induction of apoptosis (9, 24,39–41).

Resveratrol also induces the redistribution of integrinaVb3 in lipid rafts. Resveratrol binding to integrin aVb3 (8)could account for the activation of ERK1/2 (42). As resver-atrol induced a redistribution of the integrin aVb3 as well asAkt, FAK, Fyn, and Ras proteins in lipid rafts, our datasupport that these microdomains may function as micro-compartments for the assembly of signaling complexes(43). On integrin ligation, Fyn is activated and binds to

50

40

30

30

20

20

10

10

0

048 72

Time (h)

U937

SW480Co

kDaR30CoIrrWB:

FAK 125

59

25

21

170

110

Fyn

Grb2

Ras

SOS 1

Integrin β3

IP: Integrin αvβ3A

B

CRGD

R30

R30 + RGD

R30

0 2 4 6Time (min)

Upt

ake

of [3

H]-

resv

erat

rol

(dpm

/106

cel

ls)

2,500

2,000

1,500

1,000

500

0

R30 + peptide RGD

Apo

ptot

ic c

ells

(%

)

40

Figure 6. Involvement of integrinaVb3 in resveratrol uptake andproapoptotic activity in SW480cells. A, serum-starved SW480cells were left untreated (Co) ortreated with resveratrol (R30, 30mmol/L) during 10 minutes, andprotein extracts were submitted toimmunoprecipitation with ananti-integrin aVb3 Ab or anirrelevant Ab (Irr). Onerepresentative experiment of atleast 3 independent experimentsis shown. B, uptake of [3H]-resveratrol (30 mmol/L; 37�C) bySW480 colon cancer cells with (*)or without (^) peptide RGD (500nmol/L) for the indicated times. C,integrin antagonist peptide RGDdecreases resveratrol-inducedapoptosis. SW480 and U937 celllines were left untreated (Co) ortreated with resveratrol (R30, 30mmol/L), with or without RGD (500nmol/L), for indicated times beforeassessment of apoptosis byHoechst 33342 staining. Data in Band C are means � SD of arepresentative experiment from 3experiments.

Colin et al.

Cancer Prev Res; 4(7) July 2011 Cancer Prevention Research1104

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 11: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

SHC, via the SH3 domain of Fyn. It has been previouslyreported that this sequence of events is necessary to coupleintegrins to the Ras/ERK pathway (44).Taken together, these findings show that accumulation

of resveratrol in lipid rafts and active endocytosis arerequired for resveratrol to activate kinase-dependent sig-naling pathways (ERK/JNK/Akt) and to trigger caspase-dependent apoptosis in cancer cells. These results confirmthe importance of lipid rafts in the biological effects ofresveratrol. Initial reports have shown the essential roleplayed by rafts in the initiation of Fas-mediated apoptosiswith ether lipids (36, 45). In a similar manner, we havepreviously shown that lipid rafts play a role in clusteringor aggregating surface receptors and adaptor moleculesinto membrane complexes at specific sites and are shownto be essential for initiating signaling from a number ofreceptors, particularly in the initiation of Fas-mediatedapoptosis during resveratrol treatment (4). These micro-domains sequester the polyphenol, as described recentlyfor autocrine motility factor (AMF)/phosphoglucose iso-merase (PGI)-paclitaxel (46), and therefore function as aplatform that finely tunes up the tumor cell response tothis agent, either directly by inducing cell-cycle arrest orcell death (47) or indirectly by sensitizing the cells tochemotherapeutic agents (11, 15). This sensitizationcould involve the inhibition of the Hþ-ATP synthase byresveratrol (48). Similarly to the proton pump inhibitoromeprazole (49), resveratrol could affect the lysosomalfunction and reduce cell environment acidity, therebyfacilitating the drug activity (50). Interestingly, Parolini

and colleagues have described that acidity may alsochange the lipid composition of tumor cell membraneand consequently play an important role in the inter-nalization of exosomes in tumor cells (51). In the modelsof colon carcinoma used in this study, an acidification ofthe medium does not affect the antiproliferative effectof resveratrol and its uptake. In addition, a part of poly-phenol uptake involves a passive process that couldprecede intracellular specific interaction (e.g., aryl hydro-carbon and estrogen receptors). Further insights in theuptake mechanisms of resveratrol at the cytoplasmicmembrane level will provide a better understanding ofthe capability of tumor cells to accumulate the moleculeand to detail the relationships between accumulation andearly events leading to apoptosis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This study was supported by the "Conseil R�egional de Bourgogne" and"Ligue contre le Cancer", comit�e Cote d’Or et Jura. A. Jacques received apostdoctoral fellowship from the Ligue Nationale Contre le Cancer thatsupports ES group.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 10, 2010; revised March 9, 2011; accepted March 28,2011; published OnlineFirst April 5, 2011.

References1. Renaud SC, Gueguen R, Schenker J, d’Houtaud A. Alcohol and

mortality in middle-aged men from eastern France. Epidemiology1998;9:184–8.

2. Delmas D, Lancon A, Colin D, Jannin B, Latruffe N. Resveratrol as achemopreventive agent: a promising molecule for fighting cancer.Curr Drug Targets 2006;7:423–42.

3. Kundu JK, Surh YJ. Cancer chemopreventive and therapeutic poten-tial of resveratrol: mechanistic perspectives. Cancer Lett 2008;269:243–61.

4. Delmas D, Rebe C, Lacour S, Filomenko R, Athias A, Gambert P, et al.Resveratrol-induced apoptosis is associated with Fas redistribution inthe rafts and the formation of a death-inducing signaling complex incolon cancer cells. J Biol Chem 2003;278:41482–90.

5. Ahmad N, Katiyar SK, Mukhtar H. Antioxidants in chemoprevention ofskin cancer. Curr Probl Dermatol 2001;29:128–39.

6. Delmas D, Passilly-Degrace P, Jannin B, Malki MC, Latruffe N.Resveratrol, a chemopreventive agent, disrupts the cell cycle con-trol of human SW480 colorectal tumor cells. Int J Mol Med 2002;10:193–9.

7. Kuwajerwala N, Cifuentes E, Gautam S, MenonM, Barrack ER, ReddyGP. Resveratrol induces prostate cancer cell entry into s phase andinhibits DNA synthesis. Cancer Res 2002;62:2488–92.

8. Lin HY, Tang HY, Keating T, Wu YH, Shih A, Hammond D, et al.Resveratrol is pro-apoptotic and thyroid hormone is anti-apoptotic inglioma cells: both actions are integrin and ERK mediated. Carcino-genesis 2008;29:62–9.

9. She QB, Huang C, Zhang Y, Dong Z. Involvement of c-jun NH(2)-terminal kinases in resveratrol-induced activation of p53 and apop-tosis. Mol Carcinog 2002;33:244–50.

10. Bernhard D, Tinhofer I, Tonko M, Hubl H, Ausserlechner MJ, Greil R,et al. Resveratrol causes arrest in the S-phase prior to Fas-indepen-dent apoptosis in CEM-C7H2 acute leukemia cells. Cell Death Differ2000;7:834–42.

11. Delmas D, Rebe C, Micheau O, Athias A, Gambert P, Grazide S, et al.Redistribution of CD95, DR4 and DR5 in rafts accounts for thesynergistic toxicity of resveratrol and death receptor ligands in coloncarcinoma cells. Oncogene 2004;23:8979–86.

12. Dorrie J, Gerauer H, Wachter Y, Zunino SJ. Resveratrol inducesextensive apoptosis by depolarizing mitochondrial membranes andactivating caspase-9 in acute lymphoblastic leukemia cells. CancerRes 2001;61:4731–9.

13. Fulda S, Debatin KM. Sensitization for anticancer drug-inducedapoptosis by the chemopreventive agent resveratrol. Oncogene2004;23:6702–11.

14. Harikumar KB, Kunnumakkara AB, Sethi G, Diagaradjane P, AnandP, Pandey MK, et al. Resveratrol, a multitargeted agent, canenhance antitumor activity of gemcitabine in vitro and in orthotopicmouse model of human pancreatic cancer. Int J Cancer. 2009;127:257–68.

15. Reis-Sobreiro M, Gajate C, Mollinedo F. Involvement of mitochondriaand recruitment of Fas/CD95 signaling in lipid rafts in resveratrol-mediated antimyeloma and antileukemia actions. Oncogene2009;28:3221–34.

16. Jacquel A, Benikhlef N, Paggetti J, Lalaoui N, Guery L, Dufour EK,et al. Colony-stimulating factor-1-induced oscillations in phosphati-dylinositol-3 kinase/AKT are required for caspase activation in mono-cytes undergoing differentiation into macrophages. Blood 2009;114:3633–41.

Lipid Rafts Determine Resveratrol Activity

www.aacrjournals.org Cancer Prev Res; 4(7) July 2011 1105

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 12: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

17. Colin D, Lancon A, Delmas D, Lizard G, Abrossinow J, Kahn E, et al.Antiproliferative activities of resveratrol and related compounds inhuman hepatocyte derived HepG2 cells are associated with biochem-ical cell disturbance revealed by fluorescence analyses. Biochimie2008;90:1674–84.

18. Tartakoff AM. Perturbation of vesicular traffic with the carboxylicionophore monensin. Cell 1983;32:1026–8.

19. Rejman J, Bragonzi A, Conese M. Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and poly-plexes. Mol Ther 2005;12:468–74.

20. Pierce KL, Maudsley S, Daaka Y, Luttrell LM, Lefkowitz RJ. Role ofendocytosis in the activation of the extracellular signal-regulatedkinase cascade by sequestering and nonsequestering G protein-coupled receptors. Proc Natl Acad Sci U S A 2000;97:1489–94.

21. Gibson AE, Noel RJ, Herlihy JT, Ward WF. Phenylarsine oxide inhibi-tion of endocytosis: effects on asialofetuin internalization. Am JPhysiol 1989;257:C182–4.

22. KoivusaloM,Welch C, Hayashi H, Scott CC, KimM, Alexander T, et al.Amiloride inhibits macropinocytosis by lowering submembranous pHand preventing Rac1 and Cdc42 signaling. J Cell Biol 2010;188:547–63.

23. Kirchhausen T, Macia E, Pelish HE. Use of dynasore, the smallmolecule inhibitor of dynamin, in the regulation of endocytosis.Methods Enzymol 2008;438:77–93.

24. She QB, Bode AM, Ma WY, Chen NY, Dong Z. Resveratrol-inducedactivation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer Res 2001;61:1604–10.

25. Lin HY, Lansing L, Merillon JM, Davis FB, Tang HY, Shih A, et al.Integrin alphaVbeta3 contains a receptor site for resveratrol. FASEB J2006;20:1742–4.

26. Jannin B, Menzel M, Berlot JP, Delmas D, Lancon A, Latruffe N.Transport of resveratrol, a cancer chemopreventive agent, to cellulartargets: plasmatic protein binding and cell uptake. Biochem Pharma-col 2004;68:1113–8.

27. Ciolino HP, Daschner PJ, Yeh GC. Resveratrol inhibits transcription ofCYP1A1 in vitro by preventing activation of the aryl hydrocarbonreceptor. Cancer Res 1998;58:5707–12.

28. Brown VA, Patel KR, Viskaduraki M, Crowell JA, Perloff M, BoothTD, et al. Repeat dose study of the cancer chemopreventive agentresveratrol in healthy volunteers: safety, pharmacokinetics, andeffect on the insulin-like growth factor axis. Cancer Res 2010;70:9003–11.

29. Ikonen E. Roles of lipid rafts in membrane transport. Curr Opin CellBiol 2001;13:470–7.

30. Nichols BJ, Lippincott-Schwartz J. Endocytosis without clathrincoats. Trends Cell Biol 2001;11:406–12.

31. Parton RG. Caveolae and caveolins. Curr Opin Cell Biol 1996;8:542–8.32. Nichols BJ. A distinct class of endosome mediates clathrin-indepen-

dent endocytosis to the Golgi complex. Nat Cell Biol 2002;4:374–8.33. Nichols BJ, Kenworthy AK, Polishchuk RS, Lodge R, Roberts TH,

Hirschberg K, et al. Rapid cycling of lipid raft markers between the cellsurface and Golgi complex. J Cell Biol 2001;153:529–41.

34. Lamaze C, Dujeancourt A, Baba T, Lo CG, Benmerah A, Dautry-VarsatA. Interleukin 2 receptors and detergent-resistant membrane domainsdefine a clathrin-independent endocytic pathway. Mol Cell 2001;7:661–71.

35. Van Der Luit AH, Budde M, Ruurs P, Verheij M, van Blitterswijk WJ.Alkyl-lysophospholipid accumulates in lipid rafts and induces apop-tosis via raft-dependent endocytosis and inhibition of phosphatidyl-choline synthesis. J Biol Chem 2002;277:39541–7.

36. Gajate C, Del Canto-Janez E, Acuna AU, Amat-Guerri F, Geijo E,Santos-Beneit AM, et al. Intracellular triggering of Fas aggregationand recruitment of apoptotic molecules into Fas-enriched rafts inselective tumor cell apoptosis. J Exp Med 2004;200:353–65.

37. Gajate C, Gonzalez-Camacho F, Mollinedo F. Involvement of raftaggregates enriched in Fas/CD95 death-inducing signaling complexin the antileukemic action of edelfosine in Jurkat cells. PLoS One2009;4:e5044.

38. Hong J, Lu H, Meng X, Ryu JH, Hara Y, Yang CS. Stability, cellularuptake, biotransformation, and efflux of tea polyphenol (�)-epigallo-catechin-3-gallate in HT-29 human colon adenocarcinoma cells.Cancer Res 2002;62:7241–6.

39. Lin HY, Shih A, Davis FB, Tang HY, Martino LJ, Bennett JA, et al.Resveratrol induced serine phosphorylation of p53 causes apoptosisin a mutant p53 prostate cancer cell line. J Urol 2002;168:748–55.

40. Shih A, Davis FB, Lin HY, Davis PJ. Resveratrol induces apoptosis inthyroid cancer cell lines via a MAPK- and p53-dependent mechanism.J Clin Endocrinol Metab 2002;87:1223–32.

41. Patra SK. Dissecting lipid raft facilitated cell signaling pathways incancer. Biochim Biophys Acta 2008;1785:182–206.

42. Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding tointegrins. J Biol Chem 2000;275:21785–8.

43. Baillat G, Siret C, Delamarre E, Luis J. Early adhesion induces inter-action of FAK and Fyn in lipid domains and activates raft-dependentAkt signaling in SW480 colon cancer cells. Biochim Biophys Acta2008;1783:2323–31.

44. Wary KK, Mariotti A, Zurzolo C, Giancotti FG. A requirement forcaveolin-1 and associated kinase Fyn in integrin signaling and ancho-rage-dependent cell growth. Cell 1998;94:625–34.

45. Gajate C, Mollinedo F. The antitumor ether lipid ET-18-OCH(3)induces apoptosis through translocation and capping of Fas/CD95into membrane rafts in human leukemic cells. Blood 2001;98:3860–3.

46. Kojic LD,Wiseman SM, Ghaidi F, Joshi B, NedevH, Saragovi HU, et al.Raft-dependent endocytosis of autocrine motility factor/phosphoglu-cose isomerase: a potential drug delivery route for tumor cells. PLoSOne 2008;3:e3597.

47. Colin D, Gimazane A, Lizard G, Izard JC, Solary E, Latruffe N, et al.Effects of resveratrol analogs on cell cycle progression, cell cycleassociated proteins and 5fluoro-uracil sensitivity in human derivedcolon cancer cells. Int J Cancer 2009;124:2780–8.

48. Dadi PK, Ahmad M, Ahmad Z. Inhibition of ATPase activity of Escher-ichia coli ATP synthase by polyphenols. Int J Biol Macromol2009;45:72–9.

49. Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, Montinaro A,et al. Effect of proton pump inhibitor pretreatment on resistance ofsolid tumors to cytotoxic drugs. J Natl Cancer Inst 2004;96:1702–13.

50. Lugini L, Matarrese P, Tinari A, Lozupone F, Federici C, Iessi E, et al.Cannibalism of live lymphocytes by humanmetastatic but not primarymelanoma cells. Cancer Res 2006;66:3629–38.

51. Parolini I, Federici C, Raggi C, Lugini L, Palleschi S, De Milito A, et al.Microenvironmental pH is a key factor for exosome traffic in tumorcells. J Biol Chem 2009;284:34211–22.

Colin et al.

Cancer Prev Res; 4(7) July 2011 Cancer Prevention Research1106

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274

Page 13: Endocytosis of Resveratrol via Lipid Rafts and Activation ...cancerpreventionresearch.aacrjournals.org/content/canprevres/4/7/... · Research Article Endocytosis of Resveratrol via

2011;4:1095-1106. Published OnlineFirst April 5, 2011.Cancer Prev Res   Didier Colin, Emeric Limagne, Sylvie Jeanningros, et al.   Downstream Signaling Pathways in Cancer CellsEndocytosis of Resveratrol via Lipid Rafts and Activation of

  Updated version

  10.1158/1940-6207.CAPR-10-0274doi:

Access the most recent version of this article at:

  Material

Supplementary

  -10-0274.DC1

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2011/07/05/1940-6207.CAPRAccess the most recent supplemental material at:

   

   

  Cited articles

  http://cancerpreventionresearch.aacrjournals.org/content/4/7/1095.full#ref-list-1

This article cites 51 articles, 18 of which you can access for free at:

  Citing articles

  http://cancerpreventionresearch.aacrjournals.org/content/4/7/1095.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  SubscriptionsReprints and

  [email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerpreventionresearch.aacrjournals.org/content/4/7/1095To request permission to re-use all or part of this article, use this link

for Cancer Research. on May 4, 2018. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0274