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Redistribution of CD95, DR4 and DR5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells

Abstract

The natural phytoalexin resveratrol (3, 5, 4′-trihydroxystilbene) exhibits both chemopreventive and antitumor activities through a variety of mechanisms. We have shown previously that resveratrol-induced apoptosis of a human colon cancer cell line involved the redistribution of CD95 (Fas/Apo-1) into lipid rafts. Here, we show that, in colon cancer cells that resist to resveratrol-induced apoptosis, the polyphenol also induces a redistribution of death receptors into lipid rafts. This effect sensitizes these tumor cells to death receptor-mediated apoptosis. In resveratrol-treated cells, tumor necrosis factor (TNF), anti-CD95 antibodies and TNF-related apoptosis-inducing ligand (TRAIL) activate a caspase-dependent death pathway that escapes Bcl-2-mediated inhibition. Resveratrol does not enhance the number of death receptors at the surface of tumor cells but induces their redistribution into lipid rafts and facilitates the caspase cascade activation in response to death receptor stimulation. The cholesterol sequestering agent nystatin prevents resveratrol-induced death receptor redistribution and cell sensitization to death receptor stimulation. Thus, whatever its ability to induce apoptosis in a tumor cell, resveratrol induces redistribution of death receptors into lipid rafts. This redistribution sensitizes the cells to death receptor stimulation. Such a sensitizing effect may be of therapeutic interest if TRAIL agonists are introduced in clinics.

Introduction

The polyphenol resveratrol (3, 5, 4′-trihydroxystilbene) has demonstrated a cancer chemopreventive activity in animal models of carcinogenesis (Jang et al., 1997). This activity has been related to cell cycle inhibition, promotion of cell differentiation, suppression of angiogenesis and/or induction of cell death, mainly by apoptosis (Clement et al., 1998; Bernhard et al., 2000; Brakenhielm et al., 2001; Dorrie et al., 2001; Tinhofer et al., 2001; Joe et al., 2002; Delmas et al., 2003; Opipari et al., 2004). The pathways that mediate resveratrol-induced apoptosis can involve p53 (Huang et al., 1999), ceramide generation (Scarlatti et al., 2003) and the death receptor CD95 (Fas/Apo-1) (Clement et al., 1998).

Redistribution of CD95 in lipid rafts, which are plasma membrane microdomains enriched in cholesterol and glycosphingolipids, regulates the efficacy of signaling by CD95 and other death receptors (Hueber, 2003; Muppidi and Siegel, 2004). We have demonstrated recently that resveratrol-induced apoptosis of a colon cancer cell line involved the ligand-independent redistribution of CD95 into lipid rafts (Delmas et al., 2003). All the cancer cells are not equally sensitive to resveratrol-induced cell death. In the present study, we show that, in cells that resist to resveratrol-mediated apoptosis, the polyphenol still induces the redistribution of CD95 and other death receptors in lipid rafts. Although this redistribution is not sufficient to trigger cell death, it contributes to their sensitization to death receptor agonists, in accordance with the recently described synergy between reseveratrol and the death receptor ligand TRAIL (TNF-related apoptosis-inducing ligand) (Fulda and Debatin, 2004).

Results

All colon carcinoma cell lines are not equally sensitive to resveratrol-induced apoptosis

The ability of resveratrol to trigger apoptosis was studied in four human colon cancer cell lines by measuring the percentage of cells with a condensed nuclear chromatin, as identified by Hoechst 33342 staining, after a 72 h exposure to three different concentrations of the studied compound. SW620 cells appeared to be the most sensitive and HT29 the most resistant to resveratrol-induced cell death (Figure 1). Apoptosis was associated with caspase-3 activation and poly(ADP-ribose)polymerase (PARP) cleavage in sensitive cells, which was not detected in resistant cells treated in the same conditions (Figure 1, inset).

Figure 1
figure1

Differential sensitivity of colon carcinoma cell lines to resveratrol-induced apoptosis. Indicated human colon cancer cell lines were left untreated or treated for 72 h with 30 (R30), 50 (R50) or 100 (R100) μ M resveratrol before measuring the percentage of apoptosis in cells stained with Hoechst 33342 (mean ±s.d. of three independent experiments; 300 cells/point). Inset – Upper panel: Flow cytometry analysis of active caspase-3 in HT29 and SW480 cells left untreated (Co) or treated for 72 h with 30 μ M resveratrol (R30). Gray areas: Isotype-matched control Ab. White areas: Antiactive caspase-3 Ab. Lower panel: Western blot analysis of the 116 kDa PARP and its 89 kDa cleavage fragment. The 42 kDa actin was used for loading control

Resveratrol sensitizes resistant colon tumor cells to death receptor ligands

HT29 cells demonstrate limited sensitivity to death receptor-mediated cell death (Micheau et al., 1999; Lacour et al., 2001), for example, exposure to an agonistic anti-human CD95 Ab (clone CH11, 100 ng/ml) or recombinant tumor necrosis factor (TNF)α (25 ng/ml) or recombinant TRAIL (100 ng/ml) for 24 h induces apoptosis in less than 10% of these cells. Pretreatment with either 10 or 30 μ M resveratrol for 24 h dramatically enhanced HT29 cell apoptosis upon death receptor stimulation. In this synergistic effect with resveratrol, TRAIL appeared to be the most potent ligand. Similar observations were made in HCT116 cells (Figure 2).

Figure 2
figure2

Resveratrol sensitizes resistant colon cancer cells to death receptor ligands. HT29 and HCT116 cells were either left untreated (Co) or treated for 48 h with 10 (R10) or 30 (R30) μ M resveratrol. When indicated, CH11 (100 ng/ml with 0.8 μg/ml of cycloheximide) or TNFα (25 ng/ml) or TRAIL (100 ng/ml with 2 μg/ml enhancer) was added to the culture medium for the last 24 h. The percentage of cells with a condensed nuclear chromatin, as identified by Hoechst 33342 staining, was measured at the end of cell treatment (mean±s.d. of three independent experiments; 300 cells/point)

Resveratrol sensitizes colon cancer cells to CD95- and TRAIL-mediated caspase activation

Resveratrol (30 μ M for 48 h) and the death receptor agonists (CH11 anti-CD95 Abs and TRAIL at above-indicated concentrations for 24 h) demonstrated limited ability to activate caspases in HT29 cells, whereas the combination of resveratrol and a death receptor agonist generated strong caspase activation (Figure 3). This was demonstrated by studying the ability of HT29 cell lysates to cleave DEVD-AMC, LEHD-AFC and IETD-AFC fluorogenic peptides that mimic the target sites of caspase-3 (DEVD-AMC), caspase-9 (LEHD-AFC) and caspase-8 (IETD-AFC), respectively (Figure 3a), and confirmed by fluorescence microscopy (Figure 3b) and flow cytometry (Figure 3c) analysis of active caspases. The broad-spectrum caspase inhibitor z-VAD-fmk and more specific permeant caspase inhibitors prevented caspase activation induced by the resveratrol/death receptor agonist combinations (Figure 3a and b). More specific permeant caspase inhibitors prevented caspase-3 activation in HT29 cells exposed to the resveratrol/CH11 combination (Figure 3c).

Figure 3
figure3

Pretreatment with resveratrol sensitizes colon cancer cells to caspase activation by death receptor agonists. (a) HT29 cells were left untreated (Co) or treated with 30 μ M resveratrol for 48 h (R30). When indicated, CH11 Ab or TRAIL were added to the culture medium for the last 24 h in the absence or presence of 50 μ M z-VAD-fmk (zVAD), before measuring the ability of cell lysates to hydrolyse DEVD-AMC, LEHD-AFC and IETD-AFC caspase substrates, respectively (mean±s.d. of three independent experiments). Enzyme activities: ‘fold increase’ related to the control value. (b) Fluorescence microscopy analysis of active caspase-3 (C3a), caspase-9 (C9a) and caspase-8 (C8a) in HT29 cells treated as in (a). Active caspases were assigned the color green and nuclei, labeled with Hoechst 33342, were assigned the color blue. Representative cells of three independent experiments are shown. (c) HT29 cells were left untreated (Co) or treated with 30 μ M resveratrol (R30) for 48 h or CH11 for 24 h or the combination of both. In this latter case, cells were treated with resveratrol for 48 h and CH11 was added for the last 24 h, in the absence (Co) or presence of indicated permeant caspase inhibitors (50 μ M). Right panel: flow cytometry analysis of active caspase-3 (Gray areas: Isotype-matched control Ab; white areas: antiactive caspase-3 Ab). Left panel: The percentage of apoptotic cells (Hoechst 33342 staining) was measured at the end of cell treatment (mean±s.d. of three independent experiments; 300 cells/point). Similar observations were made when TRAIL was added to resveratrol-pretreated cells (not shown)

The resveratrol–death receptor agonist combination partially overcomes the mitochondria

We have shown previously that apoptosis of SW480 cells exposed to resveratrol involved cytochrome c release from the mitochondria (Delmas et al., 2003). The involvement of mitochondria in death receptor-mediated apoptosis depends on the cell type (Scaffidi et al., 1998). We show here that Bcl-2 overexpression in HT29 cells prevents apoptosis induced by the cisplatin/TRAIL combination but fails to prevent apoptosis induced by the resveratrol/CH11 or /TRAIL combination (Figure 4a and b). This was confirmed by a kinetic and comparative analysis of apoptosis induced by the resveratrol/CH11 and the cisplatin/CH11 combinations (Figure 4c). A 10 μ M concentration of resveratrol was required to sensitize HT29 cells to CH11-induced apoptosis, both in control- and Bcl-2-expressing HT29 cells (Figure 4d). Pretreatment with resveratrol did not modify the effect of CH11 or TRAIL on ROS production (Figure 4e). Altogether, these results suggest that the apoptotic pathway activated by the resveratrol/death receptor agonist combination partially overcomes the mitochondria.

Figure 4
figure4

Influence of Bcl-2 and role of ROS in resveratrol-induced apoptosis. (a) Inset: Western blot analysis of Bcl-2 protein in HT29 cells stably transfected with an empty (Co) or a bcl-2 encoding (Bcl-2) vector. The 42 kDa actin was used for loading control. (a) The percentage of apoptotic cells was determined as in Figure 1 in control (white bars) and bcl-2-transfected (black bars) HT29 cells left untreated or treated with either resveratrol (30 μ M, R30). When indicated, CH11 or TRAIL was added for the last 24 h. As a control, these cells were also treated with 16 μ M cisplatin (CDDP) alone (Co) or in combination with TRAIL (mean±s.d. of three independent experiments in duplicate). (b) Cleavage of indicated caspase peptide substrates by lysates obtained from control (white bars) and bcl-2-transfected (black bars) HT29 cells treated as above (mean±s.d. of three independent experiments; NS: not significant; *P<0.05, Student's t-test). (c) Apoptosis was measured in control (•) and bcl-2-transfected (▪) HT29 cells treated with resveratrol (30 μ M) for 24 h before adding CH11 Abs for indicated times or with the combination of CDDP (16 μ M) and CH11 for indicated times before measuring the percentage of apoptotic cells using Hoechst 33342 staining (mean±s.d. of three experiments). (d) Apoptosis was measured in control (white bars) and bcl-2-transfected (black bars) HT29 cells pretreated with indicated concentrations of resveratrol (e.g. R1: 1 μ M) for 24 h before adding CH11 for 24 h (mean±s.d. of three experiments). (e) HT29 cells were exposed either to 30 μ M resveratrol for 48 h (R30) or CH11 Ab or TRAIL or their combination (as in Figure 2) before measuring ROS production by using dihydroethidine as a substrate in a flow cytometry assay. Gray areas: untreated cells; white areas: treated cells. NS: not significant; *P<0.05 (Student's t-test)

Resveratrol facilitates the death-inducing signaling complex (DISC) formation in response to CD95-L

Exposure of HT29 and HCT116 cells to resveratrol did not induce any significant increase in the expression of CD95 and the two agonistic receptors of TRAIL (DR4 and DR5) at the plasma membrane level. In addition, neither CD95-L, nor TRAIL, nor the decoy receptors of TRAIL known as DcR1 and DcR2 could be identified on these cells, and resveratrol exposure did not induce significant expression of these proteins at their surface (data not shown). Engagement of CD95 with recombinant CD95-L induced the formation of CD95 aggregates and the time-dependent recruitment of FADD, procaspase-8 and FLIPL (DISC-associated cellular FLICE-like Inhibitory Protein) in the death-inducing signaling complex (DISC) (Figure 5a). A pretreatment with resveratrol slightly enhanced the formation of both CD95 aggregates and DISCs in response to CD95-L (Figure 5a). Engagement of death receptors with TRAIL induced the formation of a DR5-containing DISC (Figure 5b). Again, a pretreatment with resveratrol slightly enhanced the recruitment of the DISC proteins and the cleavage of procaspase-8 into active fragments (Figure 5b).

Figure 5
figure5

Resveratrol facilitates the DISC formation. (a) HT29 cells were treated with either vehicle or 30 μ M of resveratrol for 24 h before exposure to CD95-L (1 μg/ml) for indicated times. Immunoprecipitation was performed with the anti-FLAG M2 Ab (2 μg/ml), before Western blot analysis of indicated proteins. Numbers on the right indicate molecular weight in kDa and *Caspase-8 cleavage fragments. (b) HT29 cells were treated as above before exposure to TRAIL (500 ng/ml with 2 μg/ml anti-Flag Ab) for indicated times. DISCs were immunoprecipitated with protein G and analysed for indicated proteins by immunoblotting. In unstimulated controls (0), TRAIL and M2 mAb were added after lysis to immunoprecipitate nonstimulated TRAIL receptors

Resveratrol redistributes death receptors in lipid rafts

Resveratrol, but not TNFα, induced the formation of large patches of CD95 protein at the surface of HCT116 and HT29 cells (Figure 6a). These CD95 patches colocalized with caveolin-2, a protein described as a marker for raft-associated caveolae (Figure 6b). To better identify lipid rafts, cell fractions enriched in cholesterol and sphingomyelin and expressing caveolin-2 were isolated on a sucrose gradient. Exposure of HT29 cells to resveratrol triggered the redistribution of CD95, DR4, DR5, the adapter protein FADD and procaspase-8 in these fractions, whereas procaspase-3 was not redistributed. This effect was associated with a depletion of lipid rafts in cholesterol and sphingomyelin. The cholesterol-sequestering molecule nystatin suppressed resveratrol-induced changes in lipid raft components, including cholesterol, sphingomyelin and the DISC components (Figure 7).

Figure 6
figure6

Resveratrol induces the clustering of CD95 receptor at the surface of cancer cells. (a) HT29 and HCT116 cells were left untreated (Control) or treated with 30 μ M resveratrol (R30) or with 10 ng/ml TNF-α for 48 h before CD95 staining using the ZB4 monoclonal antibody (Immunotech) and confocal laser scanning microscopy analysis. (b) Cells were left untreated (Co) or treated with 30 μ M resveratrol (R30) for 48 h before confocal laser scanning microscopy analysis of caveolin-2 (Cav-2) and CD95 coexpression (in all cases: magnification × 50)

Figure 7
figure7

Resveratrol redistributes the death receptors in the rafts. Upper panels: Lipid composition of HT29 cell lysate fractions. HT29 cells were either left untreated (Control) or treated with resveratrol (R30: 30 μ M for 48 h) or nystatin (20 ng/ml for 12 h) or the combination of both (30 μ M resveratrol for 48 h with 20 ng/ml nystatin added in the last 12 h). Black bars, cholesterol; white bars, sphingomyelin. Lower panels: Western blot analysis of the expression of indicated proteins in above-defined fractions. Numbers on the right indicate molecular weight in kDa

In HT29 cells exposed to resveratrol alone for 24 h, then to the resveratrol/CH11 combination for 12 h, addition of nystatin with CH11 almost completely prevented CH11-induced apoptosis (Figure 8). In HT29 cells exposed to resveratrol alone for 24 h, then to the resveratrol/CH11 combination for 24 h, addition of nystatin during the last 12 h of CH11 treatment completely prevented further increase in apoptosis (Figure 8). Similar results were obtained with the resveratrol/TRAIL combination (not shown).

Figure 8
figure8

Nystatin prevents death receptor-mediated cell death in resveratrol-treated HT29 cells. HT29 cells were left untreated or treated with either resveratrol (R30: 30 μ M for 48 h) or CH11 antibodies (as in Figure 2) or nystatin (20 ng/ml for 12 h). Cells were also exposed to resveratrol alone for 24 h before adding CH11 antibodies (R30/CH11) for 12 or 24 h, in the absence (control) or presence (nystatin) of 20 ng/ml added for the last 12 h of treatment

Discussion

Resveratrol triggers death in some cancer cells, which has suggested that the molecule could be used as a chemotherapeutic as well as a chemopreventive drug (Pervaiz, 2003). This compound could also be used as a sensitizer, for example, it sensitizes cancer cells to death induced by cytotoxic drugs and the death receptor ligand TRAIL (Fulda and Debatin, 2004). Here, we show that, in human colon cancer cells that resist to its cytotoxic effect, resveratrol induces a redistribution of death receptors into lipid rafts. Engagement of these receptors then activates a caspase-dependent pathway to apoptosis that is not blocked by overexpression of Bcl-2. Inhibition of death receptor redistribution by nystatin demonstrates that this event contributes to the sensitizing effect of resveratrol to death receptor agonists.

CD95 (Fas/Apo-1) has become the paradigm for the study of the extrinsic pathway to apoptosis. Two cell-type-specific signaling pathways have been described to lead from CD95 engagement to apoptosis. Cells that show optimal formation of CD95 DISC initiate a direct caspase cascade that is not influenced by Bcl-2 overexpression, whereas a crosstalk with the intrinsic, mitochondria-dependent pathway to death is required by cells showing weak DISC formation. This latter pathway is blocked by Bcl-2 overexpression (Scaffidi et al., 1998). We show here that stimulation of death receptors at the surface of resveratrol-treated colon cancer cells activates a caspase-dependent pathway to apoptosis that is not affected by Bcl-2. In contrast, sensitization of cancer cells to death receptor-mediated apoptosis by exposure to DNA-damaging agents does not overcome Bcl-2 (Micheau et al., 1997; Lacour et al., 2003). The differential efficacy of resveratrol and anticancer drugs in enhancing the cancer cell response to death receptor stimulation cannot be related to the differential expression of death receptors at their surface (Sheikh et al., 1998; Rohn et al., 2001), nor by the differential redistribution of these receptors in lipid rafts (Keane et al., 1999; Lacour et al., 2004), nor by the differential recruitment and activation of caspase-8 (Donepudi et al., 2003), nor by distinct changes at the level of c-FLIPL (Micheau et al., 2002). Other parameters that could modulate DISC signaling efficacy include death receptor linkage to the actin cytoskeleton through ezrin, as demonstrated for CD95 (Lozupone et al., 2003), the stoichiometry of death receptor complexes (Thorburn, 2004) and their location within the cell (Micheau and Tschopp, 2003).

Pretreatment of HT29 colon cancer cells with resveratrol facilitates the formation of the DISC at the plasma membrane level. However, this effect remains limited as resveratrol only slightly enhances the recruitment of the DISC components and the cleavage of procaspase-8 in this complex when it forms in response to death receptor agonists. One possibility is that resveratrol facilitates caspase-8 activation at the DISC level in the absence of cleavage (Boatright et al., 2003). Another possibility is that caspase-8 activity is amplified through transactivation by other caspases, downstream of the DISC formation (Wieder et al., 2001).

Death receptor ligands bind to death receptors probably in the form of preassociated trimers (Chan et al., 2000; Siegel et al., 2000). However, a single CD95-L molecule bound to a CD95 trimeric receptor is not sufficient to induce apoptosis (Holler et al., 2003). It appears that a death receptor complex must consist of at least two ligands bound to a hexameric receptor (two preformed trimers) to signal apoptosis and other pathways (for a review see Thorburn, 2004). Actually, the multimerization of trimers could generate aggregates of activated receptors that produce high local concentration of DISC (Algeciras-Schimnich et al., 2002). Clustering of CD95 has been shown to occur in lipid rafts of T-cell plasma membrane, either in response to CD95-L (Hueber et al., 2002; Scheel-Toellner et al., 2002; Garofalo et al., 2003) or independent of the ligand (Muppidi and Siegel, 2004). We show here that resveratrol is able to trigger the redistribution of several death receptors as well as FADD and procaspase-8 in Triton X-100 insoluble fractions. This redistribution can either induce the formation of a functional DISC, as observed with CD95 in SW480 cells (Delmas et al., 2003), or sensitize the cells to the DISC activation by appropriate ligands, as observed in HT29 cells (the present study). The cholesterol sequestering molecule nystatin prevents the partial depletion of lipid rafts in cholesterol and sphingomyelin induced by resveratrol, the death receptor redistribution in lipid rafts and the sensitization to death receptor stimulation, which suggest that resveratrol-induced redistribution of death receptors in lipid rafts in an essential step in its sensitizing effect.

Resveratrol behaves as a sensitizer for classical anticancer agents such as 5-fluoro-uracil (Sun et al., 2002), paclitaxel (Kubota et al., 2003) and radiations (Zoberi et al., 2002) as well as for death receptor agonists. The clinical implications of these observations will depend on whether resveratrol will be safe in humans at pharmacologically active concentrations. Thus, the efficacy of resveratrol as a sensitizer, and the potential ability of the resveratrol/anticancer drug and resveratrol/TRAIL combinations to overcome Bcl-2-mediated resistance, now deserves to be tested in vivo.

Material and methods

Cell lines and transfection

Human colon carcinoma cell lines were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD, USA) and cultured as described (Lacour et al., 2001). Stable transfection of HT29 cells with ps-FF-Bcl2 containing full-length human bcl-2 cDNA (kindly provided by Dr J Bréard, INSERM 461, France) or the empty vector was obtained by the use of Lipofectamine Plus reagent (Gibco-BRL).

Drugs, chemical reagents and antibodies

Resveratrol was obtained from Sigma-Aldrich (Saint Quentin Fallavier, France), cisplatin from Roger Bellon Chemical Co (Neuilly, France) and soluble human recombinant TRAIL from Alexis biochemicals (Illkirch, France). Permeant caspase inhibitors were obtained from R&D Systems (Lille, France), human TNF-α from Tebu (Le Perray-en-Yvelines, France). We used rabbit polyclonal antibodies (pAb) raised against human caspase-3 and -9 active fragments and PARP from Cell Signaling (Beverly, MA, USA) and CD95 (M20) from Santa Cruz Biotech (Santa Cruz, CA, USA) and monoclonal Abs (mAbs) raised against TRAIL-R1 (M271, IgG2a), TRAIL-R2 (M413, IgG1), TRAIL-R3 (M430, IgG1) and TRAIL-R4 (M445, IgG1) kindly provided by Dr M Kubin (Immunex Corp., Seattle, WA, USA), against TRAIL (RIK2, IgG1), a kind gift from Dr H Yagita (Juntendo University, Tokyo, Japan), procaspase-8 (Immunotech, Marseille, France), caspase-8 active fragments (Cell Signaling), cytochrome c (Pharmingen, Lille, France), Smac/DIABLO (Imgenex, Lille, France), mitochondrial HSP70 (Alexis biochemicals), Bcl-2 (Dako, Denmark) and CD95 (ZB4, Immunotech; CH11, Biovalley Co, Rockville, MD, USA; DX2, Pharmingen), CD95-L (NOK1, Sigma-Aldrich), FADD and caveolin-2 (clone 65) (Transduction Lab, Erembodegen, Belgium). In immunoprecipitation experiments, we used soluble recombinant human soluble Flag-CD95L and rat anti-cFLIP mAb (Dave II, Alexis Co, San Diego, CA, USA), anti-Flag (M2) Ab from Sigma (St Louis, MO, USA), rabbit anti-CD95 pAb (C20) from Santa Cruz Biotechnology and mouse anticaspase-8 (IgG2b) mAb from MBL (Nagoya, Japan). The tested caspase inhibitors include z-VAD-fmk, z-IETD-fmk, z-LEHD-fmk, z-DEVD-fmk and z-VDVAD-fmk (R & D Systems, Lille, France).

Cytological and flow cytometry analyses

Apoptosis identification in cells stained with Hoechst 33342, flow cytometry analysis of caspases activation and ROS, and fluorescence microscopy analysis of death receptors at the surface of tumor cells were performed as described (Delmas et al., 2003).

Caspase activities

Cells were incubated in lysing buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate) for 30 min at 4°C and centrifuged (10 000 g, 20 min, 4°C) before incubating 50 μg of proteins in the presence of 100 μ M fluorogenic peptide substrate. Enzyme activities were determined as described (Lacour et al., 2003) and expressed as ‘fold increase’ compared to the control value.

Western blotting

Cells washed in DPBS were lysed in boiling buffer (1% SDS, 1 mM sodium vanadate 10 mM, Tris, pH 7.4). in the presence of 1/50 complete protease inhibitor cocktail tablet (Roche Diagnostics Corporation) for 10 min at 4°C before diluting 30 μg of proteins in loading buffer (125 mM Tris-HCl (pH 6.8), 10% β-mercaptoethanol, 4.6% SDS, 20% glycerol and 0.003% bromophenol blue). These proteins were separated on a polyacrylamide–SDS containing gel and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). After overnight incubation with 5% nonfat milk in TPBS (DPBS with 0.1% Tween 20), the membrane was incubated for 3 h at room temperature with the primary Ab, washed twice with TPBS, incubated for 30 min at room temperature with horseradish peroxidase-conjugated goat anti-mouse or -rabbit Ab (Jackson Immuno Research Labs), washed twice with TPBS and revealed using an enhanced chemiluminescence detection kit (Amersham, Les Ulis, France).

Rafts isolation and biochemical characterization

This study was performed as described (Delmas et al., 2003). Briefly, cells were lysed in 1 ml buffer containing 1% Triton X-100 for 30 min at 4°C, before passing them through an ice-cold cylinder cell homogenizer. The lysate was diluted with 2 ml buffer containing 80% sucrose (w/v), placed at the bottom of a linear sucrose gradient and centrifuged at 39 000 r.p.m. for 20 h at 4°C before collecting eleven 1-ml fractions. A measure of 60 μl of each fraction were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted. Lipids were extracted and analysed as described (Delmas et al., 2003). Concentrations were expressed in μg/mg of total proteins.

DISC analysis

DISC analysis was described previously (Micheau et al., 2002). Briefly, cells were grown in 175 cm2 dishes, stimulated or not with resveratrol and treated with CD95L (2 μg/ml) or TRAIL (500 ng/ml) in the presence of a crosslinker for indicated times. DISCs were immunoprecipitated with protein G and analysed for indicated proteins by immunoblotting. Cells were quickly cooled by adding five volume of ice-cold DPBS, lysed with 0.2% Nonidet P-40, Tris-HCl (20 mM, pH 7.4), NaCl (150 mM), 10% glycerol and the protease inhibitor cocktail (Roche Biochemicals). Cytosolic fractions were precleared with Sepharose 6B (Sigma-Aldrich) for 60 min, then incubated with protein G-coupled Sepharose beads (Amersham, Lille, France) for 3 h. Beads were washed four times with the lysis buffer. Proteins were resolved by SDS–PAGE and blotted onto nitrocellulose membranes. After blocking in PBS containing 0.5% Tween-20 and 5% (w/v) dry milk, membranes were incubated with specific primary Abs and revealed using horseradish peroxidase-conjugated goat anti-rabbit-IgG, goat anti-rat-IgG or goat anti-mouse Abs (Jackson Immunoresearch Laboratories, Lille, France) and ECL (Amersham).

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Acknowledgements

This work was supported by a grant of the Ligue Nationale Contre le Cancer (ES), the BIVB and the ‘Conseil Régional de Bourgogne’ (DD), the Ligue Bourguignonne contre le cancer (NL) and the Association pour la Recherche sur le Cancer (CR). We are grateful to C Humbert (Centre de Microscopie Appliquée à la Biologie, Dijon, France) and A Hamman for excellent technical assistance on confocal laser microscope studies.

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Correspondence to Eric Solary.

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Delmas, D., Rébé, C., Micheau, O. et al. Redistribution of CD95, DR4 and DR5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells. Oncogene 23, 8979–8986 (2004). https://doi.org/10.1038/sj.onc.1208086

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Keywords

  • apoptosis
  • death receptors
  • lipid rafts
  • resveratrol

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