British Journal of Haematology, 2002, 117, 842851

Analysis of resveratrol-induced apoptosis in human B-cell chronic leukaemia

` Viviana Roman, 1 * Christian Billard, 1 Catherine Kern, 1 He le ne Ferry-Dumazet, 2 Jean-Claude Izard, 3 Ramzi Mohammad, 4 Djavad M. Mossalayi 2 and Jean-Pierre Kolb 1 1U.365 INSERM, Institut Curie, Paris, 2Laboratoire dImmunologie et Parasitologie, UFR des Sciences Pharmaceutiques, Universite Victor Segalen, Bordeaux, 3Actichem, Montauban, France, and 4Wayne State University and Karmanos Cancer Institute, Detroit, MI, USA Received 13 August 2001; accepted for publication 18 December 2001

Summary. Trans-resveratrol was analysed for its apoptotic and growth inhibitory activity in human B-cell lines derived from chronic B-cell malignancies (WSU-CLL and ESKOL), and in leukaemic lymphocytes from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Resveratrol displayed antiproliferative activity on both B-cell lines, as estimated by the decrease in cell recovery and inhibition of thymidine uptake. Furthermore, resveratrol induced apoptosis in the two cell lines as well as in B-CLL patients cells, as evidenced by the increase in annexin V binding, caspase activation, DNA fragmentation and decrease of the mitochondrial transmembrane potential DYm. We previously reported that nitric oxide (NO), endogenously released by an

iNO synthase (iNOS) spontaneously expressed in these leukaemic cells, contributed to their resistance towards apoptosis. We show here that resveratrol inhibited both iNOS protein expression and in situ NO release in WSU-CLL, ESKOL and B-CLL patientscells. In addition, Bcl-2 expression was also inhibited by resveratrol. Thus, downregulation of the two anti-apoptotic proteins iNOS and Bcl-2 can contribute to the apoptotic effects of resveratrol in leukaemic B cells from chronic leukaemia. Our data suggest that this drug is of potential interest for the therapy of B-CLL. Keywords: resveratrol, chronic B-cell leukaemia, apoptosis, nitric oxide, Bcl-2.

Resveratrol (3,5,4-trihydroxystilbene) is a polyphenol belonging to the phytoalexin family and exists in cis and trans congurations in grapes and certain medicinal plants. Resveratrol is synthesized by the enzyme resveratrol synthase from p-coumaroyl CoA and malonyl CoA in response to stress, injury, infection or UV-irradiation. This polyphenol was initially described as an antifungicide, conferring disease resistance in plants (Hain et al, 1993). It is now well established that resveratrol favours protection against atherosclerosis through its antioxidant activity, anti-inammatory effects and inhibition of platelet aggregation (Fremont, 2000). During recent years, a number of studies on various cell lines and animal models have demonstrated that resveratrol also has cancer chemopreventive, antiproliferative and anti-

Correspondence: Dr Christian Billard, U365 INSERM, Institut Curie, 26 rue dUlm, 75248 Paris cedex 05, France. E-mail: christian.billard@curie.fr ` *Present address: Center of Immunology, Institute Stefan Nicolau, Bucharest, Romania.

tumour properties (Jang et al, 1997; Hsieh & Wu, 1999; Hsieh et al, 1999a). Among the proposed mechanims, the polyphenol was shown to act by arresting the cell cycle at the S/G2 phase transition (Ragione et al, 1998; Hsieh et al, 1999b). In addition, resveratrol was reported to exert antitumour activity by inducing apoptosis through activation of p53 (Huang et al, 1999). In connection with this, the inhibition of tumour growth by resveratrol in an in vivo rat model was also attributed to its pro-apoptotic capabilities (Carbo et al, 1999). Furthermore, resveratrol seems to regulate differentially the various types of NO synthases (NOS), the enzymes allowing the generation of nitric oxide (NO). It stimulates the activity of the endothelial NOS isoform in artery endothelial cells and in rat aorta, a mechanism that may account for the vasorelaxing and cardioprotective activity of resveratrol (Chen & Pace-Asciak, 1996; Hsieh et al, 1999b; Hung et al, 2000). In contrast, resveratrol downregulates the inducible NOS (iNOS) in macrophage-like cells (Chan et al, 2000), either by inhibiting the NF-kB binding activity required for iNOS mRNA transcription (Tsai et al, 1999) or through post-transcriptional modications (Wadsworth &
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Koop, 1999). Resveratrol was also reported to inhibit iNOS expression in Kupffer cells (Kawada et al, 1998). Recently, we described the constitutive expression of a NOS of the iNOS type in leukaemic B cells from B-CLL and hairy cell leukaemia (HCL), two chronic B-cell tumours, and we found that inhibition of endogenous NO production stimulated apoptosis of the malignant B cells. Our data suggested that NO molecules exert an anti-apoptotic action that contributes to the resistance of the leukaemic cells to programmed cell death (Zhao et al, 1998; Roman et al, 2000). While resveratrol-induced apoptosis was found in myelo-monocytic cell lines (Clement et al, 1998; Surh et al, 1999, Park et al, 2001) and in B-cell acute lymphoblastic leukaemia (Dorrie et al, 2001), no data were reported on the effects of resveratrol in B cells from B-CLL, a chronic leukaemia characterized by a deciency in apoptosis (Reed, 1998). In the present work, we investigated the in vitro effects of resveratrol on apoptotic processes in human B-cell lines derived from B-CLL and HCL, and in leukaemic cells from patients with B-CLL. Our data indicate that resveratrol induces apoptosis in these cells and that it inhibits the expression of iNOS and Bcl-2, two anti-apoptotic molecules. MATERIALS AND METHODS Cells. The ESKOL cell line was established from a patient with HCL, a preplasma B-cell leukaemia (Harvey et al, 1991), and obtained through the courtesy of Dr E. F. Srour (University of Indiana, USA). The EpsteinBarr virus (EBV)negative WSU-CLL cell line was derived from a B-CLL patient resistant to udarabine (Mohammad et al, 1996). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples of healthy volunteers by centrifugation on Ficoll Hypaque gradient (Pharmacia, Uppsala, Sweden). Leukaemic B cells were freshly puried from the peripheral blood of previously untreated hyperleucocytic patients with B-CLL (grade A, according to Binets classication), as previously described (Zhao et al, 1998). All cell cultures were carried out at 37C in an humidied atmosphere with 5% CO2 in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with l-glutamine 2 mmol/l, sodium pyruvate 1 mmol/l, antibiotics and 10% fetal calf serum (FCS) (Myoclone super plus; Gibco BRL, Cergy-Pontoise, France). Reagents. Trans-resveratrol was obtained from Sigma (Saint Louis, USA). A 5-mmol/l stock solution was prepared by dissolving resveratrol in ethanol/water (1:1). The NOS inhibitor nitro l-arginine methyl ester (L-NAME) was purchased from Sigma. The broad-specicity caspase inhibitor Z-VAD-fmk was purchased from Biomol (Plymouth Meeting, USA). Proliferation assays. Cells were seeded at 5 105/ml in 24-well plates (1 ml/well) in triplicates and the number of viable cells was counted with a Coulter counter after various times in culture. In some experiments, toxicity was monitored by trypan blue exclusion. For tritiated thymidine (3H-TdR) incorporation assay, cells were seeded at 106 cells/ml in microtitration plates (100 ll/well) in triplicates. After various times of culture, cells were labelled for 4 h with 37 kBq/well of 3H-methyl TdR (NET-027X; NEN Life

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Science Products, Belgium; specic activity 740 GBq/mmol). Cells were then collected with a multiple-automated cell harvester (Cambridge Technology, Cambridge, UK) and radioactivity was counted in a liquid scintillation spectrometer (Beckman). Results were expressed as d.p.m. SEM. Quantication of apoptosis by annexin V binding and DNA fragmentation. Two events that occur during the induction of apoptosis were studied. First, cells undergoing apoptosis were quantied by determining the percentage of cells expressing phosphatidylserine (PS) at the outer leaet of the plasma membrane. This was measured by the specic binding of annexin V labelled with uorescein isothiocyanate (FITC; Boehringer Mannheim, Mannheim, Germany or Bender Medsystems, Vienna, Austria), with or without simultaneous labelling with propidium iodide (PI), a marker of nonapoptotic necrosis, according to a modication of the technique described by Koopman et al (1994). Fluorescence data were analysed by ow cytometry on a FACScan. Results were expressed as the percentage of annexin V-FITC-positive cells. Second, DNA fragmentation was quantied by the formation of histone-associated DNA fragments in the cytoplasm (mono- and oligo-nucleosomes) using an enzyme-linked immunosorbent assay (ELISA) (Cell Death Detection ELISAPLUS, Roche Diagnostics, Indianopolis, USA). Results were expressed as the percentage of nucleosome enrichment. Mitochondrial permeability transition. The mitochondrial transmembrane potential (DYm) was evaluated by the use of the specic uorescent probe DiOC(6)3 or 3,3-dihexylocarbocyanine iodide (Calbiochem, La Jolla, USA). Briey, cells were incubated at 5 106/ml at 37C for 15 min in the presence of 40 nmol/l DiOC(6)3. After washing, cells were resuspended in 05 ml of M199 medium without phenol red and immediately analysed for uorescence intensity by ow cytometry (Fl-1 channel). Caspase 3 activity. The activity of cell lysates to cleave pDEVD-pNA, a substrate specic for caspase 3, was measured using the Caspase 3 cellular assay (Biomol) according to the manufacturers instructions. Briey, 2 106 cells/ml were lysed for 5 min at 4C {50 mmol/l HEPES, 01% 3-[(3cholamidopropyl)dimethylammonio-1-propane sulphonate] (CHAPS), 1 mmol/l dithiothreitol (DTT), 01 mmol/l EDTA, pH 74} and 20 lg of proteins were incubated with the chromogenic substrate (pDEVD-pNA). The pNA released by cleavage was recorded at 405 nm in a Victor-2 spectrometer every 10 min over 2 h. Recombinant human caspase 3 was tested in parallel. Controls included substrate alone, and the specicity of the reaction was assessed by the addition of the unlabelled DEVD-CHO caspase 3 inhibitor. Detection of Bcl-2. The Bcl-2 content in cell lysates was estimated using a classic ELISA according to the manufacturers instructions (human Bcl-2 ELISA; Bender Medsystems). Results of triplicate samples were expressed in U/ml by the aid of a standard curve of recombinant human Bcl-2. To exclude the possibility that the Bcl-2 content, as determined by ELISA, was inuenced by variations in the proportion of dead or dying cells, we also examined the expression of Bcl-2 by indirect immunouorescence and ow cytometry using a specic anti-Bcl-2 monoclonal

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antibody (mAb) after permeabilization with the Cytoperm kit (Serotec, Oxford, UK), as previously described (Zhao et al, 1998). Cells were analysed on a FACScan (Becton-Dickinson, Mountain View, USA), the viable cells being gated according to their forward-scatter/sideways-scatter (FSC/ SSC) prole, and uorescence was determined on at least 5000 cells. The percentage of uorescent cells was quantied using the lysis ii and procyt softwares. Expression of the iNOS protein. This expression was analysed by direct immunouorescence and ow cytometry. Briey, washed cells were permeabilized and incubated for 30 min at 4C with either an anti-iNOS mAb conjugated to FITC (FITC-macNOS, clone 6; BD Transduction Laboratories, Heidelberg, Germany) or a FITC-conjugated anti-IgG2a as an isotype control (Becton-Dickinson). This anti-iNOS mAb is directed against a protein fragment corresponding to the amino acids 9611144 of murine iNOS and recognizes the human enzyme. FACS analysis was then performed as above. NO production. Endogenous production of NO was measured in situ by the xation of the NO-specic probe diaminouorescein 2-diacetate (DAF-2 DA; Calbiochem) and cytouorometric analysis. Briey, cells were adjusted at 1 106/ml in culture medium and incubated with 10 lmol/l of the uorescent probe for 1 h at 37C in the dark. DAF-2 DA-labelled cells were then washed and resuspended in 1 ml of prewarmed (37C) 199 medium without phenol red (Gibco), supplemented with 1 mmol/l arginine (Sigma) in the wells of a 24-well tray (Costar, Corning, USA). Fluorescence (excitation 485 nm, emission 535 nm) was then recorded every 10 min over 2 h in a cytouorometer (Victor-2) thermostated at 37C. Results are expressed as uorescence intensity (arbitrary units) as a function of time. Changes in the slope of the curves are indicative of the different kinetics of NO production. Statistical analysis. Experiments were performed with duplicate or triplicate cell cultures, and each experiment was repeated 25 times. Statistical analysis was performed using the staview software. The unpaired two tail t-test, as modied by FisherYates for small samples (Schwartz, 1963), was used for the comparison of test and control group. RESULTS Resveratrol inhibits proliferation in leukaemic B-cell lines Treatment of WSU-CLL and ESKOL cells with resveratrol was found to result in a time- and dose-dependent inhibition of cell growth. A signicant reduction in the number of viable cells was observed after 3 d of treatment with 3 lmol/l, while an almost total inhibition of cell proliferation was reached with 12 and 25 lmol/l for, respectively, ESKOL and WSU-CLL cells (Fig 1). Assay of 3H-TdR uptake also showed signicant antiproliferative effects of resveratrol on ESKOL cells as early as 24 h with 25 lmol/l (P < 0001) and on WSU-CLL cells with 50 lmol/l (P < 001) at 72 h (data not shown). Resveratrol induces apoptosis in leukaemic B-cell lines The capability of resveratrol to stimulate apoptosis was further investigated in WSU-CLL and ESKOL cells. In both

Fig 1. Resveratrol inhibits proliferation of WSU-CLL and ESKOL cells. (A) ESKOL and (B) WSU-CLL cells were seeded at 5 105/ml in the absence or presence of varying concentrations of resveratrol. After 1 and 2 or 3 d of culture, the number of viable cells was determined using a Coulter counter. Results are means SEM of triplicate determinations from one representative experiment out of two. Comparisons between untreated cells and resveratrol-treated cells after different times of culture were performed using the modied t-test for small samples. *P < 005; **P < 001; ***P < 0001; NS, not signicant.

cell lines, resveratrol treatment was followed by a dosedependent increase in the percentage of annexin V-positive cells (Table I), whereas no effect was observed with the ethanol/water vehicle tested at the same dilutions (not shown). Resveratrol-induced apoptosis was also time dependent, as exemplied with ESKOL cells in Fig 2. The percentage of annexin V-positive/PI-negative cells was already detectable after 2 h of treatment. This percentage increased until 48 h in cells treated with 10 lmol/l of resveratrol, while cells treated with 50 lmol/l were PI positive and thus dead by 30 h. Note that similar results were obtained with the WSU-CLL cell line and that the induction of apoptosis by resveratrol was comparable to that

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Table I. Resveratrol induces apoptosis in leukaemic B-cell lines.

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% Annexin V-FITC positive cells Resveratrol (lmol/l) 3 6 12 25 50 ESKOL 10 21 33 45 52 5 12 19 17 21 WSU-CLL 75 13 9 20 9 29 13 41 15

ESKOL and WSU-CLL cells were cultured at 5 106 cell/ml for 48 h in the presence or absence of different resveratrol concentrations. Cells were then assayed for annexin V-FITC binding, as described in the Materials and methods. The percentages of annexin V-positive/PI negative cells are given, as compared with untreated cells (after subtraction of the untreated cell values). Results are expressed as means SEM from three experiments.

Fig 2. Kinetics of resveratrol-induced apoptosis in ESKOL cells. ESKOL cells were cultured at 106 cells/ml in the presence of 10 lmol/l or 50 lmol/l of resveratrol or in its absence. After various times of culture, the percentage of apoptotic cells was estimated by binding of annexin V-FITC and exclusion of the cells labelled with propidium iodide as described in Materials and methods. Results are expressed after subtraction of the untreated cell values at the corresponding times. Data are from one representative experiment of three that gave comparable results. NT, not tested.

Fig 3. Resveratrol decreases the mitochondrial transmembrane potential (A) and induces DNA fragmentation (B) in ESKOL cells. ESKOL cells were cultured at 5 105/ml in the presence or absence (control) of resveratrol. (A) After 18 h of treatment with 10 lmol/l resveratrol, 106 cells were loaded for 15 min at 37C with 40 nmol/l of the uorescent probe DiOC(6)3. Cells were then washed in PBS and immediately analysed by ow cytometry (Fl-1 channel). (B) After 48 h of incubation with varying concentrations of resveratrol, cells were lysed and analysed for the their cytoplasmic content in mono- and oligo-nucleosomes, as described in Materials and methods. Results are from one representative experiment of two and are expressed as the percentage of nucleosomes enrichment as compared with untreated cells taken as 100%.

elicited by etoposide, an inhibitor of toposisomerase II (data not shown). Resveratrol induces a dissipation of the mitochondrial transmembrane potential and DNA fragmentation in leukaemic B-cell lines Because certain stimuli such as CD47 ligation can trigger caspase-independent cell death in B-CLL, as estimated by annexin V binding without disruption of the mitochondrial

transmembrane potential (DYm) or DNA fragmentation (Mateo et al, 1999), we therefore investigated whether resveratrol could elicit mitochondrial and nuclear events of apoptosis. Analysis of the mitochondrial transmembrane potential of untreated ESKOL cells showed consistently a biphasic labelling with the DiOC(6)3 probe. Treatment with 10 lmol/l resveratrol for 18 h resulted in a dissipation of the DYm as estimated by a 34% decrease of uorescence intensity (Fig 3A). This resveratrol-associated decrease of DYm was time- and dose dependent, the optimal effect being observed after 40 h of incubation with 50 lmol/l resveratrol (data not shown). We further studied DNA fragmentation by quantifying histone-associated DNA fragments. As shown in Fig 3B, the formation of nucleosomes was induced within 18 h of treatment of ESKOL cells with resveratrol. Nucleosome enrichment, compared with untreated control cells, was already augmented with 3 lmol to reach a

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Table II. Effect of the general caspase inhibitor Z-VADfmk on resveratrol-induced apoptosis in ESKOL and WSUCLL cell lines.

% Annexin V-FITC positive cells Treatment Resveratrol Z-VAD-fmk Resveratrol + Z-VAD-fmk ESKOL 47 2 20 WSU-CLL 52 3 32

Fig 4. Resveratrol stimulates caspase 3 activity in ESKOL cells. ESKOL cells were cultured at 5 105/ml for 18 h in the presence of resveratrol 20 lmol/l or etoposide 50 lmol/l or in their absence (control). Cells were then lysed and extracts were assayed (in triplicates) for their capacity to cleave the chromogenic substrate DEVD-pNA specic for caspase 3 over a period of 2 h, as described in Materials and methods. Absorbance was read at 405 nm. Results are from one representative experiment of two.

ESKOL and WSU-CLL cells were cultured at 3 105/ml, in the absence (untreated cells) or presence of either resveratrol (50 lmol/l), or Z-VAD-fmk (10 lmol/l, a concentration that fully inhibits recombinant caspase 3 activity) or both. After 48 h of treatment, annexin V-FITC binding assay was performed and the percentage of annexin V-labelled cells were determined after subtraction of the untreated cell values. Data are from one experiment of two that gave comparable results. Table III. Resveratrol induces a decrease of iNOS expression in the ESKOL and WSU-CLL cell lines.

plateau with 25 lmol/l. Comparable results were obtained with the WSU-CLL cell line (not shown). Therefore, resveratrol promoted mitochondrial and nuclear alterations typical of the apoptotic processes. Resveratrol stimulates caspase 3 activity In order to investigate the pathways involved in resveratrolinduced apoptosis, the levels of caspase 3 activity were evaluated using the tetrapeptide DEVD-pNA, a substrate specic for caspases 3 and 7. Overnight incubation of ESKOL cells with resveratrol 20 lmol/l, or etoposide 50 lmol/l as a positive control, resulted in an increased capacity of the cell lysates to cleave the chromogenic substrate compared with untreated cells (Fig 4). Similar results were obtained with a uorogenic substrate, the DEVD-aminocoumarin (data not shown). However, the general inhibitor of caspases Z-VADfmk inhibited signicantly, but only partly, the xation of annexin V induced by resveratrol in either ESKOL or WSUCLL cells (Table II). These results indicated that resveratrolinduced apoptosis involved both caspase-dependent and independent mechanisms. Resveratrol decreases iNOS and Bcl-2 expression in leukaemic B-cell lines Our previous observation that iNOS inhibitors were able to trigger apoptosis in B-CLL and HCL cells indicated that the constitutive and functional iNOS expressed by these cells contributes to prevent programmed cell death and thus behaves like an anti-apoptotic molecule (Zhao et al, 1998; Roman et al, 2000). Interestingly, treatment of WSU-CLL and ESKOL cells with resveratrol was found here to strongly inhibit iNOS protein expression (Table III), and this effect was accompanied by a dose-dependent decrease in NO production, as measured in situ by the xation of DAF-2 DA, a specic uorescent probe (Fig 5). We also investigated the

% iNOS labelled cells Treatment Controls Resveratrol (10 lmol/l) Resveratrol (50 lmol/l) ESKOL 21 5 2 WSU-CLL 36 31 4

ESKOL and WSU-CLL cells were cultured at 2 105/ml for 24 h in the absence (controls) or presence of 10 lmol/l and 50 lmol/l resveratrol. Cells were then permeabilized, labelled with either an anti-iNOS-FITC mAb or an IgG2a-FITC as an isotypic control and analysed by ow cytometry, as described in the Materials and methods.

effect of resveratrol on the well-known anti-apoptotic protein Bcl-2. This latter belongs to a family of molecules regulating mitochondrial permeability and its expression in B-CLL was shown to be reduced by treatment with various drugs inducing apoptosis, such as avopiridol (Kitada et al, 2000). Using a quantitative ELISA, we showed that Bcl-2 content in WSU-CLL and ESKOL cell lysates was consistently reduced after resveratrol treatment (Table IV). Immunouorescence experiments with an anti-Bcl-2 mAb with permeabilized cells conrmed that Bcl-2 expression decreased upon resveratrol exposure (not shown). These results indicated that resveratrol was capable of reducing the expression of both iNOS and Bcl-2 in B-CLL and HCL cell lines. Apoptotic effects of resveratrol on leukaemic B cells from B-CLL patients Although WSU-CLL and ESKOL cell lines are representative of B-CLL and HCL, respectively, they do not express all the

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Table V. Resveratrol induces apoptosis in leukaemic cells from B-CLL patients.

% Annexin V-labelled cells in patients Resveratrol (lmol/l) 50 10 5 2 1 1 65 47 13 3 1 2 33 27 nt nt 15 3 35 20 nt nt 12 4 63 10 nt nt nt 5 51 30 nt nt nt 6 47 22 nt nt nt 7 52 30 nt nt nt

Fig 5. Resveratrol impairs endogenous NO production by ESKOL cells. ESKOL cells were cultured for 18 h at 5 105 cells/ml with medium alone (control), resveratrol 50 lmol/l or 10 lmol/l. Cells were then washed, adjusted to 106/ml and labelled with 10 lmol/l DAF-2 DA for 1 h at 37C. After washing, cells were resuspended in 199 medium and the uorescence intensity was analysed every 10 min over a period of 2 h in a Victor-2 uorimeter. Results are expressed in uorescence counts (arbitrary units) as a function of time. Table IV. Resveratrol decreases Bcl-2 expression in ESKOL and WSU-CLL cell lines.

Leukaemic cells from seven B-CLL patients were cultured at 2 106/ml for 48 h in the presence of varying concentrations of resveratrol or in its absence (untreated cells). The percentages of annexin V-FITC-labelled cells were evaluated compared with untreated cells, as described in the Materials and methods. nt, not tested.

Table VI. Resveratrol decreases iNOS expression by leukaemic cells from B-CLL patients.

% of iNOS positive cells in patients Treatment 1 53 28 (47) 2 21 1 (95) 3 62 33 (47)

Bcl-2 content (U/ml) Treatment Controls Resveratrol 50 lmol/l (% inhibition) ESKOL 27 9 (66) WSU-CLL 53 39 (26)

Controls Resveratrol 50 lmol/l (% inhibition)

ESKOL and WSU-CLL cells were cultured at 5 105/ml for 24 h in the absence (controls) or presence of 50 lmol/l of resveratrol. Bcl-2 expression was then quantied from cell lysates using an ELISA kit according to the manufacturers instructions. Results are means of triplicates from one representative experiment of two.

Leukaemic B cells from three B-CLL patients were cultured at 2 106 cells/ml for 24 h in the presence of resveratrol 50 lmol/l or in its absence (controls). iNOS expression was then evaluated on permeabilized cells with an anti-iNOS labelled with FITC and ow cytometry analysis, as described in the Materials and methods.

characteristics of leukaemic cells from patients. Experiments were thus undertaken with freshly isolated leukaemic B cells from the peripheral blood of seven untreated B-CLL patients (grade A). Unlike the cell lines, B-CLL patients cells did not spontaneously proliferate in vitro. Indeed, very low 3H-TdR uptake was found in B-CLL cells cultured for up to 3 d and no effect of resveratrol (1100 lmol/l) was observed (not shown). However, resveratrol consistently induced apoptosis in these cells, as shown by the dose-dependent augmentation of annexin V-positive cells in all seven patients tested, with a mean increase of 49% of annexin V-positive cells (range 3365%) after treatment with 50 lmol/l resveratrol for 48 h (Table V). Moreover, resveratrol also induced DNA fragmentation in B-CLL patients cells (not shown), conrming that the polyphenol could promote apoptosis not only in B-cell lines but also in B-CLL patients cells.

Further, resveratrol also showed marked inhibitory effects on iNOS expression in B-CLL cells, as exemplied with three patients in Table VI: the proportion of antiiNOS-positive cells was inhibited by 4795% after exposure to 50 lmol/l resveratrol for 18 h. This effect had functional consequences because it paralleled a decreased NO production, as shown by a representative patient in Fig 6A. Indeed, the slope of the linear xation of DAF-2 DA uorescence observed in untreated cells was signicantly reduced in cells previously treated with 50 lmol/l resveratrol for 18 h. As a control experiment, the presence of L-NAME (2 mmol/l), a NOS inhibitor, greatly reduced NO production (not shown). Finally, exposure to 50 lmol/l resveratrol also resulted in a signicant decreased Bcl-2 expression, as detected by immunouorescence (Fig 6B) as well as ELISA (not shown). Therefore, resveratrol was capable of lowering the expression of both the functional iNOS enzyme and the antiapoptotic protein Bcl-2 in B-CLL patients cells as well as in WSU-CLL and ESKOL cell lines.

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The antiproliferative properties of resveratrol were reported in a number of studies and the mechanisms of this effect have been described, involving an accumulation of p53 and of the cdk inhibitor p21WAF1/CIP1 and a cell cycle arrest at S/G2 transition (Ragione et al, 1998; Hsieh et al, 1999b). A recent report suggested the direct interaction of the polyphenol with DNA polymerases (Stivala et al, 2001). The effects of resveratrol on programmed cell death are also well documented by studies on various cellular or animal models (Clement et al, 1998; Carbo et al, 1999; Hsieh & Wu, 1999; Huang et al, 1999; Surh et al, 1999), including B-cell acute lymphoblastic leukaemia (Dorrie et al, 2001) that is not characterized by defective apoptosis. Our present results are the rst to show apoptosis induction by resveratrol in B cells from chronic leukaemias and in particular from B-CLL in which the deciency in programmed cell death is now well established, and is even responsible for the malignant development by accumulation of leukaemic B-lymphocytes (Reed, 1998). Our data are therefore relevant in B-CLL, even if resveratrol was found to exert anti-tumour effects without inducing apoptosis in some cellular models and also to either induce or attenuate cell death depending on the cellular redox status (MacCarrone et al, 1999; Bastianetto et al, 2000). Regarding the mechanisms whereby resveratrol exerts its apoptotic effects, the p53 pathway was involved in a model in which the polyphenol suppressed cell transformation and induced apoptosis (Huang et al, 1999), but was not involved in the cell cycle arrest induced by resveratrol prior to apoptosis in a T-cell line (Bernhard et al, 2000). In accordance with a previous observation on the promyelocytic HL-60 cell line (Surh et al, 1999), we found that resveratrol decreased the expression of the anti-apoptotic protein Bcl-2 in WSU-CLL and ESKOL cell lines as well as B-CLL patients cells. This molecule controls the mitochondrial membrane permeability, a crucial event in the apoptotic processes. In contrast, resveratrol was also reported to stimulate the expression of a member of the Bcl-2 family, the pro-apoptotic Bax protein (Tessitore et al, 2000). Taken together, these data suggest that resveratrol could modulate the Bcl-2/Bax ratio that is critical for in vitro apoptosis and in vivo chemoresistance in B-CLL (Pepper et al, 1997). Furthermore, we also observed a reduction of the mitochondrial transmembrane potential (DYm) by resveratrol, supporting that the mitochondrial functions are altered by the drug. Interestingly, a loss of DYm was found during resveratrol-induced apoptosis in a T-cell line (Tinhofer et al, 2001). These effects, which did not involve cytochrome c release, were associated with the activation of caspases, including caspase 3, and were antagonized by an excess of Bcl-2. Other experiments have shown that resveratroltriggered apoptosis was associated with caspase 3 activation, but with cytochrome c release in the monocytic cell line U937, and these events were attenuated by Bcl-2 overexpression (Park et al, 2001). If our present results show that caspase 3 activity was stimulated upon treatment with resveratrol, the fact that apoptosis induction was reverted but only partly by a caspase inhibitor (Z-VAD-fmk) clearly indicates that both caspase-dependent

Fig 6. Resveratrol reduces NO production (A) and Bcl-2 expression (B) in leukaemic B-cells from B-CLL patients. Freshly collected B-CLL leukaemic cells were cultured for 18 h at 2 106/ml in the presence or absence of resveratrol 50 lmol/l. (A) Cells were assayed in situ for NO production, as described in Materials and methods. Autouorescence, untreated cells not incubated with DAF-2 DA. Results are from an experiment with one patient out of three that gave similar results. (B) Permeabilized cells were tested for Bcl-2 expression by indirect immunouorescence using an anti-Bcl-2 mAb and ow cytometry analysis, as described in Materials and methods. Values are given after subtraction of the uorescence of the isotype-matched control antibody that was unmodied by resveratrol.

Effect of resveratrol on normal PBMC To determine whether leukaemic cells were preferential targets for resveratrol or whether normal quiescent cells of the peripheral blood could also be affected, PBMC from healthy donors were cultured for 24 and 48 h in the presence of ve concentrations of resveratrol ranging from 3 to 50 lmol/l. No effect on cell viability (or on 3H-TdR uptake) and no toxicity, as estimated by trypan blue assay, were detected compared with untreated cells (data not shown). DISCUSSION Our present study shows that resveratrol inhibits cell growth and induces apoptosis in B-cell lines derived from B-CLL and HCL, and also promotes apoptosis in leukaemic B-lymphocytes freshly isolated from B-CLL patients.

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and caspase-independent pathways were involved in the effects of the polyphenol on leukaemic B cells. Although resveratrol was reported to trigger Fas/CD95 signalling-dependent apoptosis in HL-60 cells (Clement et al, 1998), a number of studies on a variety of haemapoietic leukaemia cell lines of myeloid, T and B phenotypes showed that the Fas pathway was not implicated in resveratrolinduced apoptosis (Bernhard et al, 2000; Tsan et al, 2000; Dorrie et al, 2001; our unpublished observations). B-CLL cells are known to express very low or undetectable Fas levels and are resistant to Fas-mediated apoptosis. Moreover, we did not nd any Fas modulation upon treatment of B-CLL cells with resveratrol, in contrast to that observed after CD23 ligation, a positive control of stimulation of Fas expression (unpublished results). Regardless of the abilities of resveratrol, it is notable that when apoptotic processes were stimulated in B-CLL patients cells (and hairy cells as well) after treatment with iNOS inhibitors, Fas-driven apoptosis was revealed, suggesting that the NOS and Fas pathways could be competing (Zhao et al, 1998; Roman et al, 2000). Indeed, it has been demonstrated that inactive mitochondrial caspases 3 are S-nitrosylated at their active site and are denitrosylated during Fas-triggered apoptosis (Mannick et al, 2001). Inasmuch as NO is known to block caspase 3 activity through S-nitrosylation (reviewed by Kolb, 2000), it is possible that the inhibition of NO release due to a lack of iNOS activity might restore caspase activation in B-CLL cells, as previously suggested (Kolb et al, 2000). From this hypothesis, the downregulation of iNOS or inhibition of its enzymatic activity could be of particular interest for restoring decient apoptotic mechanisms in B-CLL. In connection with this, our present results indicate for the rst time that, in addition to the apoptotic effects of resveratrol, both iNOS expression and NO release were reduced, and caspase 3 activity was stimulated by treatment of B-CLL cells with resveratrol. Moreover, we also showed that the mitochondrial pathway of apoptosis was involved in the effects of resveratrol, as evidenced by our above data on Bcl-2 and mitochondrial membrane potential. Taken together, these results indicate that the drug is capable of inhibiting the functional iNOS, and that subsequent activation of caspase 3 and other apoptotic mechanisms could lead to DNA fragmentation in B-CLL. The modulation of NOS expression, including the inhibition of iNOS activity by resveratrol, has already been shown in several studies (Kawada et al, 1998). Our preliminary experiments suggested that the polyphenol could act at the transcriptional level in leukaemic cells from chronic B-cell malignancies, in agreement with the downregulation of iNOS gene expression previously found in macrophage-like cells (Chan et al, 2000). This effect of resveratrol may be mediated by inactivation of NF-jB, a transcription factor for iNOS gene (Tsai et al, 1999). However, a different mechanism involving post-transcriptional modications was reported in the same cellular model (Wadsworth & Koop, 1999). Our observations that resveratrol inhibits iNOS expression and endogenous NO release could be important in B-CLL, as (1) this leukaemia is characterized by a defective programmed cell death, and (2) iNOS behaves like an anti-

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apoptotic protein, as evidenced in our previous work describing that the NO pathway may be one of the mechanisms participating in apoptosis deciency in B-CLL cells (Zhao et al, 1998). Thus, the present study clearly showed that resveratrol induced in vitro apoptosis of leukaemic cells from B-CLL and suggests that this effect can be mediated by downregulation of two anti-apoptotic proteins, iNOS and Bcl-2. This drug therefore appears to be of potential interest for future developments in the therapy of B-CLL. We are currently investigating the effects of certain grape-derived analogues of resveratrol. In addition, the association with other polyphenols such as quercetin might be considered, as previously suggested (Elattar & Virji, 1999). Finally, we found that resveratrol did not affect the viability of human PBMC, and other data showed that longterm exposure to high concentrations of resveratrol inhibited the clonal growth of normal haematopoietic progenitor cells but did not induce apoptosis, in contrast to leukaemic cells (Gautam et al, 2000). However, activated normal PBMC and CD34+ cells might be more susceptible to the apoptotic effect of resveratrol (unpublished observations). Taken together, these data suggest that resveratrol, through its antiproliferative and apoptotic effects, could be useful for adjuvant therapy and for ex vivo purging of leukaemia cells from bone marrow autografts. ACKNOWLEDGMENTS The invaluable help of Dany Rouillard for ow cytometry analysis was greatly appreciated. We also thank Claire Mathiot (Institut Curie) and Florence Ajchenbaum-Cymbal ista (Hotel Dieu) for providing B-CLL samples. This work was supported by INSERM and by grants from ARC (#9481) and from Fondation contre la Leucemie. REFERENCES
Bastianetto, S., Zheng, W.H. & Quirion, R. (2000) Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons. British Journal of Pharmacology, 131, 711720. Bernhard, D., Tinhofer, I., Tonko, M., Hubl, H., Ausserlechner, M.J., Greil, R., Koer, R. & Csordas, A. (2000) Resveratrol causes arrest in the S-phase prior to fas-independent apoptosis in CEM-C7H2 acute leukemia cells. Cell Death and Differentiation, 7, 834842. Carbo, N., Costelli, P., Baccino, F.M., Lopez-Soriano, F.J. & Argiles, J.M. (1999) Resveratrol, a natural product present in wine, decreases tumour growth in a rat tumour model. Biochemical and Biophysical Research Communication, 254, 739743. Chan, M.Y., Mattiacci, J.A., Hwang, H.S., Shah, A. & Fong, D. (2000) Synergy between ethanol and grape polyphenols, quercetin, and resveratrol, in the inhibition of the inducible nitric oxide synthase pathway. Biochemical Pharmacology, 60, 15391548. Chen, C.K. & Pace-Asciak, C.R. (1996) Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta. General Pharmacology, 27, 363366. Clement, M.V., Hirpara, J.L., Chawdhury, S.H. & Pervaiz, S. (1998) Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells. Blood, 92, 9961002.

2002 Blackwell Science Ltd, British Journal of Haematology 117: 842851

850

V. Roman et al
MacCarrone, M., Lorenzon, T., Guerrieri, P. & Agro, A.F. (1999) Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity. European Journal of Biochemistry, 265, 2734. Mannick, J.B., Schonhoff, C., Papeta, N., Gafourifar, P., Szobor, M., Fang, K. & Gaston, B. (2001) S-Nitrosylation of mitochondrial caspases. The Journal of Cell Biology, 154, 111116. Mateo, V., Lagneaux, L., Bron, D., Biron, G., Armant, M., Delespesse, G. & Sarfati, M. (1999) CD47 ligation induces caspaseindependent cell death in chronic lymphocytic leukemia. Nature Medicine, 5, 12771284. Mohammad, R.M., Mohamed, A.N., Hamdan, M.Y., Vo, T., Chen, B., Katato, K., Abubakr, Y.A., Dugan, M.C. & al-Katib, A. (1996) Establishment of a human B-CLL xenograft model: utility as a preclinical therapeutic model. Leukemia, 10, 130137. Park, J.W., Choi, Y.J., Suh, S.I., Baek, W.K., Suh, M.H., Jin, I.N., Min, D.S., Woo, J.H., Cang, J.S., Passaniti, A., Lee, Y.H. & Kwon, T.K. (2001) Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity. Carcinogenesis, 22, 16331639. Pepper, C., Hoy, T. & Bentley, P. (1997) Bcl-2/Bas ratios in chronic lymphocytic leukaemia and their correlation with in vitro apoptosis and clinical resistance. British Journal of Haematology, 96, 935938. Ragione, F.D., Cucciolla, V., Borriello, A., Pietra, V.D., Racioppi, L., Soldati, G., Manna, C., Galletti, P. & Zappia, V. (1998) Resveratrol arrests the cell division cycle at S/G2 phase transition. Biochemical and Biophysical Research Communication, 250, 5358. Reed, J.C. (1998) Molecular biology of chronic lymphocytic leukaemia. Seminars in Oncology, 25, 1118. Roman, V., Zhao, H., Fourneau, J.M., Marconi, A., Dugas, N., Dugas, B., Sigaux, F. & Kolb, J.P. (2000) Expression of a functional inducible nitric oxide synthase in hairy cell leukemia and ESKOL cell line. Leukemia, 14, 696705. ` Schwartz, D. (1963) Methodes statistiques a lusage des medecins et biologistes. Flammarion Medecine-Sciences, Paris. Stivala, L.A., Savio, M., Carafoli, F., Perucca, P., Bianchi, L., Maga, G., Forti, L., Pagnoni, U.M., Albini, A., Prosperi, E. & Vannini, V. (2001) Specic structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. Journal of Biological Chemistry, 276, 2258622594. Surh, Y.J., Hurh, Y.J., Kang, J.Y., Lee, E., Kong, G. & Lee, S.J. (1999) Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Cancer Letters, 140, 110. Tessitore, L., Davit, A., Sarotto, I. & Caderni, G. (2000) Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21 (CIP) expression. Carcinogenesis, 21, 16191622. Tinhofer, I., Bernhard, D., Senfter, M., Anether, G., Loefer, M., Kroemer, G., Koer, R., Csordas, A. & Greil, R. (2001) Resveratrol, a tumor-suppressive compound from grapes, induces apoptosis via a novel mitochondrial pathway controlled by Bcl-2. Federation of the American Societies for Experimental Biology Journal, 15, 16131615. Tsai, S.H., Lin-Shiau, S.Y. & Lin, J.K. (1999) Suppression of nitric oxide synthase and the downregulation of the activation of NFkappaB in macrophages by resveratrol. British Journal of Pharmacology, 126, 673680. Tsan, M.F., White, J.E., Maheshwari, J.G., Bremner, T.A. & Sacco, J. (2000) Resveratrol induces Fas signalling-independent apoptosis in THP-1 human monocytic leukaemia cells. British Journal of Haematology, 109, 405412.

Dorrie, J., Gerauer, H., Wachter, Y. & Zunino, S.J. (2001) Resveratrol induces extensive apoptosis by depolarizing mitochondrial membranes and activating caspase-9 in acute lymphoblastic leukemia cells. Cancer Research, 61, 47314739. Elattar, T.M. & Virji, A.S. (1999) The effect of red wine and its components on growth and proliferation of human oral squamous carcinoma cells. Anticancer Research, 19, 54075414. Fremont, L. (2000) Biological effects of resveratrol. Life Science, 66, 663673. Gautam, S.C., Xu, Y.X., Dumaguin, M., Janakiraman, N. & Chapman, R.A. (2000) Resveratrol selectively inhibits leukemia cells: a prospective agent for ex vivo bone marrow purging. Bone Marrow Transplantation, 25, 639645. Hain, R., Reif, H.J., Krause, E., Langebartels, R., Kindl, H., Vornam, B., Wiese, W., Schmelzer, E., Schreier, P.H., Stocker, R.H. & Stenzel, K. (1993) Disease resistance results from foreign phytoalexin expression in a novel plant. Nature, 361, 153156. Harvey, W., Srour, E.F., Turner, R., Carey, R., Maze, R., Starrett, B., Kanagala, R., Pereira, D., Merchant, P., Taylor, M. & Jansen, J. (1991) Characterization of a new cell line (ESKOL) resembling hairy-cell leukemia: a model for oncogene regulation and late B-cell differentiation. Leukemia Research, 15, 733744. Hsieh, T.C. & Wu, J.M. (1999) Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines. Experimental Cell Research, 249, 109115. Hsieh, T.C., Burfeind, P., Laud, K., Backer, J.M., Traganos, F., Darzynkiewicz, Z. & Wu, J.M. (1999a) Cell cycle effects and control of gene expression by resveratrol in human breast carcinoma cell lines with different metastatic potentials. International Journal of Oncology, 15, 245252. Hsieh, T.C., Juan, G., Darzynkiewicz, Z. & Wu, J.M. (1999b) Resveratrol increases nitric oxide synthase, induces accumulation of p53 and p21 (WAF1/CIP1), and suppresses cultured bovine pulmonary artery endothelial cell proliferation by perturbing progression through S and G2. Cancer Research, 59, 25962601. Huang, C., Ma, W.Y., Goranson, A. & Dong, Z. (1999) Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway. Carcinogenesis, 20, 237242. Hung, L.M., Chen, J.K., Huang, S.S., Lee, R.S. & Su, M.J. (2000) Cardioprotective effect of resveratrol, a natural antioxidant derived from grapes. Cardiovascular Research, 47, 549555. Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beecher, C.W., Fong, H.H., Farnsworth, N.R., Kinghorn, A.D., Mehta, R.G., Moon, R.C. & Pezzuto, J.M. (1997) Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science, 275, 218220. Kawada, N., Seki, S., Inoue, M. & Kuroki, T. (1998) Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and Kupffer cells. Hepatology, 27, 12651274. Kitada, S., Zapata, J.M., Andreeff, M. & Reed, J.C. (2000) Protein kinase inhibitors avopiridol and 7-hydroxy-staurosporine downregulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood, 96, 393397. Kolb, J.P. (2000) Mechanisms involved in the pro- and anti-apoptotic role of NO in human leukemia. Leukemia, 14, 16851694. Kolb, J.P., Roman, V., Mentz, F., Zhao, H., Rouillard, D., Dugas, N., Dugas, B. & Sigaux, F. (2000) Contribution of nitric oxide to the apoptotic process in human B cell chronic lymphocytic leukemia. Leukemia and Lymphoma, 40, 243257. Koopman, G., Reutelingsperger, C.P., Kuijten, G.A., Keehnen, R.M., Pals, S.T. & van Oers, M.H. (1994) Annexin V for ow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood, 84, 14151420.

2002 Blackwell Science Ltd, British Journal of Haematology 117: 842851

Resveratrol-induced Apoptosis in B-CLL

Wadsworth, T.L. & Koop, D.R. (1999) Effects of the wine polyphenolics quercetin and resveratrol on pro-inammatory cytokine expression in RAW 264.7 macrophages. Biochemical Pharmacology, 57, 941949.

851

Zhao, H., Dugas, N., Mathiot, C., Delmer, A., Dugas, B., Sigaux, F. & Kolb, J.P. (1998) B-cell chronic lymphocytic leukemia cells express a functional inducible nitric oxide synthase displaying anti-apoptotic activity. Blood, 92, 10311043.

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