Resveratrol induces apoptosis, influences IL-6 and exerts immunomodulatory effect on mouse lymphocytic leukemia both in vitro and in vivo

International Immunopharmacology 7 (2007) 1221 – 1231 www.elsevier.com/locate/intimp

Resveratrol induces apoptosis, influences IL-6 and exerts immunomodulatory effect on mouse lymphocytic leukemia both in vitro and in vivo Tan Li a,b , Gui-Xiang Fan a , Wei Wang c , Tong Li d , Yu-Kang Yuan a,⁎ a

c

Department of Immunology and Pathogenic Biology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, PR China b Department of Immunology, Medical College of the Chinese People's Armed Police Force, Tianjin 300162, PR China Department of Nephrology, The Affiliated Hospital of Medical College of the Chinese People's Armed Police Force, Tianjin 300162, PR China d Department of Ultrasonography, Xinjiang Armed Police General Hospital, Urumqi, Xinjiang 830000, PR China Received 15 March 2007; received in revised form 17 April 2007; accepted 15 May 2007

Abstract Resveratrol, a dietary polyphenol found in grapes, has been proposed to act as a chemopreventive or anti-tumor agent in numerous epidemiologic studies. In this study, we investigate the antitumor and immunomodulation effects of resveratrol on mouse lymphocytic leukemia cells L1210 both in vitro and in vivo. Our finding indicates that resveratrol inhibits proliferation, induces apoptosis, and influences cell cycle of L1210 cells in a dose- and time-dependent manner in vitro. Furthermore, resveratrol can exert a dose-related regulatory effect on both innate and specific immune function to L1210-bearing mice. A normalization of CD4/ CD8 ratios is noted as well as an enhancement of lymphocyte proliferation, NK cell activity and anti-SRBC titers. Interleukin-6 cellular content and release are suppressed by resveratrol as well as mRNA expression. In conclusion, the data provide new findings with respect to resveratrol mechanism of action to mouse lymphocytic leukemia. © 2007 Elsevier B.V. All rights reserved. Keywords: Resveratrol; Apoptosis; Mouse lymphocytic leukemia; Immunomodulation

1. Introduction Abbreviations: PMA, phorbol-12-myristate-13-acetate; DMSO, dimethyl sulfoxide; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Con A, concanavalin A; LPS, lipopolysaccharide; FCS, fetal calf serum; PI, propidium iodide; FITC, fluorescein isothiocyanate; PE, phycoerythrin; McAb, monoclonal antibody; HRP, horseradish peroxidase; OD, optical density; FACS, fluorescence-activated cell sorting; NK, natural killer; SRBC, sheep red blood cell; IL, interleukin; RT-PCR, Reverse-Transcriptase Polymerase Chain Reaction; ELISA, enzyme linked immunosorbent assay; PBS, phosphate-buffered saline; NF-κB, nuclear factor κB. ⁎ Corresponding author. Tel.: +86 29 82655182; fax: +86 29 82656364. E-mail address: [email protected] (Y.-K. Yuan). 1567-5769/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2007.05.008

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phytoalexin presenting in grapes and a variety of medicinal plants [1,2], which can be detected in leaf epidermis and the skin of grapes (containing 50–100 μg per gram), with its concentration in wine ranging from 0.2 mg/l to 7.7 mg/l [3]. Recently, several studies have demonstrated that this molecule exhibits wide range of biological and pharmacological activities, including antioxidant property [4], anti-inflammatory property, inhibiting platelet aggregation [5] and cancer-chemopreventive activity [6]. The

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chemopreventive or anti-tumor effects of resveratrol (1) act as a phytoestrogen; (2) possess antioxidant and antimutagenic properties; (3) induce phase II drugmetabolizing enzymes (anti-initiation activity); (4) mediate anti-inflammatory effects; (5) inhibit cyclooxygenase and hydroperoxidase functions (anti-promotion activity); and (6) induce cell differentiation (anti-progression activity) [7,8]. Although resveratrol has been shown to inhibit the proliferation of a wide variety of tumor cell lines, the exact mechanism by which resveratrol exerts its effect has not yet fully elucidated. Most of the studies have focused on the beneficial effects of resveratrol in the prevention of cancer [1,6], there are only a limited number of studies considering its possible use as a therapeutic agent in vivo, especially, the concentration of resveratrol used in most studies in vitro are by far higher than those found after administration in vivo. Whereas no previous work has examined the effect of resveratrol on lymphocytic leukemia, we therefore decided to investigate the action of resveratrol on mouse lymphocytic leukemia cells L1210. The aim of this study is to elucidate the role resveratrol plays to mouse lymphocytic leukemia and to transpose the beneficial action in vitro at concentration compatible with those after oral administration in vivo. 2. Materials and methods 2.1. Reagents and cells Resveratrol, brefeldin A, PMA, ionomycin, MTT, ConA and LPS were obtained from Sigma Chemical Co.; RPMI 1640, penicillin, streptomycin and FCS from Gibco Laboratories; The anti-mouse CD3-PE-Cy5, CD4-FITC, CD8-PE, IL6-PE antibodies were purchased from eBioscience. Bcl-2 McAb was purchased from Labvision Corporation and conjugated-HRP secondary antibody was purchased from Santa Cruz Biotech. 2.2. Cell culture and MTT assay for cell viability Mouse lymphocytic leukemia L1210 cells were from ATCC (catalogue no. CCL 219), mouse fibroblast BALB/c 3T3 and YAC-1 lymphoma cell line were generous gifts from Dr YL. Wang (Life School, Xi'an Jiaotong University). The culture medium used throughout these experiments was RPMI-1640 culture medium, containing 10% heat-inactivated FCS, 2 mM L-glutamine, 200 IU/ml penicillin, 200 UG/ml streptomycin and 20 mM HEPES buffer, hereinafter referred to as the complete medium (CM). All these cell lines were maintained in CM, then seeded onto 25-cm2 culture flasks and incubated at 37 °C in humidified air with 5% CO2. Cells were divided into nine groups: the control group, and the resveratrol groups (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μM). The cellular integrity was assessed by trypan blue exclusion

(viability was always greater than 95% for all incubations). Prior to experiments, cells were resuspended in the medium described above, and plated into culture flasks at a density of 104 cells/cm2. After 24 h, the medium was removed and replaced by fresh CM with the indicated concentrations of resveratrol, and the control group was given CM without resveratrol. Cell viability was determined using the MTT cell viability assay, as described previously [9] with some modifications. In brief, approximately 104 cells per well were seeded in triplicate into 96-well plates, and then, cells were incubated with a range of drug concentrations for 12, 24 and 48 h respectively. Afterwards, 10 μl MTT (5 mg/ml) was added to a final concentration of 0.5 mg/ml in each well. 4 h later, an equal volume of DMSO was added to dissolve the blue crystals of formazan by gentle shaking of the plate. The OD was detected in the microplate reader (Multiskan MK3, Labsystems, Finland) at 570 nm, and measured values were expressed as mean ± SD. The inhibitory rate is calculated as follows: inhibitory rate (%) = [(A − B) / A] × 100, where A is the mean optical density value of the cells control, and B is that of the test samples. 2.3. Laser scanning confocal microscopy (LSCM) Apoptotic cells were detected using an ApoDETECT™ Annexin V-FITC kit (Biosea biotechnology Inc). Based on its affinity for phosphatidylserine (PS), annexin V can be used as a sensitive probe for cell surface exposure of PS. L1210 cells were treated with 25, 50 and 100 μM resveratrol for 12, 24 and 48 h. The cells (1 × 106 cells/ml) were resuspended in binding buffer(10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and then, 10 μl annexin V-FITC(20 μg/ml) and 5 μl propidium iodide (50 μg/ml) were added. The binding of annexin V-FITC to phosphatidylserine, exposed on the cell surface, was analyzed by LSCM (Bio-Rad MRC 1000) using fluorescence excitation wavelengths of 488 and 514 nm. 2.4. Flow cytometry analysis of apoptosis Cell counts were performed using a hemocytometer. Approximately 1 × 106 L1210 cells were suspended in 100 μl PBS and 200 μl 95% ethanol were added while vortexing. Cells were incubated at 4 °C for 1 h, washed with PBS and resuspended in 250 μl 1.12% (w/v) sodium citrate buffer (pH 8.4) containing 20 μg/ml of RNaseA. After incubation at 37 °C for 30 min, cellular DNA was stained by PI (50 μg/ml). Finally, the stained cells were analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) at excitation wavelength of 488 nm. Data were acquired with CellQuest acquisition software(BD Biosciences). 2.5. Western blotting analysis L1210 cells were added to the indicated concentration of resveratrol and incubated for 48 h. Monolayers were harvested and cellular lysates were prepared by suspending 1 × 106 cells

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in 100 ml lysis buffer. Cells were disrupted by sonication and extracted at 4 °C for 30 min. Protein content was determined using the method described by Lowry [10]. Samples were boiled, loaded onto 12% SDS–polyacrylamide gels, and proteins were separated by polyacrylamide gel electrophoresis (PAGE) and then electrotransferred to Immobilon-P transfer membranes (Millipore, Bedford, MA) in Tris–glycine buffer at 100 V for 1.5 h. Blots were blocked for 1 h in 5% (w/v) nonfat dry milk in PBS with 0.1% Tween-20 (PBS-T). Membranes were incubated with anti-Bcl-2 antibodies (dilution: 1/200 dilution) overnight at 4 °C with gentle shaking. Blots were then washed with PBS-T at room temperature and then probed with secondary HRP-conjugated antibodies (dilution: 1/5000), washed and visualized by enhanced chemiluminescence using autoradiography films (Hyperfilm ECL, Amersham Biosciences Corp.) according to the manufacturer's instructions. Immunoblots were analyzed by densitometry on a GelDoc 2000 system (Bio-Rad Laboratories Inc.). 2.6. Animal experiments 2.6.1. Animals Mice (BALB/c mice, male, 6–8 weeks, obtained from Center of Experiment Animal, Medical School of Xi'an Jiaotong University). Mice were housed in a pathogen-free isolation facility with a light/dark cycle of 12/12 h and fed with rodent chow and water ad libitum. The animals were allowed to acclimatize for 1 week before the experiments. 2.6.2. Ethical approval All experiments were carried out in accordance with European regulations on animal protection (Directive 86/609, OJL 358.1, December 12, 1987), the Declaration of Helsinki, and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Medical School of Xi'an Jiaotong University. 2.6.3. L1210 tumor-bearing animal model and evaluation of anti-tumor activity Prior to transplantation, 50 BALB/c mice were randomized into 5 groups: normal control group receiving the same volume of distilled water (n = 10), L1210-bearing control group injected intraperitoneally with L1210 (1 × 106 cells/ml, n = 10), other three L1210-bearing groups administered i.g. with resveratrol daily (12.5, 25, 50 mg/kg body weight (gbw), n = 10, respectively). In addition, another 30 mice were randomized into 3 groups, and received resveratrol daily as indicated above without injecting L1210 (n = 10, respectively). These 30 animals were selected for normal control to verify whether resveratrol would have bad effect on normal animals. Animal survival was monitored three times daily. After 3 weeks of treatment, all mice were sacrificed for further analyses.

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2.6.4. Determination of mitogenic activity of mice spleen lymphocytes The MTT test was used following the method described by Mosmann and Denizot [11] with modifications. Spleens of mice were squeezed and filtered over a fine nylon mesh and washed three times in Hanks' balanced salt solution. Lymphocyte suspension (2 ×106 cells/ml) was placed in a microtiter plate. 10 μg/ml ConA as a T cell mitogen was added to the cell suspension. Subsequently, cells were incubated for 68 h at 37 °C with 5% CO2. After incubation, 20 μl of the solution containing 5 mg/ml of MTT was added and the plate was incubated for another 4 h. Centrifuge the microplate and remove unreacted MTT, and then add 100 μl DMSO per well to solubilize formazan, incubating 5 min with shaking. The absorbance was read using a 570 nm filter and a reference wavelength of 690 nm in a microplate reader. Lymphocyte proliferation is expressed as a stimulation index (SI), which is defined as the mean of experimental data divided by the mean of the unstimulated control. 2.6.5. Assay of NK cells activity [12] YAC-1 cells were used as target cells (T) and seeded in 96well culture plates at 1 × 104 cells/well in RPMI-1640. Spleen cells were used as the effector cells (E) and added at 2 × 105 cells/well to give the E/T ratio 20:1. The plate was then incubated for 20 h at 37 °C in 5% CO2 atmosphere. Effector cells and target cells were incubated alone in the same condition. 10 μl MTT (5 mg/ml) was added to each well and the plate was incubated for another 4 h and subjected to MTT cellular assay. Three kinds of control measurements were performed: target cells control, blank control and effector cells control. The OD was determined at 570 nm. NK cells activity is calculated as the following equation: NK cells activityð%Þ ¼ ½ODT  ðODS  ODE Þ=ðODT Þ  100% where ODT, optical density value of target cells control, ODS, optical density value of test samples, ODE, optical density value of effector cells control. 2.6.6. Determination of the serum antibody level [13] All the mice as indicated were used to determine the complement hemolytic activity in this study. Briefly, mice were immunized intraperitoneally with 0.1 ml SRBC (about 1 × 108 SRBC) for 5 days, and then serum was separated. The serum was heat inactivated at 56 °C for 0.5 h and diluted with saline to 200-fold. Serum (1 ml), SRBC (10% v/v, 0.5 ml), and 1 ml complement were added to tubes by turns. The mixture was incubated at 37 °C for 60 min for complement-mediated SRBC hemolysis and terminated on ice. Blank control (without mice serum) and half hemolysis control were also synchronously prepared. The final mixture was kept at room temperature for 10 min and OD540 nm was read against the blank control. Hemolysin is expressed as HC50 that can be calculated as follows: HC50 ¼ OD540ðsampleÞ =OD540ð

half hemolysisÞ

 200:

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2.6.7. Purification of T lymphocytes Purified T lymphocytes were prepared from fresh peripheral blood mononuclear cells (PBMCs). Briefly, PBMCs were obtained by density gradient centrifugation. T lymphocytes were isolated from PBMCs by magnetic separation after depletion of non-T lymphocytes by negative selection with MACS mini columns (Miltenyi Biotec, Germany) according to the manufacturer's instructions. The purity of the enriched T lymphocytes routinely reached N 95% as determined by antiCD3-mAb staining and FACS analysis. 2.6.8. Flow cytometric analysis of intracellular cytokine and lymphocyte distribution T Lymphocyte subsets and their intracellular cytokine production were analyzed as described by the manufacturer (BD Biosciences). Briefly, purified T Lymphocytes were stimulated for 4 h at 37 °C in RPMI medium with 10 μg/ml brefeldin A, 25 ng/ml PMA, and 1 μg/ml ionomycin. After incubation, cells were harvested, washed and permeabilized with FACS permeabilizing solution for 10 min at room temperature. After washing with PBS, each sample was stained with monoclonal antibody to surface molecule CD3 and CD4, as well as to the intracellular cytokine IL-6. The positive control was the macrophage suspension obtained after prestimulation with LPS(1 μg/ml) and incubated for 24 h. Appropriate isotype-matched mouse immunoglobulin was used in the experiment. Intracellular cytokine expression was analyzed by the CellQuest software based on a collection of ten thousand cells, using a FACS Calibur flow cytometer. Purified T lymphocytes were incubated with CD3-PE-Cy5, CD4-FITC and CD8-PE for 15 min at RT in the dark. The samples were centrifuged and then resuspended in 500 μl of PBS. The phenotype of T lymphocytes (CD4+ and CD8+) was analyzed using a flow cytometer. The results were presented as CD4/CD8 ratio. 2.6.9. Determination of IL-6 mRNA by semiquantitative RT-PCR Semiquantitative RT-PCR was performed to investigate the expression of mRNA for IL-6 in vivo. Total RNA was extracted from the purified T lymphocytes of mice. RNA was further purified using a Qiagen RNeasy kit according to the protocol, and eluted in 50 μl of RNase-free distilled H2O. The amount of RNA was measured spectrophotometrically, 1 μg of total RNA was used for the synthesis of the first strand of cDNA using the SuperScript reverse transcriptase. Briefly, RNA, oligo(dT) primers (0.5 μg /μl) were first denatured for 5 min at 65 °C, chilled on ice for 1 min, and then incubated for 30 min at 42 °C, 5 min at 99 °C in 20 μl of reaction mixture containing 10× firststrand buffer, 10 mM dNTP mix, 0.1M DTT and 50 units of SuperScript II reverse transcriptase. Oligonucleotide primers used for RT-PCR amplification were designed according to the published sequences. Primer sequences that synthesized and purified by Shanghai Institute of Biochemistry, Chinese Academy of Science used for PCR were: IL-6 (sense, 5′-CTGGTGACAACCACGGCCTTCCCTA-3′; antisense, 5′-ATGCTTAGGCA-

TAACGCACTAGGTT-3′600 bp); actin-beta (sense, 5′ACCTCTATGCCAACACAG-3′; antisense, 5′-GTAACAGTCCGCCTAGAAG-3′, 266 bp). PCR reaction was performed as follows: denatured at 97 °C for 5 min, 93 °C for 1 min, 93 °C for 45 s, annealed at 55 °C for 45 s, with extension at 72 °C for 45 s, 30 cycles. 10 μl of each PCR production was electrophoresed in 1% agarose gel, visualized by ethidium bromide staining and scanned with a gel documentation system. The results were expressed as the ratios of intensity of the IL-6 cDNA bands to that of the β-actin bands. 2.6.10. Quantification assay of IL-6 in serum Supernatants from serum of mice were collected. The level of IL-6 was determined by sandwich ELISA according to the manufacturer's instructions (BD Biosciences, R&D Systems, Minneapolis, MN). The absorbance of each well at 490 nm was read using the microplate reader (Multiskan MK3). 2.7. Statistical analysis All data were given as the mean ±S.D. The survival curve was constructed by the Kaplan–Meier method. The data were analyzed by one way ANOVA. All statistical analyses were performed with SPSS v.13.0. Pb 0.05 was considered statistically significant. 3. Results 3.1. Anti-tumor activity in vitro 3.1.1. Morphological observation and growth inhibition of L1210 cells by resveratrol After being cultured for 12 h, 24 h and 48 h, the cells were observed. Changes in nuclear morphology of apoptosis could be seen in cells treated with resveratrol. Control cells showed intact cytoplasmic organelles, evenly distributed chromatin, and wellpreserved nuclear membranes(data not shown). Significant increases of proliferative inhibition of L1210 cells exposed in different doses of 0.78–100 μM of resveratrol were seen from 12 h to 48 h, and the maximum was 66.17% at 100 μM. The cytotoxicity of resveratrol was in a dose- and a time-dependent manner. Meanwhile, it had no influence on murine 3T3 fibroblasts cells (Fig. 1). 3.1.2. Detection and quantitation of apoptosis using the annexin V assay To obtain biochemical evidence for the induction of apoptosis by resveratrol, L1210 cells were cultured for 12, 24 and 48 h. Cells in the early stage of apoptosis spot with annexin V-FITC and the late stage of apoptosis or necrosis spot with PI. The morphology of apoptotic cells after annexin V labeling with or without simultaneous staining PI was shown in Fig. 2. According to Fig. 2C, apoptosis affected around 25% of L1210 cells after 12 h, and almost all the cells after 48 h. There was no significant difference between 50 μM and 100 μM resveratrol after 48 h. The inducing apoptosis activity of resveratrol was in a dose- and time-dependent manner.

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exhibited a significant decrease in Bcl-2 expression dosedependently compared with control cells (Fig. 4). 3.2. Anti-tumor activity in vivo 3.2.1. Survival of the tumor-bearing mice by resveratrol To elucidate whether these promising results could be also extrapolated to the “in vivo” situation, different doses resveratrol were used to treat mice receiving injection of L1210

Fig. 1. Growth inhibition of resveratrol determined by MTT assay. (A) mouse fibroblast 3T3 cells. (B) mouse lymphocytic leukemia L1210 cells.

3.1.3. Analysis of cell cycle and apoptosis After initial experiments using 0.78–100 μM resveratrol, 25– 100 μM resveratrol cultured for 48 h were found to induce significant morphological changes of apoptosis. Therefore, these concentration and time were chosen for the following experiment. In order to quantify the degree of apoptosis, cell cycle distribution was analyzed by flow cytometry. Nucleic acid staining with PI revealed typical apoptotic nuclei in resveratrol-treated cells, but control cells did not show significant features of apoptosis (Fig. 3A). After 48 h, resveratrol-treated L1210 cells showed a tendency of markedly increased cell number in the G1 phase and apoptosis accompanied with arrested cells number in the S and G2 phases. The results suggested that resveratrol could induce apoptosis and inhibit the cellular proliferation of mouse lymphocytic leukemia cells L1210 via G0/G1 phase arrest (Fig. 3, Table 1). 3.1.4. The relative expression level of Bcl-2 protein in L1210 cells We first used L1210 cells to evaluate the functional role played by Bcl-2 in preventing apoptosis using the chemotherapeutic agent resveratrol. Western analysis revealed that L1210 cells treated with resveratrol (12.5–100 μM) for 48 h

Fig. 2. Apoptosis of L1210 cells induced by resveratrol. (A–B) The morphology of apoptotic cells with annexin V-FITC/PI labeling cultured for 12 and 48 h after treated with 50 μM resveratrol (magnification of 20x). (C) The percentage of apoptosis induced by resveratrol based on data obtained by Laser scanning confocal microscopy.

1226 T. Li et al. / International Immunopharmacology 7 (2007) 1221–1231 Fig. 3. Resveratrol-induced cell cycle perturbations and apoptosis in L1210 cells cultured for 48 h. Cells stained with PI are subjected to flow cytometric analysis. (A) Control cells incubated without resveratrol. (B–D) L1210 cells treated with 25, 50, 100 μM resveratrol respectively.

T. Li et al. / International Immunopharmacology 7 (2007) 1221–1231 Table 1 Cell cycle perturbations and apoptosis induced by resveratrol treatment in L1210 cells at 48 h

Control 25 μM Resveratrol 50 μM Resveratrol 100 μM Resveratrol

Phase of cell cycle

Phase of cell cycle

Phase of cell cycle

Apoptosis

(G1 % of cells)

(S % of cells)

(G2 % of cells)

(% of cells)

34.88 ± 3.6 49.61 ± 0.9 15.52 ± 2.4 0.04 ± 0.02 55.31 ± 0.8 a 33.73 ± 1.2 a 10.96 ± 2.8 a 12.76 ± 1.1 a

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resveratrol to prolong the life span of tumor-bearing mice was evident dose-dependently. Meanwhile, toxicity was not observed in any one of the resveratrol-treated normal mice. No significant difference of survival was found between normal mice treated with three doses of resveratrol and normal mice without resveratrol (data not shown). Therefore, we used these doses of resveratrol in the following experiments.

Cell cycle distributions of control cells and resveratrol-treated cells were determined by PI staining and flow cytometric analysis. Results presented were representative of three independent experiments. Values are presented as means ± S.D. a P b 0.01 vs. control. b P b 0.05 vs. control.

3.2.2. Effect of resveratrol on Con A-induced proliferation of lymphocytes Lymphoproliferative response to Con A obtained from mice 3 weeks after resveratrol administration was measured by the MTT colorimetric assay method. Representative result was shown in Table 2. L1210 control showed significant decrease in Con A-induced proliferation compared with the normal control group. Meanwhile, lymphocytes proliferation of these mice treated with resveratrol exhibited much better response to Con A in comparison to that of the L1210 control without resveratrol, a dose-dependent increase of SI in tumor mice treated with resveratrol was seen.

cells. Kaplan–Meier curve for the survival of tumor control and tumor-bearing mice treated with intragastric administration of resveratrol was shown in Fig. 5. The ability of

3.2.3. Effect of resveratrol on NK cells activity Tumor cell elimination is known to be mediated in part by the cytotoxic activity of NK cells. We therefore measured the

52.30 ± 1.2 a 41.20 ± 2.8 b

6.50 ± 1.2 a 13.41 ± 0.6 a

49.13 ± 1.8 a 40.79 ± 3.2 b 10.03 ± 1.9 a 30.52 ± 2.7 a

Fig. 4. The relative expression level of Bcl-2 in L1210 cells in vitro. (A) Proteins were detected by ECL. (B)The groups treated by resveratrol were all decreased compared to L1210 cells control (P b 0.01). 1: Control group cells without resveratrol. 2–5: Four groups of L1210 cells treated by 12.5, 25, 50 and 100 μM resveratrol. Data presented as mean ± S.D.

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Fig. 5. Antitumor effect of resveratrol for treatment of L1210-bearing mice. The significant differences were (log-rank test): control vs. resveratrol (12.5 mg/kg/day), P = 0.268; control vs. resveratrol (both 25 and 50 mg/kg/day), P b 0.01.

cytotoxic activity of splenocytes against NK-sensitive (YAC-1) tumor cells. Resveratrol could increase NK cells activity significantly in a dose-dependent manner compared with the L1210 control (Table 2). 3.2.4. Effect of resveratrol on humoral immune response The level of a specific antibody in the serum can be used as a measure of the functional status of all the three development phases of the humoral immune response-antigen recognition, activation, and expression [14]. Hemolysin level can reflect humoral immunity. To determine whether resveratrol could affect the regulation of hemolysin, mice were immunized with SRBC. The serum antibody level in response to SRBC of those

Fig. 6. Flow cytometric analyses CD4+ and CD8+ lymphocyte subsets. (A) Normal control group. (B) L1210-bearing control group. (C–E) Three groups of L1210-bearing mice treated with 12.5, 25 and 50 mg/kg resveratrol.

Table 2 Effect of resveratrol to L1210-bearing mice SI Normal 1.12 ± 0.134 control L1210 0.65 ± 0.164a control 12.5 mg/kg 1.01 ± 0.165b Resveratrol 25 mg/kg 1.58 ± 0.12b Resveratrol 50 mg/kg 1.83 ± 0.172b Resveratrol

Activity of NK (%) 29.18 ± 3.77

HC50 102 ± 3.51

10.52 ± 2.23a 79.6 ± 2.94a

CD4/CD8 ratios 3.26 ± 0.22 1.38 ± 0.43a

18.76 ± 3.93c

120 ± 5.66b 1.78 ± 0.59c

39.67 ± 5.04b

207 ± 10.23b 2.26 ± 0.64c

54.32 ± 2.17b 239.7 ± 8.74b 3.07 ± 0.28b

CD4/CD8 ratios was increased as well as an enhancement of lymphocyte proliferation, NK cell activity and anti-SRBC titers. Lymphocyte proliferation was expressed as a stimulation index (SI). Effect of resveratrol on humoral immunity assessed by quantitative hemolysis of SRBC assay was expressed as HC50. Values are presented as means±S.D. a P b 0.01 vs. Normal control. b P b 0.01 vs. L1210 control. c P b 0.05 vs. L1210 control.

mice was shown in Table 2. Decreasing absorbance of L1210bearing control group compared to normal control group at the time 50% hemolysin taking place (HC50) showed decreasing in hemolysin and reduction in humoral immunity. All the three L1210-bearing mice administered with resveratrol increased significantly in hemolysin level dose-dependently compared to the L1210 control. It indicated that resveratrol could promote the humoral immune response in L1210-bearing mice and enhance the formation of antibody cells. 3.2.5. Resveratrol enhances CD4/CD8 ratios After 3 weeks of treatment, all mice were sacrificed, and the peripheral blood T lymphocytes of mice were collected for flow cytometric analysis. CD4/CD8 ratios of the L1210bearing mice treated with two doses of resveratrol (25 and 50 mg/kg) were significantly higher than that of the L1210 control without resveratrol. No significant difference was found between the dose of the 12.5 mg/kg group and the L1210 control (Table 2, Fig. 6).

T. Li et al. / International Immunopharmacology 7 (2007) 1221–1231 Table 3 Effect of resveratrol on the production of IL-6 in vivo Fluorescence Relative level intensity of of mRNA intracellular IL-6 Normal control L1210 control 12.5 mg/kg Resveratrol 25 mg/kg Resveratrol 50 mg/kg Resveratrol

Serum values (pg/ml)

2.04 ± 1.32 37.67 ± 5.68 a 30.02 ± 2.92 b

0.017 ± 0.0021 110.23 ± 6.89 0.102 ± 0.014 a 562.78 ± 15.63 a 0.087 ± 0.013 c 463.65 ± 12.94 b

15.58 ± 4.33 c

0.052 ± 0.014 c 277.26 ± 14.78 c

10.26 ± 3.69 c

0.031 ± 0.011 c 189.72 ± 13.56 c

Values are presented as means ± S.D. a P b 0.01 vs. normal control. b P b 0.05 vs. L1210 control. c P b 0.01 vs. L1210 control.

3.2.6. Resveratrol inhibits intracellular IL-6 production Since resveratrol was discovered to modulate lymphocytes proliferation and the production of IL-6 was involved in modulating immunological response, especially in some hematonosis [15,16], we tested whether resveratrol could affect the production of intracellular IL-6 in mouse lymphocytic leukemia. The results showed that significant reduction in the amounts of intracellular IL-6 releasing were observed in the groups with resveratrol compared with the L1210-bearing control without resveratrol in a dose-dependent manner (Table 3). 3.2.7. Effect of resveratrol on IL-6 mRNA expression and release By using semiquantitative RT-PCR, IL-6 mRNA expression of mice T lymphocytes were measured as shown in Fig. 7. IL-6 mRNA of the three groups after being treated with resveratrol were decreased dose-dependently compared to the tumor control (Table 3). ELISA for quantification of IL-6 in mice serum revealed that the IL-6 release of mice treated with resveratrol were significantly

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decreased in a dose-dependent manner compared with the L1210bearing control (Table 3).

4. Discussion Apoptosis is essential for development, maintenance of tissue homeostasis and elimination of the unwanted or damaged cells from multicellular organisms. The aberrant regulation of apoptosis has been observed in many forms of tumor [17]. Several studies have revealed that resveratrol is capable of inducing apoptosis in a multitude of tumor cell lines [18–20]. Recently, one of the probable mechanisms by which resveratrol exercises its anti-tumor property is through the suppression of the NF-κB signaling pathway [21]. Bcl-2 is one of the most important factors among NF-κB-regulated genes [22]. Studies have reported that Bcl-2 inhibits apoptosis by preventing cytochrome c release from the mitochondria and inhibiting caspase activation [23]. In our study, the antiproliferative effect of resveratrol is possibly related to the ability of inducing apoptosis. It is possible that downregulation of Bcl-2 by resveratrol could lead to the suppression of mouse lymphocytic leukemia L1210 cells proliferation in vitro. Furthermore, we and other authors previously have reported that resveratrol have no cytotoxicity on normal cells [13]. It indicates that whereas synthetic antineoplasic drugs cause nonspecific killing of cells that limit their efficacy in anticancer therapy [24], resveratrol obtained from natural products offers protective and therapeutic actions with low cytotoxicity and is beneficial in producing nutrient repletion to compromised people [25]. Interestedly, there also have been contradictory results on resveratrol-induced apoptosis. For example, it has been not only claimed that resveratrol is a powerful [26] apoptotic agent, which is compatible with

Fig. 7. Effects of resveratrol on the relative expression level of IL-6 mRNA in mice T lymphocytes detected by a gel documentation system.

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ours result, but also that resveratrol is able to prevent apoptosis [27]. A possible reason may be the differences in levels of specific cellular receptors for resveratrol and/ or metabolism of resveratrol in different tumor cells. Numerous reports have indicated either that resveratrol could inhibit proliferation of tumor cells by causing cell cycle arrest at different stages of the cell cycle [7,28]. We have found that apoptosis induced by resveratrol is closely linked to the perturbation of cell cycle. Our data indicate that the accumulation of L1210 cells in G1 phase could imply the suppression of cellular proliferation via G0/G1 phase arrest. Since resveratrol has been shown to inhibit ribonucleotide reductase and DNA polymerase activation, two key enzymes involved in DNA synthesis [29], the S phase suppression of resveratrol to L1210 cells may be involved in these processes. Besides, the effect of resveratrol on the G2 phase of L1210 cells could be attributed to the reported action of resveratrol on the cytoskeleton [30]. It is clear that the effects of resveratrol on cell cycle are highly variable, depending on the cell line studied or the culture conditions. The cell cycle signaling conflict may partly explain resveratrol's apoptosis inducing and anti-proliferative effects. Although resveratrol has been implicated in chemoprevention and chemotherapeutic of some kinds of cancer, whether the mechanism by which resveratrol exerts its action involves modulation of the immune system is not very clear. Besides, Gao et al. [31] found that resveratrol, despite its antileukemic effects in vitro, was not very effective in preventing the progression of murine myeloid leukemia in vivo. In order to ascertain if resveratrol can suppress, retard, or reverse the carcinogenic processes in vivo and the relationship between resveratrol and immunomodulation effect, we have investigated additional studies to evaluate the activity of administering resveratrol orally in mouse lymphocytic leukemia cells L1210-bearing mice. Immunerelated cells including CD3+CD4+ and CD3+CD8+ cells play pivotal role in the surveillance, detection and destruction of tumor cells in mammals and can independently defend against cancer cells [32]. Consequently, the CD4/CD8 ratio is a major indicator for assessing the function of T cell mediated immunity. In the present study, resveratrol not only increases the CD4/CD8 ratio and Con A-induced T lymphocytes proliferation, but also enhances the B cell mediated immune response which may be reflected from the higher frequency of the serum antibody level. Furthermore, resveratrol has also been found to significantly stimulate NK cells activity. Recently, IL-6 has been reported to link with tumorigenesis [33], and acts as a paracrine growth factor for multiple myeloma, non-Hodgkin's lymphoma, colorec-

tal cancer and so on [15,16]. Our results presented here indicate that the level of IL-6 has rapidly increased after mice injected with L1210 cells, however, IL-6 cellular content and release are suppressed evidently by resveratrol as well as mRNA expression in L1210-bearing mice. Flow cytometry-based intracellular cytokine staining (ICS) assays can provide means to characterize cytokine expression in individual cells and a more illuminating approach to estimate cell-mediated immune response [34]. Thus, it could be pointed out that our data from intracellular cytokine detection reflect the quantity of T lymphocytes capable to produce IL-6, whereas decreased levels of the mRNA illustrate the situation at the transcriptional level. Consequently, the cancer-chemotherapeutic activity of resveratrol to mouse lymphocytic leukemia may be partly due to the suppression of IL-6. Especially, Kaplan–Meier curves for the survival reveal that resveratrol could prolong the life span of tumorbearing mice evidently in a dose-dependent manner. Therefore, it is suggested that resveratrol is an immunostimulant and could reverse the reduction of immune response of mouse lymphocytic leukemia in vivo. In conclusion, our results indicate that resveratrol inhibits cell growth, induces apoptosis and reduces the expression of Bcl-2 in L1210 cells. Moreover, resveratrol also enhances the anti-tumor immune activity and modulates IL-6 level in the L1210-bearing mice. Our results provide new findings with respect to resveratrol mechanism of action and indicate that resveratrol might have chemotherapeutic potential to lymphocytic leukemia. Acknowledgements This work was supported by a grant from the foundation of Medical college of the Chinese People's Armed Police Force (No. WY2005-2). References [1] Fremont L. Biological effects of resveratrol. Life Sci 2000;66:663–73. [2] Soleas GJ, Diamandis EP, Goldberg DM. Resveratrol: a molecule whose time has come to gone? Clin Biochem 1997;30:91–113. [3] Creasy L, Coffee M. Phytoalexin production potential of grape berries. J Am Soc Hortic Sci 1988;113:230–4. [4] Fauconneau B, Waffo-Teguo P, Huguet F, Barrier L, Decendit A, Merillon JM. Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests. Life Sci 1997;61:2103–10. [5] Pace-Asciak CR, Hahn S, Diamandis EP, Soleas G, Goldberg DM. The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. Clin Chim Acta 1995;235: 207–14.

T. Li et al. / International Immunopharmacology 7 (2007) 1221–1231 [6] Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997;275:218–20. [7] Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 2004;24:2783–840. [8] Aziz MH, Kumar R, Ahmad N. Cancer chemoprevention by resveratrol: in vitro and in vivo studies and the underlying mechanisms. Int J Oncol 2003;23:17–28. [9] Storch A, Burkhardt K, Ludolph AC, Schwarz, Johannes. Protective effects of riluzole on dopamine neurons: involvement of oxidative stress and cellular energy metabolism. J Neurochem 2000;75:2259–69. [10] Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193: 265–75. [11] Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modification to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986;89: 271–7. [12] Si CP. Medical immunological experiment. Beijing, China: People's Medical Publishing House; 1999. p. 21–2. [13] Yang Y, Liu W, Han B, Wang C, Fu C, Liu B. The antioxidative and immunostimulating properties of D-glucosamine. Int Immunopharmacol 2007;7:29–35. [14] Silkworth JB, Loose LD. Assessment of environmental contaminant-induced lymphocyte dysfunction. Environ Health Perspect 1981;39:105–28. [15] Voorzanger N, Touitou R, Garcia E, Delecluse HJ, Rousset F, Joab I, et al. Interleukin (IL)-10 and IL-6 are produced in vivo by non-Hodgkin's lymphoma cells and act as cooperative growth factors. Cancer Res 1996;56:5499–505. [16] Landi S, Moreno V, Gioia-Patricola L, Guino E, Navarro M, de Oca J, et al. Association of common polymorphisms in inflammatory genes interleukin (IL)6, IL8, tumor necrosis factor alpha, NFKB1, and peroxisome proliferator-activated receptor gamma with colorectal cancer. Cancer Res 2003;63:3560–6. [17] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456–62. [18] Ferry-Dumazet H, Garnier O, Mamani-Matsuda M. Resveratrol inhibits the growth and induces the apoptosis of both normal and leukemic hematopoietic cells. Carcinogenesis 2002;23:1327–33. [19] Joe AK, Liu H, Suzui M, Vural ME, Xiao D, Weinstein IB. Resveratrol induces growth inhibition, S-phase arrest, apoptosis, and changes in biomarker expression in several human cancer cell lines. Clin Cancer Res 2002;8:893–903. [20] Park JW, Choi YJ, Suh SI. Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity. Carcinogenesis 2001;22:1633–9.

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[21] Aggarwal BB, Shishodi S. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol 2006;71: 1397–421. [22] Aggarwal BB. Nuclear factor-kappaB: the enemy within. Cancer Cell 2004;6:203–8. [23] Kirkin V, Joos S, Zornig M. The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 2004;1644:229–49. [24] Lançon A, Delma D, Osman H, Thénot JP, Jannin B, Latruffe N. Human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process. Biochem Biophys Res Commun 2004;316:1132–7. [25] Reddy L, Odhav B, Bhoola KD. Natural products for cancer prevention: a global perspective. Pharmacol Ther 2003;99:1–13. [26] Clement MV, Hirpara JL, Vhawdhury SH, Pervaiz S. Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells. Blood 1998;92:996–1002. [27] Maccarrone M, Lorenzon T, Guerrieri P, Finazzi-Agrò A. Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity. Eur J Biochem 1999;265:27–34. [28] Kotha A, Sekharam M, Cilenti L, Siddiquee K, Khaled A, Zervos AS, et al. Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein. Mol Cancer Ther 2006;5:621–9. [29] Sun NJ, Woo SH, Cassady JM, Snapka RM. DNA polymerase and topoisomerase II inhibitors from Psoralea corylifolia. J Nat Prod 1998;61:362–6. [30] Schneider Y, Chabert P, Stutzmann J, Coelho D, Fougerousse A, Gosse F, et al. Resveratrol analog (Z)-3,5,4-trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin polymerization. Int J Cancer 2003;107:189–96. [31] Gao XH, Xu YX, Divine G, Janakiraman N, Chapman RA, Gautam SC. Disparate in vitro and in vivo antileukemic effects of resveratrol, a natural polyphenolic compound found in grapes. J Nutr 2002;132:2076–81. [32] Mille JS. The biology of natural killer cells in cancer, infection, and pregnancy. Exp Hematol 2001;29:1157–68. [33] Aggarwal BB, Shishodia S, Sandur SK, Pandey K, Sethi Gautam. Inflammation and cancer: how hot is the link? Biochem Pharmacol 2006;72:1605–21. [34] Godoy-Ramirez K, Franck K, Mahdavifar S, Andersson L, Gaines H. Optimum culture conditions for specific and nonspecific activation of whole blood and PBMC for intracellular cytokine assessment by flow cytometry. J Immunol Methods 2004;292:1–15.