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Resveratrol-induced cellular apoptosis and cell cycle arrest in neuroblastoma cells and antitumor effects on neuroblastoma in mice
Resveratrol-induced cellular apoptosis and cell cycle arrest in neuroblastoma cells and antitumor effects on neuroblastoma in mice Yun Chen, MD, Sheng-Hong Tseng, MD, PhD, Hong-Shiee Lai, MD, PhD, and Wei-Jao Chen, MD, PhD, Taipei, Taiwan
Background. The prognosis of neuroblastoma patients remains unsatisfactory. Therefore, developing an effective treatment strategy is important. Resveratrol, a natural polyphenol, possesses chemopreventive and antitumor effects. We investigated the effects of resveratrol on the proliferation, apoptosis, and cell cycle alteration of neuroblastoma cells and determined its effects on neuroblastoma tumors in mice. Methods. Cytotoxic effects, cellular apoptosis, and alterations in the cell cycle were determined in neuro2a neuroblastoma cells exposed for varying lengths of time to a series of resveratrol concentrations. Expression of associated cell cycle regulatory proteins, cyclin E and p21, was detected by Western blot analysis, and the antitumor effects of resveratrol were investigated by treating subcutaneous neuroblastoma tumors with intraperitoneal injections of 40 mg/kg resveratrol daily for 28 days. Results. Resveratrol exerted cytotoxic effects on neuroblastoma cells. After resveratrol treatment, the apoptosis rate of the neuroblastoma cells significantly increased, a significant accumulation of cells occurred at the S phase of the cell cycle, p21 was downregulated, and cyclin E was upregulated. In addition, resveratrol treatment suppressed the growth rate of subcutaneous neuroblastomas, resulting in 70% long-term survival. Conclusion. Resveratrol caused significant cytotoxicity and increased apoptosis and S-phase accumulation of neuroblastoma cells. S-phase accumulation was related to the down-regulation of p21 and up-regulation of cyclin E. In addition, resveratrol exerted antitumor effects on neuroblastomas in mice. Thus, resveratrol shows promise for the treatment of neuroblastoma. (Surgery 2004;136:57-66.) From the Department of Surgery, Far Eastern Memorial Hospital, and the Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
NEUROBLASTOMA IS THE MOST FREQUENT solid malignancy in children.1 The prognosis of neuroblastoma patients is poor, and long-term survival of patients with advanced disease is rare despite recent advances in combination therapy.1-3 For these reasons, more effective treatments are needed. Resveratrol (3,4,59-trihydroxy-trans-stilbene), a natural polyphenol present in grapes and other foods, has been shown to exert anticancer effects in different systems based on its ability to inhibit
Accepted for publication January 24, 2004. Reprint requests: Dr. Hong-Shiee Lai, MD, PhD, Division of Pediatric Surgery, Department of Surgery, National Taiwan University Hospital, 7 Chung-Shan S. Rd., Taipei 100, Taiwan. 0039-6060/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.surg.2004.01.017
diverse cellular events associated with tumor initiation, promotion, and progression.4,5 Resveratrol has antioxidant activity and inhibits cyclooxygenase (COX) activity in different cancer models at the levels of cancer initiation and promotion.4,6-9 In addition, resveratrol inhibits the growth of several cancer cell lines, which suggests that it also has an inhibitory effect on cancer progression.5,10 Reports have shown resveratrol possesses antiestrogen properties;5-11 inhibits ribonucleotide reductase,12 DNA polymerase,13 and COX activities;8,9 disrupts cell cycle transition at the G1-S phase;14,15 and induces apoptosis.16 Thus, resveratrol has a wide range of potential targets. However, the underlying mechanisms of resveratrol action are not well understood, and the effects of resveratrol on neuroblastoma cells have not been investigated. In this study, we investigated the chemotherapeutic effect of resveratrol on neuroblastoma cells and on SURGERY 57
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neuroblastoma tumors in mice and explored the possible mechanism of the cytotoxicity effect of resveratrol on neuroblastoma cells. MATERIAL AND METHODS Cell line and cell culture. Neuro-2a cells, a subclone of the C1300 murine neuroblastoma derived spontaneously in the A/J mouse (American Type Culture Collection, Rockville, Md), were maintained in a humidified 378C, 5% carbon dioxide incubator. Cells underwent serial passage as monolayers in Eagle’s minimal essential medium, which was supplemented with nonessential amino acids, 100 IU/ml penicillin, 100 lg/ml streptomycin, 2 mmol/L L-glutamine, and 10% fetal calf serum. Cytotoxicity effects of resveratrol on the neuro2a cells. The sensitivity of the neuro-2a cells to resveratrol (Sigma Chemical Co. St. Louis, Mo) was determined in vitro by MTT (3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide)-based colorimetric assay (Boehringer Mannheim, Indianapolis, Ind).17 Briefly, 53103 neuro-2a cells were seeded in triplicate wells in a flat-bottomed 24-well microtiter plate. The cells were exposed to increasing concentrations (0, 0.1, 1, 2.5, 10, 25, and 100lmol/L) of resveratrol for 24, 48, or 72 hours. For the in vitro experiments throughout the whole study, the resveratrol was dissolved in dimethyl sulfoxide (DMSO), and the control well (0 lmol/L) was treated with DMSO. After removal of the drug, the cells were incubated for a total of 5 days after cell seeding. Cell proliferation and viability were then determined by MTT assay. All procedures were conducted according to the manufacturer’s instructions. The concentration at which 50% of the cells were killed was designated as LD50. Analysis of resveratrol-induced apoptosis by flow-activated cell sorter (FACS) flow cytometry. The apoptotic fraction of the neuroblastoma cells after exposure to resveratrol was studied by FACS analysis. Briefly, after treatment with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol for 24, 48, or 72 hours, 106 neuro-2a cells were trypsinized and washed twice with PBS. Then the cells were stored in 1 ml 80% alcohol/PBS at ÿ208C for subsequent experiments. For FACScan analysis, the cells were centrifuged at 14,000 rpm for 5 minutes and washed with PBS. Then the cells were incubated with 0.5 ml 0.5% Triton X-100/PBS and 5 lg RNase A for 30 minutes. The cells were stained with 0.5 ml 50 lg/ml propidium iodide/ PBS in the dark. The stained cells were analyzed
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using FACScan flow cytometry (FACSCalibur; Becton Dickson Immunocytometry System, San Jose, Calif). Effects of caspase 3 inhibitor on the resveratrolinduced apoptosis of the neuroblastoma cells. To investigate the effects of a caspase 3 inhibitor on resveratrol-induced apoptosis, the 106 neuro-2a cells were pretreated with the caspase 3 inhibitor (DEVD-CHO,100lmol/L,cellpermeable)(Biosource International, Camarillo, Calif) for 2 hours before resveratrol treatment, followed by concomitant treatment with DEVD-CHO and 25 lmol/L resveratrol for 48 hours. Then the apoptosis fraction of these cells was analyzed by FACScan flow cytometry as described above and compared with that of the neuro-2a cells treated with DMSO, DEVD-CHO, or 25 lmol/L resveratrol only. In addition to the FACScan analyses, these cells were subjected to terminal deoxynucleotidyl transferase mediated dUTP nick-end label (TUNEL) staining. For TUNEL staining, one million neuro-2a cells cultured on Lab-Tek chamber slides (Nunc, Inc, Naperville, Ill) received treatment as described above, after which the adherent cells were stained using MEBSTAIN Apoptosis Kit Direct (Immunotech, Marseille, France). All the procedures were conducted according to the manufacturer’s instructions. The slides were observed on a Zeiss Axioskopz epifluorescence microscope (Carl Zeiss Jena GmbH, Zeiss Gruppe, Germany). Analysis of cell cycle alteration after resveratrol treatment. The neuroblastoma cells were treated with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol for 24, 48, or 72 hours and stained by propidium iodide as previously described. Data were acquired using CellQuest software (Becton Dickinson Labware, Franklin Lakes, NJ) with a FACSCalibur (Becton) flow cytometry system using 10,000 cells per analysis. Cell cycle distribution was calculated using ModFit LT 2.0 software (Verity Software House, Topsham, Me). Western blot analysis. After treatment with 25 lmol/L resveratrol for various time exposures (0, 15, and 30 minutes, and 1, 3, 6, and 24 hours), approximately 1 3 107 cells were washed twice with PBS and lysed in 100 ll of ice-cold lysis buffer (20 mmol/L HEPES at pH 7.6, 75 mmol/L NaCl, 2.5 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.1% Triton X-100, 0.1 mmol/L Na3VO4, 50 mmol/L NaF, 0.5 lg/ml leupeptin, 1 lg/ml aprotinin, and 100 lg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride) at 48C for 30 minutes, then centrifuged at 10,000 rpm for 10 minutes. The precipitates were discarded and the solutions stored at ÿ708C. The
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concentration of protein was determined by the BCA protein assay kit (Pierce, Rockford, Ill) using bovine serum albumin as a standard. Western blot analysis was conducted by a method described previously.18-20 Briefly, cellular protein (20-50 lg) was loaded onto 10% SDS-polyacrylamide gels. The protein bands were transferred electrophoretically to PVDF membranes (Micron Separations Inc, Westborough, Mass). Membranes were probed with anti-p21, anti-cyclin E antibody (Santa Cruz Biotechnology, Santa Cruz, Calif), followed by a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, Calif). Detection of the antibody reactions was performed with Western blotting reagent ECL (Amersham Pharmacia, Piscataway, NJ), and chemiluminescence was exposed by the filters of Kodak X-Omat films (Eastman Kodak Co, Rochester, NY). The bands were analyzed by densitometry. Animal experiments. Animal experiments were approved by the Department of Clinical Research at the National Taiwan University Hospital, Taiwan, and conducted according to the guidelines of the Laboratory Animal Center of the National Taiwan University College of Medicine. Syngeneic A/J mice, weighing 15-20 g each, were used for the experiments. The mice were housed with free access to food and water on a 12-hour day, 12-hour night cycle (lights on between 6:00 A.M. and 6:00 P.M.) and with room temperature maintained at 208C. The mice were anesthetized with an intraperitoneal injection of 0.3 ml of ketamine (10 mg/ml) and xylazine (0.5 mg/ml) before experimentation. Treatment of subcutaneous neuroblastoma in mice with resveratrol. Cultured log phase neuro-2a cells (1 3 106) in 10 ll PBS were injected subcutaneously into the shaved right flanks of male A/J mice divided into 3 groups of 10 mice each. The control group received no treatment. The vehicle and experimental groups were given an intraperitoneal injection of propylene glycol (vehicle) or 40 mg/kg resveratrol (in 0.2 ml propylene glycol), respectively, once daily for 28 days, and animal survival was followed. Growth rates of the subcutaneous tumors were monitored, and tumor size was measured by a blinded observer twice weekly until the mouse died. Tumor volume was calculated from the formula: V = 1/2 (d1 d2 d3), where d1, d2, and d3 represent tumor diameters measured with calipers in mutually perpendicular directions.21 Average daily tumor volumes from each group were compared throughout the course of the experiment. Group averages were not compared after 1 animal in the group died. Survival
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time and rate of the mice were recorded. Animals surviving more than 70 days were considered longterm survivors. Survival rates and survival times were compared between the 2 groups. Histopathological studies of the tumors treated or not treated with resveratrol. The mice with subcutaneous neuroblastomas receiving various treatments as described above were killed at weeks 2, 3, and 4 after tumor cell inoculation, with 3 mice killed at each of the 3 weeks. The tumors were removed and then embedded in AMES ornithine carbamyl transferase embedding compound (Miles, Elkhart, Ind) and frozen at ÿ708C. For histopathologic (H&E stain) examination, 10-l m cryostat sections of the brains were fixed in acetone at ÿ208C for 1 minute, then washed with PBS. Then the sections were stained with hematoxylin at room temperature for 3.5 minutes, washed, and then stained with eosin for several seconds. After being washed, the sections were air dried, mounted, coverslipped, and viewed under microscope. Statistical analysis. Comparisons of cytotoxicity and apoptosis between groups were made using the Student t test. Average tumor volumes from each group were compared throughout the course of the experiment using ANOVA and post hoc Scheffe´ multiple comparison. The Kaplan-Meier method was used to assess the survival of mice inoculated with murine neuroblastoma cells, and the log-rank statistic was used to test differences between groups. The Fisher exact test was used for the analysis of animal survival rate. P values < .05 were considered statistically significant. RESULTS Cytotoxicity effects of resveratrol on neuroblastoma cells. To determine the inhibitory effects of resveratrol on the neuro-2a neuroblastoma cells, a cytotoxicity assay was conducted as described above. Figure 1 shows the survival curves of neuroblastoma cells treated with various concentrations of resveratrol for different exposure times. The relative viability of the cells subsequent to resveratrol treatment proved to be dependent on both concentration and time. Significant correlations existed between the percentage of cells killed and resveratrol dosage and drug exposure time (P < .05). Neuroblastoma cells became rounded and lost adherent characteristics after resveratrol treatment. Survival curves shifted to the left with longer drug exposures. The LD50 values for resveratrol treatment were 20.2, 14.1, and 7.5 lmol/L for 24, 48, and 72 hours of resveratrol treatment, respectively. Neuroblastoma survival
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Fig 1. Cytotoxicity effects of resveratrol on the neuro-2a neuroblastoma cells. neuro-2a cells (5 3 103) were seeded in triplicate wells in flat-bottomed 24-well microtiter plates. Subsequently, the cells were exposed to increasing concentrations (0, 0.1, 1, 2.5, 10, 25, 100 lmol/L) of resveratrol (dissolved in dimethyl sulfoxide [DMSO]) for 24, 48, or 72 hours. The control well (0 lmol/L) was treated with DMSO. After removal of the drug, the cells were incubated for a total of 5 days after cell seeding. Cell proliferation and viability were then determined by MTT assay. Percent survival is defined as the optical density at a given drug concentration divided by the optical density for control treated with DMSO alone, multiplied by 100. Each point is the average of 3 independent trials (9 determinations for each concentration) and presented as mean ± standard deviation. Significant correlations existed between the percentage of cells killed and resveratrol dosage and drug exposure time (P < .05, ANOVA). The concentration at which 50% of the cells were killed was designated as the LD50. The LD50 was 20.2, 14.1, and 7.5 lmol/L for 24, 48, and 72 hours of resveratrol treatment, respectively.
rates decreased to 36.8 ± 3.3%, 15.5 ± 2.5%, and 3.1 ± 1.1% after 24, 48, and 72 hours, respectively, of 25 lmol/L resveratrol treatment. These data indicate that resveratrol exerts a significant cytotoxic effect on the neuroblastoma cells in a concentration-dependent and time-dependent manner. Induction of apoptosis of neuroblastoma cells by resveratrol. To explore the antiproliferative properties of resveratrol, the fraction of neuroblastoma cells undergoing apoptosis after treatment with resveratrol was determined by FACScan. The apoptosis fraction of the neuro-2a cells was directly related to both resveratrol concentration and exposure period (Fig 2). The apoptosis rate rose slightly after 10 lmol/L resveratrol treatment and increased further after 25 lmol/L resveratrol treatment. Neuroblastoma cells treated with DMSO (control) for 24, 48, or 72 hours exhibited apoptosis rates of 6.1 ± 0.2%, 5.2 ± 1.0%, and 5.7 ± 0.6%, respectively. In contrast, treatment with
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Fig 2. Apoptosis of the neuro-2a neuroblastoma cells subsequent to resveratrol treatment. neuro-2a cells (106) were treated with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol (dissolved in dimethyl sulfoxide [DMSO]) for 24, 48, or 72 hours. The control well (0 lmol/L) was treated with DMSO. Then the apoptosis fraction of the cells was analyzed by FACScan. Each point is the average of 3 independent trials (9 determinations for each concentration) and is presented as mean ± standard deviation. The resveratrol induced apoptosis in neuroblastoma cells, especially when the cells were treated at high concentrations and for long drug exposure times. *P < .05 vs concentration 0 lmol/L (ANOVA).
25 lmol/L resveratrol for 24, 48, or 72 hours induced apoptosis of 11.7 ± 0.7%, 16.6 ± 0.5%, and 21.3 ± 1.3%, respectively (P < .05). The resveratrol induced apoptosis in neuroblastoma cells, especially when the cells were treated at high concentrations and for long drug exposure times. However, results of the previous cytotoxicity assay indicate that cellular apoptosis after resveratrol treatment only partially explains the cytotoxicity effect of resveratrol. Caspase 3 inhibitor suppressed the resveratrolinduced apoptosis of the neuroblastoma cells. To elucidate the role of caspase 3 in the resveratrolinduced apoptosis process, FACScan was used to analyze the apoptosis fraction of the neuro-2a cells treated with DMSO, DEVD-CHO (caspase 3 inhibitor), 25 lmol/L resveratrol for 48 hours, or pretreated with DEVD-CHO for 2 hours and followed by concomitant treatment with DEVDCHO and 25 l mol/L resveratrol for 48 hours. The apoptosis fractions were 5.58 ± 0.2% for the control cells (treated with DMSO) and 5.90 ± 0.34% for the cells treated with caspase 3 inhibitor. The apoptosis fraction between these 2 groups was not different (P > .1). By contrast, the cells treated with 25 lmol/L resveratrol for 48 hours had an
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Fig 3. Effects of caspase 3 inhibitor on the resveratrol-induced apoptosis of the neuroblastoma cells. neuro2a cells (106) received various treatments, including, A, dimethyl sulfoxide (DMSO); B, 100 lmol/L DEVDCHO (caspase 3 inhibitor); C, 25 lmol/L resveratrol; and D, pretreatment with DEVD-CHO for 2 hours, followed by concomitant treatment with DEVD-CHO and 25 lmol/L resveratrol for 48 hours. Then the apoptosis fraction of these cells was analyzed by FACScan flow cytometry. Three independent experiments were performed, and the representative data are shown here. The results showed that the extent of resveratrol-induced apoptosis was alleviated by the caspase 3 inhibitor.
apoptosis fraction of 17.23 ± 0.81%, which was significantly higher than those of the control group and the cells treated with caspase 3 inhibitor (P < .005). When the cells were pretreated with DEVD-CHO followed by concomitant treatment with DEVD-CHO and resveratrol, the apoptosis fraction of the neuroblastoma cells was decreased to 10.44 ± 0.52%, which was significantly lower than that of the cells treated with resveratrol only (P < .005) (Fig 3). The TUNEL staining also showed that no significantly apoptotic cells were noted in the neuroblastoma cells treated with DMSO or caspase 3 inhibitor (data not shown). By contrast, apoptotic cells were noted in the neuroblastoma cells treated with resveratrol; however, the relative proportion of the apoptotic cells to total cells was decreased when the neuroblastoma cells were pretreated with caspase 3 inhibitor before resveratrol treatment. The results indicated that caspase 3 inhibitor could inhibit the resveratrolinduced apoptosis of neuroblastoma cells, and thus, resveratrol-induced apoptosis of neuroblastoma cells was related to the activation of caspase 3.
Effects of resveratrol on cell cycle accumulation at the S phase. To further elucidate other mechanisms of cytotoxicity after resveratrol treatment, flow cytometric analysis of the cell cycle was performed. The distribution of cells in different phases of the cell cycle after resveratrol treatment is illustrated in Fig 4. Treatment with 25 lmol/L resveratrol for 24, 48, and 72 hours yielded Sphase fractions of 22.54 ± 1.01%, 20.60 ± 2.14%, and 31.32 ± 1.27%, respectively, which is a 2-fold elevation compared to the control values of 10.98 ± 0.12%, 11.79 ± 1.5%, and 11.71 ± 1.62%, respectively (P < .05). Resveratrol promoted cell cycle accumulation at the S phase, especially at high concentrations and for long drug exposure times. However, such cell cycle alteration only partially explains the antiproliferative effect of resveratrol. Alteration of p21 and cyclin E levels after resveratrol treatment. To assess the role of cell cycle regulatory proteins in resveratrol-induced cell cycle accumulation at the S phase, we examined neuroblastoma cell expression of p21 and cyclin E after 25 lmol/L resveratrol treatment at various
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Fig 5. The expression of cyclin E and p21 of the neuro-2a cells after resveratrol treatment. After treatment with 25 lmol/L of resveratrol for 0, 15, and 30 minutes and 1, 3, 6, and 24 hours, the expression of cyclin E and p21 of the neuro-2a neuroblastoma cells was studied by Western blot analysis. The upper panel represents the expression of the cyclin E, the middle panel represents the p21, and the lower panel represents the a-tubulin (control).
Fig 4. Cell cycle alteration subsequent to resveratrol treatment. The neuro-2a cells were treated with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol (dissolved in dimethyl sulfoxide [DMSO]) for 24, 48, and 72 hours. The control well (0 lmol/L) was treated with DMSO. Then the cell cycle of the neuro-2a neuroblastoma cells was analyzed using flow-activated cell sorter (FACS) flow cytometry. The data are presented as mean ± standard deviation. Resveratrol promoted cell cycle accumulation at the S phase, especially at high concentration and for long drug exposure times. *P < .05 vs concentration 0 lmol/L (ANOVA).
time points (Fig 5). Western blot analysis showed that the p21 level increased 1.5-fold at 1 hour, reached 3.5-fold at 3 hours, and then decreased to 0.3-fold at 24 hours. Cyclin E protein level increased gradually after 3 hours and reached a 3-fold elevation after 24 hours of resveratrol treatment. The decrease in expression of p21 and increase in expression of cyclin E after resveratrol treatment may be related to the entry of the neuro-2a cells into the S phase. Antitumor effects of resveratrol on subcutaneous neuroblastomas in mice. Because resveratrol
had cytotoxic effects on the neuroblastoma cells, we studied its antitumor effects on subcutaneous neuroblastomas in mice. A/J mice were subcutaneously inoculated with 106 neuro-2a cells in the right flank followed by no treatment (control), intraperitoneal injection of propylene glycol (vehicle), or a resveratrol concentration of 40 mg/kg each day for 28 days. During the 4-week treatment period, the animals treated with resveratrol showed good activity with normal food and water intake and had no body-weight loss, which indicated the mice tolerated the treatment well. However, the animals had body-weight loss and decreased appetite when the tumor became large. Figure 6 shows tumor growth rate of the neuroblastomas under resveratrol treatment. Tumor growth rate in the resveratrol group was significantly slower than that in the vehicle and control groups (P < .05), indicating a resveratrol concentration of 40 mg/kg daily suppressed the growth of subcutaneous neuroblastomas; however, histopathologically, the tumors showed no significant difference among various groups. Figure 7 shows survival curves of the mice carrying neuroblastomas that were treated with various regimens. All mice in the control and vehicle groups died, with survival times of 31.8 ± 2.9 and 33.4 ± 2.6 days, respectively. In contrast, 70% of the mice in the resveratrol group achieved long-term survival. The survival rate of the mice in the resveratrol group was significantly higher than that in the vehicle and control groups (P = .0015, Fisher exact test). We further analyzed the animal survival time in various groups and found there was no difference in the animal survival time between the control group and the group treated with propylene glycol (P = .27). By contrast, the group treated with resveratrol showed significantly longer survival time than did the
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Fig 6. The tumor growth rate of subcutaneous neuroblastomas under resveratrol treatment. neuro-2a cells (106) were injected subcutaneously into the shaved right flanks of male A/J mice divided into 3 groups of 10 mice each. The control group received no treatment. The vehicle and experimental groups were given an intraperitoneal injection of propylene glycol (vehicle) or a resveratrol concentration of 40 mg/kg (in 0.2 ml propylene glycol), respectively, once daily for 28 days. The tumor size was measured twice a week. The volume of the tumor was calculated from the formula V = 1/2 (d1 d2 d3), where d1, d2, and d3 were mutually perpendicular diameters measured by calipers. Each point represents the average volumes (cm3) of the tumors in each of the 3 groups. Daily average tumor volumes from each group were compared throughout the course of the experiment using ANOVA. Group averages were not compared after 1 or more animals in the group died. *P < .05 vs both vehicle and control groups.
control group or the group treated with propylene glycol (P < .00001). The results indicated that treatment with a resveratrol concentration of 40 mg/kg daily exerted antitumor effects on neuroblastoma tumors in mice. DISCUSSION Resveratrol is a chemopreventive phytochemical that effectively inhibits 3 major stages of carcinogenesis: initiation, promotion, and progression.4 In the literature, resveratrol has been found to inhibit the proliferation of a variety of cancer cells.16,22-25 However, no article has reported the effect of resveratrol on neuroblastomas. Therefore, we investigated the cytotoxic effects of resveratrol on neuroblastoma cells and demonstrated the dosedependent and duration-dependent inhibitory effects of resveratrol on the proliferation of neuroblastoma cells. Resveratrol was cytotoxic at concentrations ranging from 10 to 100 lmol/L
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Fig 7. The survival curve of the mice treated by resveratrol. neuro-2a cells (106) were injected subcutaneously into the shaved right flanks of male A/J mice divided into 3 groups of 10 mice each. The control group received no treatment. The vehicle and experimental groups were given an intraperitoneal injection of propylene glycol (vehicle) or a resveratrol concentration of 40 mg/kg (in 0.2 ml propylene glycol), respectively, once daily for 28 days. The survival curves were analyzed by the Kaplan-Meier method, and the log-rank statistic was used to test differences between groups. *P < .05 for both vehicle and control groups.
with an LD50 at the micromolar level. The LD50 at the micromolar level did not differ markedly from that reported for other tumors, such as leukemia, prostate, breast, and colon cancers.11,22,23,26,27 The anti-initiation and antipromotion properties of resveratrol have been attributed to its antioxidant and anticyclooxygenase properties.4 However, the mechanism underlying prevention of tumor progression is not well understood. Therefore, we studied possible mechanisms controlling the antiproliferative effect of resveratrol on neuroblastoma cells. Defective control of apoptosis has been suggested to play a central role in the pathogenesis of neoplasm.28 We investigated the effect of resveratrol on the induction of apoptosis of neuroblastoma cells and noted that resveratrol did induce apoptosis, as seen for other types of cancer cells.16,22-25 This resveratrol-induced apoptosis of neuroblastoma cells was most dramatic after treatment with 25 lmol/L resveratrol. Prolonged exposure of neuroblastoma cells to 25 lmol/L resveratrol elicited significantly more cellular apoptosis than did shorter periods of treatment. For the induction of apoptosis, activation of caspase is a key event.29 There have been at least 13 caspases identified from mammalian cells, and they function in a variety of fashions.30 Of these caspases, caspase 3 is a downstream caspase and plays a determining role in the execution phase of apoptosis.31 After the
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activation of the apoptosis process by upstream caspases, caspase 3 will cleave a variety of substrates; thus caspase 3 is considered to constitute a general terminal mediator of apoptosis.31 We therefore studied the role of caspase 3 in the resveratrolinduced apoptosis of neuroblastoma cells and found caspase 3 inhibitor significantly alleviated the extent of resveratrol-induced apoptosis of the neuroblastoma cells. Thus, the resveratrol-induced apoptosis of neuroblastoma cells is considered to be mediated, at least partially, through the activation of caspase 3. Cell cycle regulation is another mechanism of anti-proliferation in neoplasms.32 We studied the alteration in cell cycle after treatment of resveratrol in neuroblastoma cells and found that resveratrol induced cell cycle accumulation at the S phase. We observed a 2-fold increase in the percentage of neuroblastoma cells accumulated at the S phase after treatment with 25 lmol/L resveratrol for 24 hours. Cell cycle accumulation at the S phase also had been observed in prostate cells and HL-60 cells after resveratrol treatment.14,16,27 We further explored changes in the cell cycle regulator proteins cyclin E and p21 in neuroblastoma cells treated with resveratrol. Cyclin E is a rate-limiting component of the machinery that controls entry into the S phase.33 The expression of cyclin E is periodic and reaches a maximum value at the G1-S transition.33 The 3-fold elevation of cyclin E level in neuroblastoma cells after 25 lmol/L resveratrol treatment for 24 hours was consistent with the phenomenon of neuroblastoma cell accumulation at the S phase. Cyclin E associates with cyclin-dependent kinase 2 (CDK2) to form a cyclin E-CDK2 complex, which phosphorylates the retinoblastoma (Rb) protein to maintain normal cell cycling.34 In addition to the cyclin E-CDK2 complex, the cell cycle progression through the G1 into S phase is also under multiple levels of control by p21.35 p21 plays a dual role in both positive and negative effects on the G1 progression because p21 is required for cyclin/CDK complexes to assemble and be active, and p21 exerts positive effect on the G1 progression; in contrast, p21 may also block CDK activity, especially the cyclin E/CDK2 complex.35 Therefore, we studied the change in p21 expression and found p21 expression increased initially but subsequently decreased to 0.3-fold after 25 lmol/L resveratrol treatment for 24 hours. The early increase of p21 expression may be due to some mitogenic signals activated by resveratrol during early G1 phase progression.36 The subsequent decrease in p21 level might relieve the inhibition on the cyclin E/CDK2 complex and thus
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facilitate the phosphorylation of Rb protein and release of the E2F, and finally promote the progression from the G1 to S phase.27,35 As a whole, the downregulation of p21, associated with the upregulation of cyclin E, might account for the increased entry of neuroblastoma cells into the S phase; however, the limitations that prevent cells from passing into the G2-M phase might be related to other mechanisms, such as the inhibitory effect of resveratrol on DNA synthesis, which occurs at concentrations above 15 lmol/L.12,27 The inhibition of the DNA synthesis is probably related to the effects of resveratrol on the enzymes of DNA synthesis, such as DNA polymerase a and d and ribonucleotide reductase.12,13,37 Thus resveratrol was considered to foster a collision course between 2 important processes in cell cycle progression by activating signaling pathways required for the entry of cells into the S phase and at the same time limiting their progression through the S phase by inhibiting DNA synthesis.27 The unique ability of resveratrol to recruit neuroblastoma cells into the S phase suggests resveratrol might exert a radiosensitization effect; however, further studies are necessary to clarify this point. Because resveratrol had significant cytotoxic effects on neuroblastoma cells, we investigated in vivo antitumor effects on neuroblastoma tumors in mice. We found intraperitoneal injections of resveratrol concentrations of 40 mg/kg daily had antitumor effects on subcutaneous neuroblastomas in mice, as demonstrated by reduced tumor growth rate, prolonged animal survival time, and increased animal survival rate (70% vs 0% long-term survival rate). The in vitro cytotoxicity and in vivo growth inhibition of neuroblastoma tumors suggest resveratrol may be effective for the treatment of neuroblastoma. The dosage of a resveratrol concentration of 40 mg/kg daily was effective against neuroblastoma tumors. However, we do not know the serum level of resveratrol achieved by this dosage in the mice. One report mentions that oral administration of 28 lg resveratrol to male rats achieved a peak plasma level of resveratrol greater than 20 mg/ml after 1 hour.38 Although the animals used for this study were different from ours, we estimate a treatment dose of 40 mg/kg may produce a level of 25 lmol/L of resveratrol in the serum.38 In summary, this study demonstrated that resveratrol caused concentration-dependent and time-dependent cytotoxicity and induction of apoptosis in neuroblastoma cells. The resveratrolinduced apoptosis of the neuroblastoma cells could be alleviated by the caspase 3 inhibitor. In addition,
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resveratrol induced accumulation of neuroblastoma cells at the S phase of the cell cycle. The cyclin E upregulation and p21 downregulation in neuroblastoma cells after resveratrol treatment were compatible with induction of cells entering the S phase. Finally, resveratrol exerted an antitumor effect on neuroblastoma tumors as shown by inhibition of tumor growth and increase of animal survival rate and survival time. To the best of our knowledge, this is the first report demonstrating the antitumor effects of resveratrol on neuroblastoma cells and subcutaneous neuroblastoma tumors in mice. Additional clinical studies are needed before the use of resveratrol in the treatment of neuroblastoma can be recommended.
REFERENCES 1. Yoshida H, Enomoto H, Kawamura K, Takenaga K, Tanabe M, Ohnuma N, et al. Antitumor vaccine effect of irradiated murine neuroblastoma cells producing interleukin-2 or granulocyte macrophage-colony stimulating factor. Int J Oncol 1998;13:73-8. 2. Davidoff AM, Kimbrough SA, Ng CY, Shochat SJ, Vanin EF. Neuroblastoma regression and immunity induced by transgenic expression of interleukin-12. J Pediatr Surg 1999; 34:902-7. 3. Chen Y, Lin SM, Lai HS, Tseng SH, Chen WJ. Effects of irradiated tumor vaccine and continuous localized infusion of granulocyte-macrophage colony-stimulating factor on neuroblastomas in mice. J Pediatr Surg 2002; 37:1298-304. 4. 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. 5. Mgbonyebi OP, Russo J, Russo H. Antiproliferative effect of synthetic resveratrol on human breast epithelial cells. Int J Oncol 1998;12:865-9. 6. Belguendouz L, Fremont L, Linard A. Resveratrol inhibits metal ion-dependent and independent peroxidation of porcine low-density lipoproteins. Biochem Pharmacol 1997;53:1347-55. 7. 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. 8. Maccarrone M, Lorenzon T, Guerrieri P, Agro AF. Resveratrol prevents apoptosis in K562 cells by inhibiting lipooxygenase and cyclooxygenase activity. Eur J Biochem 1999;265:27-34. 9. Subbaramaiah K, Chung WJ, Michaluart P, Telang N, Tanabe T, Inoue H, et al. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 1998; 273:21875-82. 10. Elattar TM, Virji AS. Modulating effect of resveratrol and quercetin on oral cancer cell growth and proliferation. Anticancer Drugs 1999;10:187-93. 11. Lu R, Serrero G. Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the
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growth of human breast cancer cells. J Cell Physiol 1999; 179:297-304. Fontcave M, Lepoivre M, Elleingand E, Gerez C, Guittet O. Resveratrol, a remarkable inhibitor of ribonucleotide reductase. FEBS Lett 1998;421:277-9. Sun NJ, Woo SH, Cassady JM, Snapka RM. DNA polymerase and topoisomerase II inhibitors form Psoralea corylifolia. J Natl Prod 1998;61:362-6. Ragione FD, Cucciolla V, Borriello A, Pietra VD, Racioppi L, Soldati G, et al. Resveratrol arrests the cell division cycle at S/G2 phase transition. Biochem Biophys Res Commun 1998;250:53-8. Zou J, Huang Y, Chen Q, Wang N, Cao K, Hsieh TC, et al. Suppression of mitogenesis and regulation of cell cycle traverse by resveratrol in cultured smooth muscle cells. Int J Oncol 1999;15:647-51. Hsieh TC, Wu JM. Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines. Exp Cell Res 1999;249: 109-15. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Methods 1983;65:55-63. Chau YP, Shiah SG, Don MJ, Kuo ML. Involvement of hydrogen peroxide in topoisomerase inhibitor b-lapachoneinduced apoptosis and differentiation in human leukemia cells. Free Radical Biol Med 1998;24:660-70. Shiah SG, Chuang SE, Chau YP, Shen SC, Kuo ML. Activation of c-Jun NH2-terminal kinase and subsequent CPP32/Yama during topoisomerase inhibitor belapachoneinduced apoptosis through an oxidation-dependent pathway. Cancer Res 1999;59:391-8. Chuang SM, Wang IC, Yang JL. Roles of JNK, p38 and ERK mitogen-activated protein kinase in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 2000; 21:1423-32. Weizsaecker M, Deen DF, Rosenblum ML, Hoshino T, Gutin PH, Barker M. The 9L rat brain tumor: description and application of an animal model. J Neurol 1981;224: 183-92. Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ. Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cell. Cancer Lett 1999;140:1-10. Clement MV, Hirpara JL, Chawdhury 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. She QB, Bode AM, Ma WY, Chen NY, Dong Z. Resveratrolinduced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer Res 2001;61:1604-10. Huang C, Ma WY, Goranson A, Dong Z. Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway. Carcinogenesis 1999; 20:237-42. Schneider Y, Vincent F, Duranton B, Badolo L, Gosse F, Bergmann C, et al. Antiproliferative effect of resveratrol, a natural component of grapes and wine, on human colonic cancer cells. Cancer Lett 2000;158:85-91. Kuwajerwala N, Cifuentes E, Gautam S, Menon M, Barrack ER, Reddy GPV. Resveratrol induces prostate cancer cell entry into S phase and inhibits DNA synthesis. Cancer Res 2002;62:2488-92. Hickman JA. Apoptosis and tumourigenesis. Curr Opin Genet Dev 2002;12:67-72.
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29. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell 1997;88:347-54. 30. Thornberry N, Lazebnik Y. Caspases: enemies within. Science 1998;281:1312-6. 31. Mandlekar S, Yu R, Tan TH, Kong AH. Activation of caspase3 and c-Jun NH2-terminal kinase-1 signaling pathways in tamoxifen-induced apoptosis of human breast cancer cells. Cancer Res 2000;60:5995-6000. 32. Carnero A. Targeting the cell cycle for cancer therapy. Br J Cancer 2002;87:129-33. 33. Sherr CJ. Cancer cell cycles. Science 1996;274:1672-7. 34. Matsushime H, Quelle DE, Shurtleff SA, Shibuya M, Sherr CJ, Kato JY. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol 1994;14:2066-76.
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35. Weinberg WC, Denning MF. P21WAF1 control of the epithelial cell cycle and cell fate. Crit Rev Oral Biol Med 2002;13:453-64. 36. Sherr CJ, Roverts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999; 13:1501-12. 37. Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, et al. Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. J Biol Chem 2001;276:22586-94. 38. Bertelli AA, Giovannini L, Stradi R, Urien S, Tillement JP, Bertelli A. Kinetics of trans- and cis-resveratrol (3,49, 5-trihydroxystilbene) after red wine oral administration in rats. Int J Clin Pharmacol Res 1996;16:77-81.
Background. The prognosis of neuroblastoma patients remains unsatisfactory. Therefore, developing an effective treatment strategy is important. Resveratrol, a natural polyphenol, possesses chemopreventive and antitumor effects. We investigated the effects of resveratrol on the proliferation, apoptosis, and cell cycle alteration of neuroblastoma cells and determined its effects on neuroblastoma tumors in mice. Methods. Cytotoxic effects, cellular apoptosis, and alterations in the cell cycle were determined in neuro2a neuroblastoma cells exposed for varying lengths of time to a series of resveratrol concentrations. Expression of associated cell cycle regulatory proteins, cyclin E and p21, was detected by Western blot analysis, and the antitumor effects of resveratrol were investigated by treating subcutaneous neuroblastoma tumors with intraperitoneal injections of 40 mg/kg resveratrol daily for 28 days. Results. Resveratrol exerted cytotoxic effects on neuroblastoma cells. After resveratrol treatment, the apoptosis rate of the neuroblastoma cells significantly increased, a significant accumulation of cells occurred at the S phase of the cell cycle, p21 was downregulated, and cyclin E was upregulated. In addition, resveratrol treatment suppressed the growth rate of subcutaneous neuroblastomas, resulting in 70% long-term survival. Conclusion. Resveratrol caused significant cytotoxicity and increased apoptosis and S-phase accumulation of neuroblastoma cells. S-phase accumulation was related to the down-regulation of p21 and up-regulation of cyclin E. In addition, resveratrol exerted antitumor effects on neuroblastomas in mice. Thus, resveratrol shows promise for the treatment of neuroblastoma. (Surgery 2004;136:57-66.) From the Department of Surgery, Far Eastern Memorial Hospital, and the Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
NEUROBLASTOMA IS THE MOST FREQUENT solid malignancy in children.1 The prognosis of neuroblastoma patients is poor, and long-term survival of patients with advanced disease is rare despite recent advances in combination therapy.1-3 For these reasons, more effective treatments are needed. Resveratrol (3,4,59-trihydroxy-trans-stilbene), a natural polyphenol present in grapes and other foods, has been shown to exert anticancer effects in different systems based on its ability to inhibit
Accepted for publication January 24, 2004. Reprint requests: Dr. Hong-Shiee Lai, MD, PhD, Division of Pediatric Surgery, Department of Surgery, National Taiwan University Hospital, 7 Chung-Shan S. Rd., Taipei 100, Taiwan. 0039-6060/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.surg.2004.01.017
diverse cellular events associated with tumor initiation, promotion, and progression.4,5 Resveratrol has antioxidant activity and inhibits cyclooxygenase (COX) activity in different cancer models at the levels of cancer initiation and promotion.4,6-9 In addition, resveratrol inhibits the growth of several cancer cell lines, which suggests that it also has an inhibitory effect on cancer progression.5,10 Reports have shown resveratrol possesses antiestrogen properties;5-11 inhibits ribonucleotide reductase,12 DNA polymerase,13 and COX activities;8,9 disrupts cell cycle transition at the G1-S phase;14,15 and induces apoptosis.16 Thus, resveratrol has a wide range of potential targets. However, the underlying mechanisms of resveratrol action are not well understood, and the effects of resveratrol on neuroblastoma cells have not been investigated. In this study, we investigated the chemotherapeutic effect of resveratrol on neuroblastoma cells and on SURGERY 57
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neuroblastoma tumors in mice and explored the possible mechanism of the cytotoxicity effect of resveratrol on neuroblastoma cells. MATERIAL AND METHODS Cell line and cell culture. Neuro-2a cells, a subclone of the C1300 murine neuroblastoma derived spontaneously in the A/J mouse (American Type Culture Collection, Rockville, Md), were maintained in a humidified 378C, 5% carbon dioxide incubator. Cells underwent serial passage as monolayers in Eagle’s minimal essential medium, which was supplemented with nonessential amino acids, 100 IU/ml penicillin, 100 lg/ml streptomycin, 2 mmol/L L-glutamine, and 10% fetal calf serum. Cytotoxicity effects of resveratrol on the neuro2a cells. The sensitivity of the neuro-2a cells to resveratrol (Sigma Chemical Co. St. Louis, Mo) was determined in vitro by MTT (3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide)-based colorimetric assay (Boehringer Mannheim, Indianapolis, Ind).17 Briefly, 53103 neuro-2a cells were seeded in triplicate wells in a flat-bottomed 24-well microtiter plate. The cells were exposed to increasing concentrations (0, 0.1, 1, 2.5, 10, 25, and 100lmol/L) of resveratrol for 24, 48, or 72 hours. For the in vitro experiments throughout the whole study, the resveratrol was dissolved in dimethyl sulfoxide (DMSO), and the control well (0 lmol/L) was treated with DMSO. After removal of the drug, the cells were incubated for a total of 5 days after cell seeding. Cell proliferation and viability were then determined by MTT assay. All procedures were conducted according to the manufacturer’s instructions. The concentration at which 50% of the cells were killed was designated as LD50. Analysis of resveratrol-induced apoptosis by flow-activated cell sorter (FACS) flow cytometry. The apoptotic fraction of the neuroblastoma cells after exposure to resveratrol was studied by FACS analysis. Briefly, after treatment with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol for 24, 48, or 72 hours, 106 neuro-2a cells were trypsinized and washed twice with PBS. Then the cells were stored in 1 ml 80% alcohol/PBS at ÿ208C for subsequent experiments. For FACScan analysis, the cells were centrifuged at 14,000 rpm for 5 minutes and washed with PBS. Then the cells were incubated with 0.5 ml 0.5% Triton X-100/PBS and 5 lg RNase A for 30 minutes. The cells were stained with 0.5 ml 50 lg/ml propidium iodide/ PBS in the dark. The stained cells were analyzed
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using FACScan flow cytometry (FACSCalibur; Becton Dickson Immunocytometry System, San Jose, Calif). Effects of caspase 3 inhibitor on the resveratrolinduced apoptosis of the neuroblastoma cells. To investigate the effects of a caspase 3 inhibitor on resveratrol-induced apoptosis, the 106 neuro-2a cells were pretreated with the caspase 3 inhibitor (DEVD-CHO,100lmol/L,cellpermeable)(Biosource International, Camarillo, Calif) for 2 hours before resveratrol treatment, followed by concomitant treatment with DEVD-CHO and 25 lmol/L resveratrol for 48 hours. Then the apoptosis fraction of these cells was analyzed by FACScan flow cytometry as described above and compared with that of the neuro-2a cells treated with DMSO, DEVD-CHO, or 25 lmol/L resveratrol only. In addition to the FACScan analyses, these cells were subjected to terminal deoxynucleotidyl transferase mediated dUTP nick-end label (TUNEL) staining. For TUNEL staining, one million neuro-2a cells cultured on Lab-Tek chamber slides (Nunc, Inc, Naperville, Ill) received treatment as described above, after which the adherent cells were stained using MEBSTAIN Apoptosis Kit Direct (Immunotech, Marseille, France). All the procedures were conducted according to the manufacturer’s instructions. The slides were observed on a Zeiss Axioskopz epifluorescence microscope (Carl Zeiss Jena GmbH, Zeiss Gruppe, Germany). Analysis of cell cycle alteration after resveratrol treatment. The neuroblastoma cells were treated with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol for 24, 48, or 72 hours and stained by propidium iodide as previously described. Data were acquired using CellQuest software (Becton Dickinson Labware, Franklin Lakes, NJ) with a FACSCalibur (Becton) flow cytometry system using 10,000 cells per analysis. Cell cycle distribution was calculated using ModFit LT 2.0 software (Verity Software House, Topsham, Me). Western blot analysis. After treatment with 25 lmol/L resveratrol for various time exposures (0, 15, and 30 minutes, and 1, 3, 6, and 24 hours), approximately 1 3 107 cells were washed twice with PBS and lysed in 100 ll of ice-cold lysis buffer (20 mmol/L HEPES at pH 7.6, 75 mmol/L NaCl, 2.5 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.1% Triton X-100, 0.1 mmol/L Na3VO4, 50 mmol/L NaF, 0.5 lg/ml leupeptin, 1 lg/ml aprotinin, and 100 lg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride) at 48C for 30 minutes, then centrifuged at 10,000 rpm for 10 minutes. The precipitates were discarded and the solutions stored at ÿ708C. The
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concentration of protein was determined by the BCA protein assay kit (Pierce, Rockford, Ill) using bovine serum albumin as a standard. Western blot analysis was conducted by a method described previously.18-20 Briefly, cellular protein (20-50 lg) was loaded onto 10% SDS-polyacrylamide gels. The protein bands were transferred electrophoretically to PVDF membranes (Micron Separations Inc, Westborough, Mass). Membranes were probed with anti-p21, anti-cyclin E antibody (Santa Cruz Biotechnology, Santa Cruz, Calif), followed by a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, Calif). Detection of the antibody reactions was performed with Western blotting reagent ECL (Amersham Pharmacia, Piscataway, NJ), and chemiluminescence was exposed by the filters of Kodak X-Omat films (Eastman Kodak Co, Rochester, NY). The bands were analyzed by densitometry. Animal experiments. Animal experiments were approved by the Department of Clinical Research at the National Taiwan University Hospital, Taiwan, and conducted according to the guidelines of the Laboratory Animal Center of the National Taiwan University College of Medicine. Syngeneic A/J mice, weighing 15-20 g each, were used for the experiments. The mice were housed with free access to food and water on a 12-hour day, 12-hour night cycle (lights on between 6:00 A.M. and 6:00 P.M.) and with room temperature maintained at 208C. The mice were anesthetized with an intraperitoneal injection of 0.3 ml of ketamine (10 mg/ml) and xylazine (0.5 mg/ml) before experimentation. Treatment of subcutaneous neuroblastoma in mice with resveratrol. Cultured log phase neuro-2a cells (1 3 106) in 10 ll PBS were injected subcutaneously into the shaved right flanks of male A/J mice divided into 3 groups of 10 mice each. The control group received no treatment. The vehicle and experimental groups were given an intraperitoneal injection of propylene glycol (vehicle) or 40 mg/kg resveratrol (in 0.2 ml propylene glycol), respectively, once daily for 28 days, and animal survival was followed. Growth rates of the subcutaneous tumors were monitored, and tumor size was measured by a blinded observer twice weekly until the mouse died. Tumor volume was calculated from the formula: V = 1/2 (d1 d2 d3), where d1, d2, and d3 represent tumor diameters measured with calipers in mutually perpendicular directions.21 Average daily tumor volumes from each group were compared throughout the course of the experiment. Group averages were not compared after 1 animal in the group died. Survival
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time and rate of the mice were recorded. Animals surviving more than 70 days were considered longterm survivors. Survival rates and survival times were compared between the 2 groups. Histopathological studies of the tumors treated or not treated with resveratrol. The mice with subcutaneous neuroblastomas receiving various treatments as described above were killed at weeks 2, 3, and 4 after tumor cell inoculation, with 3 mice killed at each of the 3 weeks. The tumors were removed and then embedded in AMES ornithine carbamyl transferase embedding compound (Miles, Elkhart, Ind) and frozen at ÿ708C. For histopathologic (H&E stain) examination, 10-l m cryostat sections of the brains were fixed in acetone at ÿ208C for 1 minute, then washed with PBS. Then the sections were stained with hematoxylin at room temperature for 3.5 minutes, washed, and then stained with eosin for several seconds. After being washed, the sections were air dried, mounted, coverslipped, and viewed under microscope. Statistical analysis. Comparisons of cytotoxicity and apoptosis between groups were made using the Student t test. Average tumor volumes from each group were compared throughout the course of the experiment using ANOVA and post hoc Scheffe´ multiple comparison. The Kaplan-Meier method was used to assess the survival of mice inoculated with murine neuroblastoma cells, and the log-rank statistic was used to test differences between groups. The Fisher exact test was used for the analysis of animal survival rate. P values < .05 were considered statistically significant. RESULTS Cytotoxicity effects of resveratrol on neuroblastoma cells. To determine the inhibitory effects of resveratrol on the neuro-2a neuroblastoma cells, a cytotoxicity assay was conducted as described above. Figure 1 shows the survival curves of neuroblastoma cells treated with various concentrations of resveratrol for different exposure times. The relative viability of the cells subsequent to resveratrol treatment proved to be dependent on both concentration and time. Significant correlations existed between the percentage of cells killed and resveratrol dosage and drug exposure time (P < .05). Neuroblastoma cells became rounded and lost adherent characteristics after resveratrol treatment. Survival curves shifted to the left with longer drug exposures. The LD50 values for resveratrol treatment were 20.2, 14.1, and 7.5 lmol/L for 24, 48, and 72 hours of resveratrol treatment, respectively. Neuroblastoma survival
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Fig 1. Cytotoxicity effects of resveratrol on the neuro-2a neuroblastoma cells. neuro-2a cells (5 3 103) were seeded in triplicate wells in flat-bottomed 24-well microtiter plates. Subsequently, the cells were exposed to increasing concentrations (0, 0.1, 1, 2.5, 10, 25, 100 lmol/L) of resveratrol (dissolved in dimethyl sulfoxide [DMSO]) for 24, 48, or 72 hours. The control well (0 lmol/L) was treated with DMSO. After removal of the drug, the cells were incubated for a total of 5 days after cell seeding. Cell proliferation and viability were then determined by MTT assay. Percent survival is defined as the optical density at a given drug concentration divided by the optical density for control treated with DMSO alone, multiplied by 100. Each point is the average of 3 independent trials (9 determinations for each concentration) and presented as mean ± standard deviation. Significant correlations existed between the percentage of cells killed and resveratrol dosage and drug exposure time (P < .05, ANOVA). The concentration at which 50% of the cells were killed was designated as the LD50. The LD50 was 20.2, 14.1, and 7.5 lmol/L for 24, 48, and 72 hours of resveratrol treatment, respectively.
rates decreased to 36.8 ± 3.3%, 15.5 ± 2.5%, and 3.1 ± 1.1% after 24, 48, and 72 hours, respectively, of 25 lmol/L resveratrol treatment. These data indicate that resveratrol exerts a significant cytotoxic effect on the neuroblastoma cells in a concentration-dependent and time-dependent manner. Induction of apoptosis of neuroblastoma cells by resveratrol. To explore the antiproliferative properties of resveratrol, the fraction of neuroblastoma cells undergoing apoptosis after treatment with resveratrol was determined by FACScan. The apoptosis fraction of the neuro-2a cells was directly related to both resveratrol concentration and exposure period (Fig 2). The apoptosis rate rose slightly after 10 lmol/L resveratrol treatment and increased further after 25 lmol/L resveratrol treatment. Neuroblastoma cells treated with DMSO (control) for 24, 48, or 72 hours exhibited apoptosis rates of 6.1 ± 0.2%, 5.2 ± 1.0%, and 5.7 ± 0.6%, respectively. In contrast, treatment with
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Fig 2. Apoptosis of the neuro-2a neuroblastoma cells subsequent to resveratrol treatment. neuro-2a cells (106) were treated with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol (dissolved in dimethyl sulfoxide [DMSO]) for 24, 48, or 72 hours. The control well (0 lmol/L) was treated with DMSO. Then the apoptosis fraction of the cells was analyzed by FACScan. Each point is the average of 3 independent trials (9 determinations for each concentration) and is presented as mean ± standard deviation. The resveratrol induced apoptosis in neuroblastoma cells, especially when the cells were treated at high concentrations and for long drug exposure times. *P < .05 vs concentration 0 lmol/L (ANOVA).
25 lmol/L resveratrol for 24, 48, or 72 hours induced apoptosis of 11.7 ± 0.7%, 16.6 ± 0.5%, and 21.3 ± 1.3%, respectively (P < .05). The resveratrol induced apoptosis in neuroblastoma cells, especially when the cells were treated at high concentrations and for long drug exposure times. However, results of the previous cytotoxicity assay indicate that cellular apoptosis after resveratrol treatment only partially explains the cytotoxicity effect of resveratrol. Caspase 3 inhibitor suppressed the resveratrolinduced apoptosis of the neuroblastoma cells. To elucidate the role of caspase 3 in the resveratrolinduced apoptosis process, FACScan was used to analyze the apoptosis fraction of the neuro-2a cells treated with DMSO, DEVD-CHO (caspase 3 inhibitor), 25 lmol/L resveratrol for 48 hours, or pretreated with DEVD-CHO for 2 hours and followed by concomitant treatment with DEVDCHO and 25 l mol/L resveratrol for 48 hours. The apoptosis fractions were 5.58 ± 0.2% for the control cells (treated with DMSO) and 5.90 ± 0.34% for the cells treated with caspase 3 inhibitor. The apoptosis fraction between these 2 groups was not different (P > .1). By contrast, the cells treated with 25 lmol/L resveratrol for 48 hours had an
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Fig 3. Effects of caspase 3 inhibitor on the resveratrol-induced apoptosis of the neuroblastoma cells. neuro2a cells (106) received various treatments, including, A, dimethyl sulfoxide (DMSO); B, 100 lmol/L DEVDCHO (caspase 3 inhibitor); C, 25 lmol/L resveratrol; and D, pretreatment with DEVD-CHO for 2 hours, followed by concomitant treatment with DEVD-CHO and 25 lmol/L resveratrol for 48 hours. Then the apoptosis fraction of these cells was analyzed by FACScan flow cytometry. Three independent experiments were performed, and the representative data are shown here. The results showed that the extent of resveratrol-induced apoptosis was alleviated by the caspase 3 inhibitor.
apoptosis fraction of 17.23 ± 0.81%, which was significantly higher than those of the control group and the cells treated with caspase 3 inhibitor (P < .005). When the cells were pretreated with DEVD-CHO followed by concomitant treatment with DEVD-CHO and resveratrol, the apoptosis fraction of the neuroblastoma cells was decreased to 10.44 ± 0.52%, which was significantly lower than that of the cells treated with resveratrol only (P < .005) (Fig 3). The TUNEL staining also showed that no significantly apoptotic cells were noted in the neuroblastoma cells treated with DMSO or caspase 3 inhibitor (data not shown). By contrast, apoptotic cells were noted in the neuroblastoma cells treated with resveratrol; however, the relative proportion of the apoptotic cells to total cells was decreased when the neuroblastoma cells were pretreated with caspase 3 inhibitor before resveratrol treatment. The results indicated that caspase 3 inhibitor could inhibit the resveratrolinduced apoptosis of neuroblastoma cells, and thus, resveratrol-induced apoptosis of neuroblastoma cells was related to the activation of caspase 3.
Effects of resveratrol on cell cycle accumulation at the S phase. To further elucidate other mechanisms of cytotoxicity after resveratrol treatment, flow cytometric analysis of the cell cycle was performed. The distribution of cells in different phases of the cell cycle after resveratrol treatment is illustrated in Fig 4. Treatment with 25 lmol/L resveratrol for 24, 48, and 72 hours yielded Sphase fractions of 22.54 ± 1.01%, 20.60 ± 2.14%, and 31.32 ± 1.27%, respectively, which is a 2-fold elevation compared to the control values of 10.98 ± 0.12%, 11.79 ± 1.5%, and 11.71 ± 1.62%, respectively (P < .05). Resveratrol promoted cell cycle accumulation at the S phase, especially at high concentrations and for long drug exposure times. However, such cell cycle alteration only partially explains the antiproliferative effect of resveratrol. Alteration of p21 and cyclin E levels after resveratrol treatment. To assess the role of cell cycle regulatory proteins in resveratrol-induced cell cycle accumulation at the S phase, we examined neuroblastoma cell expression of p21 and cyclin E after 25 lmol/L resveratrol treatment at various
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Fig 5. The expression of cyclin E and p21 of the neuro-2a cells after resveratrol treatment. After treatment with 25 lmol/L of resveratrol for 0, 15, and 30 minutes and 1, 3, 6, and 24 hours, the expression of cyclin E and p21 of the neuro-2a neuroblastoma cells was studied by Western blot analysis. The upper panel represents the expression of the cyclin E, the middle panel represents the p21, and the lower panel represents the a-tubulin (control).
Fig 4. Cell cycle alteration subsequent to resveratrol treatment. The neuro-2a cells were treated with various concentrations (0, 2.5, 10, and 25 lmol/L) of resveratrol (dissolved in dimethyl sulfoxide [DMSO]) for 24, 48, and 72 hours. The control well (0 lmol/L) was treated with DMSO. Then the cell cycle of the neuro-2a neuroblastoma cells was analyzed using flow-activated cell sorter (FACS) flow cytometry. The data are presented as mean ± standard deviation. Resveratrol promoted cell cycle accumulation at the S phase, especially at high concentration and for long drug exposure times. *P < .05 vs concentration 0 lmol/L (ANOVA).
time points (Fig 5). Western blot analysis showed that the p21 level increased 1.5-fold at 1 hour, reached 3.5-fold at 3 hours, and then decreased to 0.3-fold at 24 hours. Cyclin E protein level increased gradually after 3 hours and reached a 3-fold elevation after 24 hours of resveratrol treatment. The decrease in expression of p21 and increase in expression of cyclin E after resveratrol treatment may be related to the entry of the neuro-2a cells into the S phase. Antitumor effects of resveratrol on subcutaneous neuroblastomas in mice. Because resveratrol
had cytotoxic effects on the neuroblastoma cells, we studied its antitumor effects on subcutaneous neuroblastomas in mice. A/J mice were subcutaneously inoculated with 106 neuro-2a cells in the right flank followed by no treatment (control), intraperitoneal injection of propylene glycol (vehicle), or a resveratrol concentration of 40 mg/kg each day for 28 days. During the 4-week treatment period, the animals treated with resveratrol showed good activity with normal food and water intake and had no body-weight loss, which indicated the mice tolerated the treatment well. However, the animals had body-weight loss and decreased appetite when the tumor became large. Figure 6 shows tumor growth rate of the neuroblastomas under resveratrol treatment. Tumor growth rate in the resveratrol group was significantly slower than that in the vehicle and control groups (P < .05), indicating a resveratrol concentration of 40 mg/kg daily suppressed the growth of subcutaneous neuroblastomas; however, histopathologically, the tumors showed no significant difference among various groups. Figure 7 shows survival curves of the mice carrying neuroblastomas that were treated with various regimens. All mice in the control and vehicle groups died, with survival times of 31.8 ± 2.9 and 33.4 ± 2.6 days, respectively. In contrast, 70% of the mice in the resveratrol group achieved long-term survival. The survival rate of the mice in the resveratrol group was significantly higher than that in the vehicle and control groups (P = .0015, Fisher exact test). We further analyzed the animal survival time in various groups and found there was no difference in the animal survival time between the control group and the group treated with propylene glycol (P = .27). By contrast, the group treated with resveratrol showed significantly longer survival time than did the
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Fig 6. The tumor growth rate of subcutaneous neuroblastomas under resveratrol treatment. neuro-2a cells (106) were injected subcutaneously into the shaved right flanks of male A/J mice divided into 3 groups of 10 mice each. The control group received no treatment. The vehicle and experimental groups were given an intraperitoneal injection of propylene glycol (vehicle) or a resveratrol concentration of 40 mg/kg (in 0.2 ml propylene glycol), respectively, once daily for 28 days. The tumor size was measured twice a week. The volume of the tumor was calculated from the formula V = 1/2 (d1 d2 d3), where d1, d2, and d3 were mutually perpendicular diameters measured by calipers. Each point represents the average volumes (cm3) of the tumors in each of the 3 groups. Daily average tumor volumes from each group were compared throughout the course of the experiment using ANOVA. Group averages were not compared after 1 or more animals in the group died. *P < .05 vs both vehicle and control groups.
control group or the group treated with propylene glycol (P < .00001). The results indicated that treatment with a resveratrol concentration of 40 mg/kg daily exerted antitumor effects on neuroblastoma tumors in mice. DISCUSSION Resveratrol is a chemopreventive phytochemical that effectively inhibits 3 major stages of carcinogenesis: initiation, promotion, and progression.4 In the literature, resveratrol has been found to inhibit the proliferation of a variety of cancer cells.16,22-25 However, no article has reported the effect of resveratrol on neuroblastomas. Therefore, we investigated the cytotoxic effects of resveratrol on neuroblastoma cells and demonstrated the dosedependent and duration-dependent inhibitory effects of resveratrol on the proliferation of neuroblastoma cells. Resveratrol was cytotoxic at concentrations ranging from 10 to 100 lmol/L
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Fig 7. The survival curve of the mice treated by resveratrol. neuro-2a cells (106) were injected subcutaneously into the shaved right flanks of male A/J mice divided into 3 groups of 10 mice each. The control group received no treatment. The vehicle and experimental groups were given an intraperitoneal injection of propylene glycol (vehicle) or a resveratrol concentration of 40 mg/kg (in 0.2 ml propylene glycol), respectively, once daily for 28 days. The survival curves were analyzed by the Kaplan-Meier method, and the log-rank statistic was used to test differences between groups. *P < .05 for both vehicle and control groups.
with an LD50 at the micromolar level. The LD50 at the micromolar level did not differ markedly from that reported for other tumors, such as leukemia, prostate, breast, and colon cancers.11,22,23,26,27 The anti-initiation and antipromotion properties of resveratrol have been attributed to its antioxidant and anticyclooxygenase properties.4 However, the mechanism underlying prevention of tumor progression is not well understood. Therefore, we studied possible mechanisms controlling the antiproliferative effect of resveratrol on neuroblastoma cells. Defective control of apoptosis has been suggested to play a central role in the pathogenesis of neoplasm.28 We investigated the effect of resveratrol on the induction of apoptosis of neuroblastoma cells and noted that resveratrol did induce apoptosis, as seen for other types of cancer cells.16,22-25 This resveratrol-induced apoptosis of neuroblastoma cells was most dramatic after treatment with 25 lmol/L resveratrol. Prolonged exposure of neuroblastoma cells to 25 lmol/L resveratrol elicited significantly more cellular apoptosis than did shorter periods of treatment. For the induction of apoptosis, activation of caspase is a key event.29 There have been at least 13 caspases identified from mammalian cells, and they function in a variety of fashions.30 Of these caspases, caspase 3 is a downstream caspase and plays a determining role in the execution phase of apoptosis.31 After the
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activation of the apoptosis process by upstream caspases, caspase 3 will cleave a variety of substrates; thus caspase 3 is considered to constitute a general terminal mediator of apoptosis.31 We therefore studied the role of caspase 3 in the resveratrolinduced apoptosis of neuroblastoma cells and found caspase 3 inhibitor significantly alleviated the extent of resveratrol-induced apoptosis of the neuroblastoma cells. Thus, the resveratrol-induced apoptosis of neuroblastoma cells is considered to be mediated, at least partially, through the activation of caspase 3. Cell cycle regulation is another mechanism of anti-proliferation in neoplasms.32 We studied the alteration in cell cycle after treatment of resveratrol in neuroblastoma cells and found that resveratrol induced cell cycle accumulation at the S phase. We observed a 2-fold increase in the percentage of neuroblastoma cells accumulated at the S phase after treatment with 25 lmol/L resveratrol for 24 hours. Cell cycle accumulation at the S phase also had been observed in prostate cells and HL-60 cells after resveratrol treatment.14,16,27 We further explored changes in the cell cycle regulator proteins cyclin E and p21 in neuroblastoma cells treated with resveratrol. Cyclin E is a rate-limiting component of the machinery that controls entry into the S phase.33 The expression of cyclin E is periodic and reaches a maximum value at the G1-S transition.33 The 3-fold elevation of cyclin E level in neuroblastoma cells after 25 lmol/L resveratrol treatment for 24 hours was consistent with the phenomenon of neuroblastoma cell accumulation at the S phase. Cyclin E associates with cyclin-dependent kinase 2 (CDK2) to form a cyclin E-CDK2 complex, which phosphorylates the retinoblastoma (Rb) protein to maintain normal cell cycling.34 In addition to the cyclin E-CDK2 complex, the cell cycle progression through the G1 into S phase is also under multiple levels of control by p21.35 p21 plays a dual role in both positive and negative effects on the G1 progression because p21 is required for cyclin/CDK complexes to assemble and be active, and p21 exerts positive effect on the G1 progression; in contrast, p21 may also block CDK activity, especially the cyclin E/CDK2 complex.35 Therefore, we studied the change in p21 expression and found p21 expression increased initially but subsequently decreased to 0.3-fold after 25 lmol/L resveratrol treatment for 24 hours. The early increase of p21 expression may be due to some mitogenic signals activated by resveratrol during early G1 phase progression.36 The subsequent decrease in p21 level might relieve the inhibition on the cyclin E/CDK2 complex and thus
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facilitate the phosphorylation of Rb protein and release of the E2F, and finally promote the progression from the G1 to S phase.27,35 As a whole, the downregulation of p21, associated with the upregulation of cyclin E, might account for the increased entry of neuroblastoma cells into the S phase; however, the limitations that prevent cells from passing into the G2-M phase might be related to other mechanisms, such as the inhibitory effect of resveratrol on DNA synthesis, which occurs at concentrations above 15 lmol/L.12,27 The inhibition of the DNA synthesis is probably related to the effects of resveratrol on the enzymes of DNA synthesis, such as DNA polymerase a and d and ribonucleotide reductase.12,13,37 Thus resveratrol was considered to foster a collision course between 2 important processes in cell cycle progression by activating signaling pathways required for the entry of cells into the S phase and at the same time limiting their progression through the S phase by inhibiting DNA synthesis.27 The unique ability of resveratrol to recruit neuroblastoma cells into the S phase suggests resveratrol might exert a radiosensitization effect; however, further studies are necessary to clarify this point. Because resveratrol had significant cytotoxic effects on neuroblastoma cells, we investigated in vivo antitumor effects on neuroblastoma tumors in mice. We found intraperitoneal injections of resveratrol concentrations of 40 mg/kg daily had antitumor effects on subcutaneous neuroblastomas in mice, as demonstrated by reduced tumor growth rate, prolonged animal survival time, and increased animal survival rate (70% vs 0% long-term survival rate). The in vitro cytotoxicity and in vivo growth inhibition of neuroblastoma tumors suggest resveratrol may be effective for the treatment of neuroblastoma. The dosage of a resveratrol concentration of 40 mg/kg daily was effective against neuroblastoma tumors. However, we do not know the serum level of resveratrol achieved by this dosage in the mice. One report mentions that oral administration of 28 lg resveratrol to male rats achieved a peak plasma level of resveratrol greater than 20 mg/ml after 1 hour.38 Although the animals used for this study were different from ours, we estimate a treatment dose of 40 mg/kg may produce a level of 25 lmol/L of resveratrol in the serum.38 In summary, this study demonstrated that resveratrol caused concentration-dependent and time-dependent cytotoxicity and induction of apoptosis in neuroblastoma cells. The resveratrolinduced apoptosis of the neuroblastoma cells could be alleviated by the caspase 3 inhibitor. In addition,
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resveratrol induced accumulation of neuroblastoma cells at the S phase of the cell cycle. The cyclin E upregulation and p21 downregulation in neuroblastoma cells after resveratrol treatment were compatible with induction of cells entering the S phase. Finally, resveratrol exerted an antitumor effect on neuroblastoma tumors as shown by inhibition of tumor growth and increase of animal survival rate and survival time. To the best of our knowledge, this is the first report demonstrating the antitumor effects of resveratrol on neuroblastoma cells and subcutaneous neuroblastoma tumors in mice. Additional clinical studies are needed before the use of resveratrol in the treatment of neuroblastoma can be recommended.
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