Cannot find the PDF, published in Molecular Carcinogenesis
Growth-stimulatory effect of resveratrol in human cancer cells
Masayuki Fukui, Noriko Yamabe, Ki Sung Kang, Bao Ting Zhu *
Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, KS
*Correspondence to Bao Ting Zhu, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 2146 W. 39th Street, Kansas City, KS 66160.
Masayuki Fukui and Noriko Yamabe contributed equally to this work.
Keywords
resveratrol • mitogenic effect • growth hormone-like effect • NF-B • tumor promotion
Abstract
Earlier studies have shown that resveratrol could induce death in several human cancer cell lines in culture. Here we report our observation that resveratrol can also promote the growth of certain human cancer cells when they are grown either in culture or in athymic nude mice as xenografts. At relatively low concentrations (5 µM), resveratrol exerted a significant growth-stimulatory effect in the MDA-MB-435s human cancer cells, but this effect was not observed in several other human cell lines tested. Analysis of cell signaling molecules showed that resveratrol induced the activation of JNK, p38, Akt, and NF-B signaling pathways in these cells. Further analysis using pharmacological inhibitors showed that only the NF-B inhibitor (BAY11-7082) abrogated the growth-stimulatory effect of resveratrol in cultured cells. In athymic nude mice, resveratrol at 16.5 mg/kg body weight enhanced the growth of MDA-MB-435s xenografts compared to the control group, while resveratrol at the 33 mg/kg body weight dose did not have a similar effect. Additional analyses confirmed that resveratrol stimulated cancer cell growth in vivo through activation of the NF-B signaling pathway. Taken together, these observations suggest that resveratrol at low concentrations could stimulate the growth of certain types of human cancer cells in vivo. This cell type-specific mitogenic effect of resveratrol may also partly contribute to the procarcinogenic effect of alcohol consumption (rich in resveratrol) in the development of certain human cancers. © 2010 Wiley-Liss, Inc.
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Received: 5 January 2010; Revised: 16 April 2010; Accepted: 22 April 2010
Digital Object Identifier (DOI)
10.1002/mc.20650 About DOI
Article Text
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Abbreviations:
PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; NF-B, nuclear factor-kappaB; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun NH2-terminal kinase; H/E, hematoxylin and eosin; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling; ER, estrogen receptor ; SIRT1, silent mating type information regulation 2 homolog 1.
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INTRODUCTION
Resveratrol (trans-3,4,5-trihydroxystilbene), a naturally occurring polyphenolic compound, is highly enriched in a variety of food sources, such as grapes, peanuts, and red wine [1-3]. A number of previous studies have investigated many of its unique beneficial effects, such as lifespan prolongation, cancer prevention, and anti-inflammatory effect. It was reported that resveratrol could significantly extend the lifespan of the yeast (Saccharomyces cerevisiae), worm (Caenoehabditis elegans), and fruit fly (Dorosophila melanogaster) [4][5], and it could also improve the survival of mice on a high-energy diet [6]. In addition, studies have shown that resveratrol has a strong chemoprotective effect against the development of cancers of the skin, breast, prostate, and lung (reviewed in [7-9]). The evidence for the cancer chemoprotective effect of resveratrol was rather convincing, because it was shown to prevent tumorigenesis in a number of animal models (summarized in [9]). In addition to these studies, it was also reported that resveratrol can inhibit the growth of human cancer cells in vitro when it was present alone at rather high concentrations (usually >50 µM) or when it was used in combination with other anticancer drugs [10-19]. This anticancer effect of resveratrol was thought to be largely due to its ability to induce cell cycle arrest and/or apoptosis [10-19].
In the present study, we sought to further examine the modulating effect of resveratrol on the growth of human cancer cells. To our surprise, we found that resveratrol could significantly stimulate the growth of some (but not all) of the human cancer cell lines both in vitro and in vivo (as cancer xenografts). To gain insights into the molecular mechanism of resveratrol's mitogenic effect, we probed several major intracellular signaling pathways (phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinases (MAPKs), and nuclear factor-kappaB (NF-B)) for their potential involvement. These signaling pathways were selected for our initial investigation because they are among the best-known players involved in the regulation of cell growth and survival [20-24]. The MAPKs are a family of protein serine/threonine kinases, including ERK1/2, the c-Jun NH2-terminal kinase (JNK) and p38 MAPKs [20], and they play an important role in regulation of cell growth and death. Similarly, PI3K/Akt and NF-B are the other two vitally important signaling molecules that are involved in regulating cancer cell proliferation, survival, metastasis, invasion, and death (reviewed in [24]). Among these cellular signaling pathways, we found that activation of NF-B signaling pathway by resveratrol contributed importantly to the mitogenic effect of resveratrol seen in vitro and in vivo.
MATERIALS AND METHODS
Chemicals
Resveratrol was purchased from Sigma (St. Louis, MO). Iscove's modified minimum essential medium was obtained from Life Technology (Rockville, MD). The antibiotics solution (containing 10 000 U/mL penicillin and 10 mg/mL streptomycin) was obtained from Invitrogen (Carlsbad, CA), and trypsin-EDTA mixture (containing 0.25% trypsin and 0.02% EDTA) and fetal bovine serum (FBS) from Sigma. Pharmacological inhibitors of BAY11-7082, SP600125, PD98059, SB202190, LY294002, and Akt Inhibitor II were purchased from Calbiochem (La Jolla, CA).
Cell Lines and Cell Culture Conditions
All cancer cell lines used in this study were purchased from the American Type Culture Collection (Manassas, VA). MDA-MB-435s and MDA-MB-231 human breast cancer cells were maintained in Iscove's modified minimum essential medium, MCF-7 human breast cancer cells and DU145 human prostate cancer cells were maintained in Minimum Essential Medium Eagle, supplemented with 10% (v/v) FBS, 3.024 g/L NaHCO3 and incubated at 37°C under 5% CO2. Cells were subcultured every 3-4 d.
Here it is worth a brief note regarding the lineage controversy over MDA-MB-435s cells. This cell line was originally reported to be derived from the pleural effusion of a female patient with breast cancer [25], and has been widely used as an in vitro model in studying human breast cancer. However, analysis of gene expression patterns of this cell line has revealed its unique resemblance to melanoma cells [26-29]. These features are very distinct from other human breast cancer cell lines, including MCF-7 and MDA-MB-231 cells [27].
Analysis of Cell Viability and Cell Cycle
For determining cell viability and growth, the MTT assay and [3H]-thymidine incorporation assay were used as described previously [15]. Cells were seeded in 96-well plates at a density of 5000 cells per well. The stock solution of resveratrol (dissolved in pure ethanol) was diluted in the culture medium immediately before addition to each well at the desired final concentrations, and the treatment usually lasted for 2-3 d. For MTT assay, 10 µL of MTT (at 5 mg/mL) was added to each well at a final concentration of 500 µg/mL, and the mixture was further incubated for 1 h, and the liquid in the wells was removed thereafter. DMSO (100 µL) was then added to each well, and the absorbance was read with a UV max microplate reader (Molecular Device, Palo Alto, CA) at 560 nm. The relative cell growth was expressed as a percentage of the control that was treated with vehicle only. For thymidine incorporation assay, cells were pulsed with [methyl-3H]thymidine (0.1-1 µCi/well, PerkinElmer, Boston, MA) for the last 24 h. Cells were then harvested onto glass fiber filters using the FilterMate Harvester (PerkinElmer), while the unincorporated [methyl-3H]thymidine was removed with multiple washes according the procedures recommended by manufactures. The incorporated radioactivity was then counted using a -scintillation counter (Microbeta Trilux, PerkinElmer).
For assaying cell cycle change, cells were harvested by trypsinization and washed once with phosphate-buffered saline (PBS, pH 7.4). After centrifugation, the cells resuspended in 1 mL of 0.9% NaCl, followed by addition of 2.5 mL of ice-cold 90% ethanol. After incubation at room temperature for 30 min, cells were centrifuged and the supernatant was removed. The cells were resuspended in 1 mL of PBS containing 50 µg/mL propidium iodide (PI; Sigma) and 100 µg/mL ribonuclease A (Sigma) and incubated at 37°C for 30 min. After centrifugation, cells were resuspended in PBS. Flow cytometric analyses were performed on a flow cytometer (model BD LSR II, BD Bioscience, San Jose, CA).
Western Blotting
Western blotting analysis was performed as reported previously [30]. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). An equal amount of proteins was loaded in each lane. The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred to a polyvinylidene difluoride membrane (Bio-Rad). After blocking the membrane using 5% skim milk, target proteins were immunodetected using specific antibodies. All primary antibodies, phospho-I-B antibody (Cat. no.: #2859), I-B antibody (Cat. no.: 9242), JNK antibody (Cat. no.: 9252), phospho-extracellular signal-regulated protein kinase (ERK) antibody (Cat. no.: 9101), ERK antibody (Cat. no.: 9102), phospho-p38 antibody (Cat. no.: 9211), p38 antibody (Cat. no.: 9212), phospho-Akt antibody (Cat. no.: 9271), Akt antibody (Cat. no.: 9272), GAPDH antibody (Cat. no.: 2118), were obtained from Cell Signaling Technology (Beverly, MA) except the anti-JNK1/2 phospho-specific antibodies, which were obtained from Biosource (Cat. no.: 44-682G, Camarillo, CA). Thereafter, the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cat. no.: 81-612, Invitrogen) was applied as the secondary antibody, and the positive bands were detected using Amersham ECL Plus Western blotting detection reagents (GE Health Care, Piscataway, NJ). For determination of NF-B subcellular localization, the nuclear fractions were isolated using the Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA). The NF-B p65 antibody (Cat. no.: 4764) was obtained from the Santa Cruz Biotechnology (Santa Cruz, CA) and the TATA binding protein (TBP) antibody (Cat. no.: ab818) was from Abcam (Cambridge, MA).
Reporter Assay
The plasmid pNF-B-Luc carrying a firefly luciferase cDNA driven by 5 × NF-B-binding sites was purchased from Stratagene (La Jolla, CA), and pBIND carrying the Renilla luciferase cDNA driven by the SV40 promoter was purchased from Promega (Madison, WI). MDA-MB-435s cells were transfected with pNF-B-Luc and pBIND, 0.05 µg each in 24-well culture plate using Lipofectamine 2000 reagent (Invitrogen). Twenty-four hours after transfection, cells were treated with resveratrol with or without BAY11-7082 for 48 h. Then cells were harvested and the luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega) in TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). Firefly luciferase activity was normalized to the Renilla luciferase activity.
Growth of Human Cancer Xenografts in Athymic Mice
Female athymic nu/nu mice, 4-5 wk of age, were obtained from Harlan Laboratories (Indianapolis, IN). The animal diet (9604 Teklad Rodent Diet) was purchased from Harlan Laboratories. All procedures were approved by the Institutional Animal Care and Use Committee of our university and strictly followed the NIH guidelines for humane treatment of animals. MDA-MB-435s cells (5 × 106 cells/100 µL PBS) were injected s.c. into the right and left flank of the mice. Two weeks after inoculation, mice were randomly grouped according to body weight and tumor size. The treatment groups were as follows: control (2% ethanol in PBS, i.p., n = 10) and resveratrol (16.5 or 33 mg/kg BW, i.p., 3 times a week, n = 10). The main reason that we chose to use the route of i.p. administration of resveratrol in this initial study was to reduce experimental variations so that the number of animals needed for each group to achieve the same level of statistical significance would be reduced.
To determine the tumor size during the experiment, the maximum and minimum diameters of the tumor were measured twice a week using a slide caliper. Tumor volume was calculated using the formula [/6 × d3], where d is the mean diameter. At the end of the experiment, the mice were killed with CO2 overdose followed by cervical dislocation. Tumor tissues from each mouse were removed and trimmed of the surrounding connective tissue. The tumor tissue samples were used for various histological and histochemical analyses as described below.
Histopathological and Histochemical Analyses of Tumor Tissues
The collected tumor tissues were fixed in 10% buffered formalin phosphate, dehydrated, embedded in paraffin, sectioned in 5-µm thickness, and stained with hematoxylin and eosin (H/E) for histopathological analysis.
Cell proliferation in tumor tissues was assessed using the immunohistochemical staining of the proliferating cell nuclear antigen (PCNA)-positive cells [31]. Specific antibody against PCNA (Cat. no.: ab2426) was obtained from Abcam. For determining apoptotic cell death, terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) assay was used (In Situ Apoptosis Detection Kit, Chemicon International, Temecula, CA).
NF-B expression in tumor tissues was determined using the immunohistochemical staining. Specific antibody against the NF-B p65 (Cat. no.: 4764) was obtained from the Santa Cruz Biotechnology. All slides were counterstained with Mayer's hematoxylin.
HPLC Analysis of Resveratrol in Plasma
Following light anesthetization with isoflurane (IsoFlos, Abbot Laboratories, North Chicago, IL), blood samples (100 µL) were drawn from mice at 5, 30, 120, and 240 min after resveratrol administration. The plasma concentrations of resveratrol were measured using HPLC as reported previously. The relative standard deviation of the intra-day repeatability was <5%.
Reproducibility of Experiments and Statistical Analysis
All experiments described in the present study were repeated multiple times. The data are presented as mean ± SD. For the in vitro cell culture experiments, statistics were analyzed using two-way ANOVA. For the animal experiments, statistical significance was analyzed using two-way ANOVA and multiple comparisons with Dunnett's test (SPSS software). A P-value of less than 0.05 was considered statistically significant.
RESULTS
Growth-Stimulatory Effect of Resveratrol in MDA-MB-435s Cells in Culture
We first tested the proliferative effect of resveratrol in vitro on selected human cancer cell lines of the breast (MCF-7, MDA-MB-435s, and MDA-MB-231) and prostate (DU145). Resveratrol promoted the growth of MDA-MB-435s and DU145 cells, but this effect was not observed in MCF-7 and MDA-MB-231 cells (Figure 1A and B). The mitogenic effect of resveratrol in MDA-MB-435s and DU145 cells was observed at relatively low concentrations (<10 µM), but it suppressed their proliferation at higher concentrations (>40 µM). A similar mitogenic effect was observed in MDA-MB-435s cells using the 3H-thymidine incorporation assay (Figure 1C). Notably, the mitogenic effect described above for resveratrol was not observed with several commonly used anticancer agents, including paclitaxel (1-20 nM), doxorubicin (50-800 nM), etoposide (2-40 µM), and 5-fluorouracil (1-20 µM) (data not shown).
Figure 1. Effect of resveratrol on the growth of human cancer cell lines in vitro. (A) MDA-MB-435s cells were treated with resveratrol for 4 d using 10% regular FBS or 10% charcoal-stripped FBS (DCC FBS). Cell culture medium was changed at Day 2 once and cell growth was determined using the MTT assay. (B) DU145, MCF-7, and MDA-MB-231 cells were treated with resveratrol at indicated concentrations for 4 d using 10% regular FBS. Cell culture medium was changed at Day 2 once and cell growth was determined using the MTT assay. © MDA-MB-435s cells were treated with resveratrol for 4 d. Cell culture medium was changed at Day 2 once. DNA synthesis was determined using the 3H-thymidine incorporation assay. (D) MDA-MB-435s and MCF-7 cells were treated with resveratrol for 24 h, and then stained with PI for the analysis of cell cycles. Experiments were repeated three times. Each data point in panels A, B and C is the mean ± SD (n = 3). *P < 0.05; **P < 0.01 versus respective controls.
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Since resveratrol was reported to induce S-phase cell cycle arrest and cell death in several cancer cell lines [11][12], we also investigated these effects of resveratrol in MDA-MB-435s and MCF-7 cells. Resveratrol induced significant S-phase cell cycle arrest in both cancer cell lines when present at relatively high concentrations (Figure 1D). While resveratrol did not induce cell death (based on the presence of sub-G1 population that reflects cells with fragmented DNA) in MDA-MB-435s cells, it induced cell death in MCF-7 cells when present at 10 µM concentrations (Figure 1D).
Alterations of the Growth-Signaling Molecules by Resveratrol
To gain insights into the molecular mechanism(s) by which resveratrol exerted its mitogenic actions, we analyzed the effect of resveratrol on MAPK, PI3K/Akt, and NF-B signaling pathways. MAPKs (JNK and p38, but not ERK) and Akt were found to be phosphorylated after 24-h incubation with 5 µM resveratrol (Figure 2). I-B was also phosphorylated, suggesting that resveratrol may initiate NF-B activation. Next we investigated the effect of chemical inhibitors of these signaling molecules on resveratrol's mitogenic effect. Specifically, we used PD98059, U0125, SP600125, and SB202190 as inhibitors of the MAPK signaling pathways; Akt Inhibitor II and LY294002 as inhibitors of the PI3K/Akt signaling pathway; and BAY11-7082 as an inhibitor of the NF-B signaling pathway. Only BAY 11-7082 significantly and consistently diminished the mitogenic effect of resveratrol (Figure 3). Because other inhibitors did not show an appreciable effect, we then focused on studying the role of the NF-B signaling pathway. After treatment with 5 µM resveratrol, NF-B was detected in the nuclear fraction after 36-h treatment with resveratrol (Figure 4A). Next we conducted immunocytochemical analysis to confirm the translocation of NF-B into the nuclei. As shown in Figure 4B, an increase in NF-B translocation into the nuclei was seen following resveratrol treatment. Furthermore, we also examined the transcriptional activity of NF-B after resveratrol treatment by using a luciferase reporter assay. Forty-eight hours after the NF-B-Luc reporter gene-transfected cells were treated with resveratrol, the transcriptional activity of NF-B was increased in a dose-dependent manner, and the presence of BAY11-7082 attenuated this effect (Figure 4C).
Figure 2. Effect of resveratrol on intracellular signaling molecules in MDA-MB-435s cells. (A) Cells were treated with 5 µM resveratrol for indicated length of time and cell extracts were analyzed using Western blotting. (B) The relative mean densitometry value ± SD is calculated from three separate experiments, with the mean of the control group arbitrarily set at 1.0. *P < 0.05; **P < 0.01 versus respective controls.
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Figure 3. Effect of specific inhibitors for signaling molecules on resveratrol's mitogenic action in MDA-MB-435s cells. Cells were pre-treated with BAY11-7082 (BAY; 0.2 µM: NF-B inhibitor), SP600125 (5 µM: JNK inhibitor), PD98059 (5 µM: MEK1/2 inhibitor), SB202190 (5 µM: p38 inhibitor), LY294002 (0.5 µM; phosphatidylinositol 3-kinase inhibitor), or Akt Inhibitor II (Akt I; 5 µM) for 2 h and then incubated with resveratrol at indicated concentrations for 4 d. Cell culture medium was changed at Day 2 once. Cell growth was determined using the MTT assay. Experiments were repeated three times. Each data point is the mean ± SD (n = 3). **P < 0.01.
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Figure 4. Effect of resveratrol on NF-B subcellular localization and transcriptional activity in MDA-MB-435s cells. (A) Cells were treated with 5 µM resveratrol for indicated length of time, and then the nuclear fraction was isolated and subjected to Western blot analysis. TATA binding protein (TBP) was used as nuclear loading control. The relative mean densitometry value ± SD is shown in (A, lower part), with the mean of the control group arbitrarily set at 1.0. (B) Cells were incubated with 5 µM resveratrol for 4 d, and stained with anti-NF-B p65-specific antibody and PI. White arrowheads show the nuclear translocation of NF-B p65. © After transiently transfected with the NF-B-luciferase reporter gene and the pBIND-luciferase expression gene, cells were treated with resveratrol with or without BAY11-7082 for 48 h. Luciferase activity in these cells was then measured. Experiments were repeated three times. Each data point is the mean ± SD (n = 3). *P < 0.05; **P < 0.01. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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Growth-Stimulatory Effect of Resveratrol in MDA-MB-435s Cells in Athymic Nude Mice
In this study, MDA-MB-435s cells (at 5 × 106 cells in 100 µL PBS) were injected s.c. into the right and left flanks of the mice. Athymic nude mice received vehicle or i.p. injections of resveratrol (16.5 or 33 mg/kg BW). A significant increase in tumor volume was observed in mice treated with 16.5 mg/kg resveratrol, but this effect was not observed when the animals were treated with 33 mg/kg resveratrol (Figure 5A). At the higher dose, resveratrol slightly decreased the tumor volume (but not statistically significant). At the end of the experiment, each tumor was removed and weighed. The mean tumor weight in animals treated with the lower-dose resveratrol was higher than the tumor weight of the control group (P < 0.05; Figure 5A, inset). No significant changes in body weight were observed among different experimental groups (Figure 5B).
Figure 5. Effect of resveratrol on human breast cancer xenograft growth in female athymic nu/nu mice. MDA-MB-435s cells (at 5 × 106 cells in 100 µL PBS) were s.c. injected into the right and left flank of the animals. The animals received resveratrol (at 16.5 or 33 mg/kg, 3 times a week, i.p., n = 10) or vehicle alone (2% ethanol in PBS, i.p., n = 10). (A) Tumor size was measured twice a week. At the end of the experiment, each tumor was removed, trimmed, and the dehydrated tumor weight was determined (data shown in panel A inset). Note that the dehydrated tumor tissue weight was determined immediately after the tumor specimen had gone through the regular ethanol-xylene dehydration steps and then blotted with a paper towel to remove excess xylene. (B) The body weight was measured three times a week. Statistical analysis was performed using two-way ANOVA along with multiple comparisons with Dunnett's test (SPSS software). *P < 0.05 versus control vehicle group. © H/E, TUNEL, PCNA, and NF-B p65 stainings of the tumor tissue sections. Scale bar shows 20 µm. Quantitative data for the positive cells (TUNEL, PCNA and NF-B staining) are shown in the lower part. (D) Plasma concentrations (mean ± SD) of resveratrol as measured by HPLC. The animal experiments were repeated twice, and similar results were obtained (only one representative data set was shown). *P < 0.05; **P < 0.01. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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Histological examination of dissected tumor sections (H/E staining) revealed that the morphology and density of the tumor cells were not significantly different (Figure 5C, H/E staining). Histochemical staining of TUNEL-positive cells (i.e., apoptotic cells) and PCNA-positive cells (proliferating cells) in tumor sections are shown in Figure 5C (TUNEL and PCNA). The apoptotic indices in tumors from animals treated with a higher-dose resveratrol were higher than tumors from the lower-dose group (Figure 5C, TUNEL). Inversely, the PCNA-labeling indices of tumors from animals treated with the lower-dose resveratrol were higher than tumors from the vehicle or higher-dose group (Figure 5C, PCNA). These data confirmed that there was an increase in the rate of cancer cell proliferation in the lower-dose resveratrol group. Lastly, to confirm the relationship of NF-B activation with resveratrol's growth stimulatory effect in vivo, immunohistochemical analysis for NF-B p65 was performed in all tumor sections. NF-B p65 nuclear expression was readily detected in cancer cells of the lower-dose resveratrol group, but largely undetectable in cancer cells of the vehicle group or the higher-dose resveratrol group (Figure 4C, NF-B p65). Altogether, these data confirmed that resveratrol at a relatively lower dose could enhance cell growth in vivo via activation of the NF-B signaling pathway.
Resveratrol Concentrations
To probe what effective concentrations of resveratrol in vivo, we determined the plasma concentrations of resveratrol in control athymic mice. The plasma concentrations of resveratrol were 13.7 ± 0.4 and 20.6 ± 2.6 µM, respectively, at 5 min after administration of the low and high doses of resveratrol (Figure 5D). At 30 min after resveratrol administration, the plasma concentrations rapidly decreased to levels below 5 µM.
DISCUSSION
Many earlier studies have shown that resveratrol had a strong growth-inhibitory effect when present alone at relatively high concentrations [10-12]. Also, it was reported that resveratrol could sensitize tumor cells to anticancer drug-induced or death receptor-mediated apoptosis [12-14]. However, the results of our present study unequivocally showed that resveratrol had a concentration-dependent stimulatory and inhibitory dual effect on the growth of certain types of human cancer cells in vitro and also in vivo. The growth-stimulatory action is cell type-specific because only certain cell lines are sensitive to the growth-stimulatory actions of resveratrol.
A few earlier studies reported that resveratrol has weak estrogenic activity (as a phytoestrogen) and can stimulate the growth of the estrogen receptor (ER)-positive MCF-7 human breast cancer cells in vitro through an ER-mediated mechanism [10][32][33]. In these studies, the stimulatory effect of resveratrol was usually seen between 10 and 20 µM [10][32][33]. Since MDA-MB-435s cells are ER-negative, it is apparent that the growth-stimulatory effect of resveratrol observed in this cell line at relatively lower concentration (usually <5-10 µM) is not mediated by the ER system. Notably, we did not observe a growth-stimulatory effect of resveratrol in the ER-positive MCF-7 cells in the present study, likely because the weak estrogenic effect of resveratrol was masked by the endogenous estrogens that were contained in the fetal bovine serum used in this study.
Earlier studies have shown that resveratrol could modulate the activity of some of intracellular signaling pathways [11-19]. To gain insights into the molecular mechanism underlying resveratrol's mitogenic effect, we investigated several major intracellular signaling molecules involved in regulating cell growth and survival. We found that treatment of cells with 5 µM resveratrol resulted in the activation (increased phosphorylation) of JNK, p38, and Akt. To further probe their contribution to the growth-stimulatory effect of resveratrol in MDA-MB-435s cells, we tested the effect of selective pharmacological inhibitors of each signaling molecule. We found that the presence of the Akt inhibitor or the PI3K inhibitor (LY294002) did not affect the mitogenic effect of resveratrol in this cell line, and thus it appeared to us that the growth stimulatory effect observed in this study most likely was not mediated by the PI3K signaling pathway. Interestingly, it was reported earlier that resveratrol could activate the PI3K activity through activation of the ER [34][35]. Since, the cell line (MDA-MB-435s) used in our present study was estrogen receptor-negative, it was believed that the activation of the estrogen receptor-mediated signaling pathway by resveratrol did not contribute to the mitogenic effect observed in this study.
Similarly, when the cells were treated with the inhibitors of JNK, ERK, and p38-MAPK, the growth-stimulatory effect of resveratrol was not altered, likely suggesting that these signaling molecules were also not involved in mediating the mitogenic actions of resveratrol. However, when BAY11-7082, an inhibitor of the NF-B signaling pathway, was present, it attenuated the mitogenic effect of resveratrol seen at low concentrations (2.5-5 µM), but it did not affect resveratrol's growth-inhibitory effect seen at high concentrations (10 µM). To further confirm the role of NF-B activation in mediating resveratrol's mitogenic action, we investigated the nuclear translocation of NF-B p65 and also its transcriptional activity following resveratrol treatment. As expected, resveratrol induced the nuclear translocation of NF-B p65, and it also increased its transcriptional activity in a concentration-dependent manner. Moreover, the effects of resveratrol on NF-B p65 nuclear translocation and transcriptional activity were abrogated in the presence of the NF-B inhibitor. Activation of NF-B was also observed in MDA-MB-435s xenografts in athymic mice when they were treated with a growth-stimulatory low dose of resveratrol (Figure 5C).
Notably, resveratrol was recently reported to be able to activate SIRT1 (Sirtuin 1: silent mating type information regulation 2 homolog 1), and its activation has been suggested to mediate some of resveratrol's actions [4][5]. Since activation of SIRT1 by resveratrol would inhibit NF-B signaling by promoting deacetylation of Lys310 of the p65 protein [36-38], it appeared that SIRT1 was not a mediator of resveratrol's mitogenic effect seen in MAD-MB-435s cells.
It has been reported that resveratrol induces p53 activation and subsequently alters the ratio of anti- and pro-apoptotic bcl-2 family proteins [39][40]. In the human cancer cell lines tested in the present study, only MCF-7 cells express wild-type p53 protein, while other cell lines lack the functional p53 protein [41-43]. Since p53 is activated by treatment with resveratrol [39], it is possible that its activation may mask the growth-stimulatory effect in cells that express the wild-type p53 protein. However, this possibility does not appear to be supported by the observation that resveratrol does not have a mitogenic effect in MDA-MB-231 cells, which express a mutant, functionally inactive p53 protein [43]. More studies are needed to determine the relationship, if any, between the p53 functionality and resveratrol's mitogenic actions.
A large number of studies in recent years have collectively led to the suggestion that resveratrol may have a number of health-promoting beneficial effects, such as lifespan prolongation [4][5], improvement of general health and athletic performance [6][44], and perhaps even cancer prevention [7]. The results of our present study showed that resveratrol at pharmacologically achievable concentrations in vivo showed a significant growth-stimulatory effect in certain types of cancer cells. This mitogenic effect may partially contribute to the procarcinogenic effect of alcohol consumption (rich in resveratrol) in certain types of human cancers where resveratrol may exert a particularly strong, cell type-specific growth stimulation. Lastly, given the fact that many cancer patients nowadays are taking resveratrol as part of the dietary supplements in hopes of improving the treatment outcomes for their cancer, our results are very timely and call for cautions in its use for this purpose. More studies are needed to further assess the overall benefits of resveratrol in cancer patients.References
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Edited by drmz, 26 June 2010 - 07:01 PM.
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