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Introduction
Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of long-survival plasma cells in
the bone marrow. The median survival from diagnosis among patients treated with conventional chemotherapy is 3_4 years.
Introduction of high-dose chemotherapy with stem cell support has led to improved survival, but these patients eventually
relapse from their disease and it remains an incurable disease with the currently available therapeutic
modalities[1]. Clearly, more effective and less toxic treatment
options are needed in the battle against MM.
Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin found in the skin of red grapes, various other
fruits, and root extracts of the weed Polygonum
cuspidatum, has recently attracted considerable interest because of its
inhibitory activity on multiple cellular and molecular events associated with tumor development.
Resveratrol has been shown to have potent inhibitory effects on the growth of leukemic cells, breast and colon cancer cells, cervical tumor cells
and gastric adenocarcinoma cells[2_4].
In vivo, resveratrol inhibits three major steps of carcinogenesis: initiation, promotion,
and progression[5]. Resveratrol has been considered as one of the most promising cancer chemopreventive or
chemotherapeutic agents. However, the molecular mechanism by which resveratrol
exerts its anticancer effect is largely unknown.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases capable of degrading many
components of the extracellular matrix (ECM) and basement
membranes[6]. Recently, studies found that MMPs have a
multifunctional role in the pathogenesis of MM. MMPs can contribute to cancer growth, invasion, angiogenesis, bone
degradation and other processes important in the pathogenesis of MM. MMP inhibition not only resulted
in reduction of tumor growth, but also had a significant effect
on neovascularization and bone disease
development[7]. Thus, the purpose of the present study was to examine if resveratrol affects MMPs and to evaluate the antitumor activity of resveratrol against
MM cells.
Materials and methods
Materials Human MM cell lines RPMI 8226 and U266 were obtained from the American Type Culture Collection (ATCC;
Manassas, VA). The KM3 cell line was kindly provided by Prof Jian HOU (Senond Military Medical University, Shanghai,
China). All the tumor cell lines were maintained in culture in RPMI-1640 supplemented with 10% heat-inactivated fetal
bovine serum in an atmosphere of 5%
CO2 at
37 °C. Resveratrol,
3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and gelatin were purchased from
Sigma Chemical Co (St Louis, MO, USA). Rabbit anti-MMP-2, anti-MMP-9, anti-Bax, anti-Bcl-2, and
anti-Bcl-xL polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti- X-linked inhibitor of apoptosis
protein (XIAP) polyclonal antibodies were purchased from R&D systems (Minneapolis, MN, USA). Goat anti-rabbit
horseradish peroxidase (HRP) conjugate was purchased from Zhongshan Company (Beijing, China). Chemiluminescence (ECL)
reagent was obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). A stock solution of resveratrol was made in
dimethylsulfoxide (DMSO) at a concentration of 100 mmol/L and stored at -20
°C. RPMI-1640 and FBS were obtained from Gibco
Life Technologies (Burlington, Ontario, Canada). Annexin V-PI Staining Kit (BMC306FI) was obtained from Bender Medsystems
Inc (Burlingame, CA, USA). The transwell plate was obtained from Corning Costar (Cambridge, MA, USA). All other
chemicals were purchased from authentic sources and were of the highest grade and purity.
MTT assay The effect of resveratrol on cell proliferation was measured using an MTT based assay. Briefly, the cells
(5000/well) were incubated in triplicate in a 96-well plate in the presence of various concentrations of resveratrol (12.5, 25, 50,
100, 200 µmol/L) in a final volume of 0.2 mL for the
indicated times. Thereafter, 0.025 mL of MTT solution (5
g/L) was added to each well and then incubated for 4 h. After centrifugation, the supernatant was removed from each well. The colored
formazan crystal produced from MTT was dissolved in 0.15 mL of DMSO and then the optical density
(OD) value was measured at 490 nm by a multiscanner autoreader (Dynatech MR 5000, Chantilly, VA). The following formula was used:
percentage cell viability=(OD of the experimental
samples/OD of the control) ×100.
Flow cytometric analysis
To determine the apoptosis, resveratrol-treated cells were washed in PBS and resuspended in
binding buffer at a concentration of
1×109 cells/L. After incubation, 195 µL of the solution was transferred to a 5 mL culture
tube with 5 µL annexin V-FITC added. The tube was then incubated for 30 min at room temperature in the
dark. The cells were washed with binding buffer and resuspended in 190 µL binding buffer, with 10 µL PI added. Finally, the tube was gently
vortexed and incubated for another 30 min in the dark, and then the cells were analyzed immediately by flow cytometry.
Cell invasion Assay The inhibitory effect of resveratrol on VEGF-induced invasion was demonstrated in 24-well transwell
cell culture chambers with the upper chamber containing filters of 8.0-µm pore size. The upper surface of the filters was
coated with a mixture of basement membrane components (Matrigel matrix, 25 µg/filter) and dried overnight at 37 °C. RPMI
8226 cells were serum-starved in RPMI-1640 with 1% FBS for 12 h and then
collected. After diluting cells in RPMI-1640 containing 1% FBS, 100 000 cells were seeded on the upper chamber wells together with or without resveratrol (12.5, 25, 50,
100 µmol/L). RPMI-1640 medium containing 1% FBS plus 25 µg/L VEGF was placed in the lower chamber as a chemotactant.
After 24 h of incubation at 37 °C, cells that invaded into the lower compartment were counted by means of a Coulter counter
ZBII (Coulter Electronics, Bedfordshire, England).
Gelatin Zymography Gelatin zymography was performed in KM3 conditioned media to visualize the gelatinolytic activity
of secreted MMP-2 and -9. 1×106 cells/well were plated and treated with various concentrations of resveratrol (25, 50, 100,
200, and 400 µmol/L) in 6-well plates and incubated for 24 h at 37 °C. After incubation, the cells were washed with serum-free
medium, replated in 0.5 mL serum-free medium and incubated for 24 h at 37 °C. The conditioned media were collected and
centrifuged at 4000 rpm for 10 min to remove cell debris. A total of 10 µg of proteins from each conditioned medium were
applied in duplicate to 10% SDS-PAGE gels copolymerized with type A gelatin at a final concentration of 0.1% under
non-reducing conditions. After electrophoresis, gels were washed in 2.5% Triton X-100 for
1 h to remove SDS, incubated for 18 h
at 37 °C, and stained in 0.1% Coomassie brilliant blue. The gelatinolytic regions were observed as white bands against a blue
background. The levels of MMP activity were assessed by scoring the
OD of the bands by a computerized image analysis
Gel Pro
3.0 (Media Cybernetics, LP Silver Spring, MD).
Western blot analysis Conditioned medium (30 µL) was used for MMP-2 and -9 analysis. For the analysis of Bcl-2,
Bcl-xL, Bax and XIAP, whole cell extracts were prepared by lysing the resveratrol-treated cells in lysis buffer (50 mmol/L Tris-HCl,
pH 8.0; 150 mmol/L NaCl; 1 mmol/L EDTA, 1% Triton-X 100; 1 mg/mL aprotinin; 1mg/mL leupeptin and 100 µg /mL PMSF).
Lysates were then spun at 14 000 rpm for 10 min to remove insoluble material. 30 to 60 µg protein extracts were separated by
10% SDS-PAGE under reducing conditions and then transferred to nitrocellulose membranes.
After blocking with 5% nonfat milk, membranes were incubated overnight at 4 °C with respective primary antibody. Levels
of GAPDH or β-actin were confirmed to ensure equal loading of the samples. After washing, membranes were incubated with
HRP-conjugated secondary antibodies at room temperature for 1 h. Blots were then developed using ECL. Densitometric
analysis of protein bands was performed using Gel Pro 3.0.
Statistical analysis Data were expressed as mean±SD. Statistical significance of difference observed in
resveratrol-treated versus control cultures was determined by means of Student
t-test. The minimal level of significance was
P<0.05.
Results
Resveratrol suppresses the proliferation of MM cells
Resveratrol had significant growth inhibition effects on MM cells.
MM cell lines RPMI 8226, U266, and KM3 were cultured in complete medium in the absence (control) or presence of various
concentrations of resveratrol for indicated time periods. Untreated cells (control) were considered as the baseline (100%) for
the analysis. Resveratrol inhibited cell growth of all 3 MM cell lines in a dose- and time- dependent manner (Figure 1A, 1B).
The concentrations of resveratrol needed to inhibit cell growth of RPMI8226, U266,
and KM3 were quite similar. Their IC50 values at 48 h were
72.3±
9.6 µmol/L, 74.1±7.8 µmol/L, and 80.3±6.5 µmol/L, respec-tively.
Resveratrol induces apoptosis in MM cells
To study whether resveratrol induced apoptosis in MM cells, we examined
the expression of annexin V and exclusion of PI using two-color flow cytometry. Resveratrol induced apoptosis in RPMI 8226
cells in a dose-dependent fashion, the percentage of cells undergoing apoptotic cell death increased from 7.3% in the control
culture to 36.9% after exposure to 100 µmol/L resveratrol for 24 h (Table 1). In the case of KM3 and U266 cell lines, similar
results were obtained (data not shown).
Resveratrol down-regulates Bcl-2,
Bcl-xL, and XIAP and up-regulates Bax protein levels
Analysis of the expression of the proteins involved in the apoptotic pathway revealed that resveratrol reduced the expression of the antiapoptotic proteins
Bcl-2, Bcl-xL, and XIAP and induced the expression of the proapoptotic protein Bax (Figure
2). Resveratrol reduced the levels of Bcl-2,
Bcl-xL, and XIAP proteins in a dose-depedent manner. A complete decline can be seen at 100 µmol/L resveratrol
treatment. Likewise, resveratrol up-regulated Bax protein level in a dose-dependent manner. The expression of Bax was
up-regulated at 12.5 µmol/L treatment and the effect was even more evident at 50 µmol/L.
Resveratrol inhibits MMPs in MM cells
MM cell line KM3 was shown to constitutively release MMP-2 and
MMP-9 with gelatinolytic activities at 62 kDa and 88 kDa, respectively, indicating that both enzymes are present in their cleaved, activated
form. The levels of MMP-2 were much higher than those of MMP-9. 24 h treatment of KM3 cells with resveratrol reduced the
gelatinolytic activity of MMP-2 as well as of MMP-9 in a concentration-dependent manner. The effect of resveratrol on
MMP-9 appeared more dramatic than its effect on MMP-2 (Figure 3A).
To investigate whether the resveratrol-mediated inhibition of MMP-2 and MMP-9 gelatinolytic activities could be
attributable to the inhibition of the release of MMP-2 and MMP-9 protein, the amount of MMP-2 and MMP-9 proteins released by
KM3 cells was further assessed by Western blotting. As shown in Figure 3B, resveratrol treatment markedly reduced the
amount of MMP-2 and MMP-9 protein in the conditioned
medium.
Resveratrol inhibits invasion of MM
cells The effect of resveratrol on cell invasion was tested using Matrigel
matrix-coated Boyden chambers. The addition of VEGF (25
µg/L) to the lower chamber induced transmigration of RPMI 8226, U266,
and KM3 cells through the Matrigel (Figure 4A_4C).
The VEGF-induced response was inhibited by resveratrol in a
concentration-dependent manner with an averge
IC50 value of 64±8 µmol/L, 93±11 µmol/L, and 153±11 µmol/L,
respectively.
Discussion
Resveratrol is a naturally occurring phytoalexin and a polyphenolic compound. The molecule was found to be present in
various fruits and vegetables and is abundant in grapes and red wine.
Recently, resveratrol has been the fo
cus of numerous research investigations due to its anti-oxidative, anti-inflammatory, estrogenic effects as well as
chemopreventive and antitumor
activities[5,8,9]. Resveratrol acts on the process of carcinogenesis by affecting the three
phases: tumor initiation, promotion and progression phases and suppresses the final steps of carcinogenesis, ie
angiogenesis and metastasis. It is also able to activate apoptosis, to arrest the cell cycle or to inhibit kinase pathways. Intere-stingly,
resveratrol does not present any cytotoxicity in animal
models[4,10,11]. Recent studies in humans have revealed that resveratrol
is pharmacologically quite safe[4,12]. Thus, the molecule has been considered a promising drug for cancer
chemoprevention.
In the present study we showed that resveratrol inhibited the proliferation of all tested MM cells in a dose- and time-
dependent manner, in agreement with similar findings recently reported by Jazirehi and
Bonavida[13]. FACS analysis revealed that treatment of RPMI 8226 cells with resveratrol for 24 h increased annexin V-positive and PI-negative populations as well
as annexin V and PI-positive populations in a dose-dependent fashion, suggesting that at least part of the
resveratrol-induced suppression as observed in the present study is mediated through the induction of apoptosis. Resveratrol exerts its
apoptotic effects by down-regulation of Bcl-2,
Bcl-xL, and XIAP antiapoptotic proteins along with significant up-regulation
of Bax proapoptotic protein.
Degradation of ECM is crucial for malignant tumor growth, invasion, metastasis, and angiogenesis. Matrix degradation
is due to the secretion of a variety of enzymes, the most important belonging to the family of the MMPs. MMPs are a family
of zinc-dependent neutral endopeptidases with proteolytic antivity for a large range of components of the ECM. Elevated
levels of distinct MMPs can be detected in tumor tissue or serum of patients with advanced cancer, and their role as
prognostic indicators in cancer has been widely
examined[14]. In myeloma, it has been shown that tumor cells from patients
with MM and from 5T33MM mice, bearing murine myeloma cells, secretes MMP-9 and that this secretion is induced by the
interaction of MM cells with the BM microenvironment. BM stromal cells from patients with MM are also an important
source of MMPs, mainly MMP-2[7]. Morever, secretion of MMPs by plasma cells parallel the progression of human
MM[15]. In the present study, the KM3 expressed and secreted high levels of MMP-2, and sizable levels of MMP-9 were coexpressed
and cosecreted. Our results are consistent with the previous studies that U266 cells predominantly secreted MMP-2 and to
a less extent, MMP-9[16]. Resveratrol suppressed the gelatinolytic activity of MMP-2 as well as of MMP-9 in a
concentration-dependent manner. As reported, MMPs family is involved in tumor progression, and inhibition of MMPs may suppress the
invasion of tumor cells. We tested the effect of resveratrol on MM invasion. We found that treatment of MM cells with
resveratrol abolished the VEGF-induced invasion. Our results corroborate those of Krishna et al, who found that inhibition
of constitutive expression of MMP-9 protein by METVAN inhibited leukemic cell invasion through Matrigel
matrix [17].
However, the concentrations of resveratrol that have been shown to elicit bioactivities in vitro are difficult to achieve
in vivo in the intact mammalian target
organ[18,19]. Results from preclinical studies in rats suggested that peak plasma levels of
unmetabolized resveratrol are well below 10 µmol/L even after a high oral dose of 50 mg/kg, and its elimination is rather rapid.
In contrast, resveratrol conjugates seem to reach much higher plasma levels than the parent
agent[18]. Bioavailability studies in humans also demonstrated that the detected amounts of free resveratrol in plasma were very low (few µg/L or less) after
moderate consumption of red wine or large amount of pure resveratrol
consumption[19]. Therefore, the new routes of
administration of resveratrol leading to high tissue concentrations in target organ should be explored and the potential
biological activity of resveratrol metabolites should be considered for future investigations.
In conclusion, our results demonstrate that resveratrol is an effective inhibitor of MMPs in human MM cells. Resveratrol
plays a role in suppressing the proliferation and invasion of MM cells and in inducing apoptosis. Validation of our in vitro
findings with an in vivo model system is warranted for the potential clinical application in the management of patients with
MM.
Acknowledgments
We thank Prof Jian HOU (Senond Military Medical University, Shanghai) for providing the multiple myeloma cell line
KM3 and Prof Tang-chun WU (Institute of Occupational Medicine, School of Public Health, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan) for offering relevant experimental facilities and technical support.
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