Suppression of 7,12-Dimethylbenz(a)anthracene-induced ...cancerres.aacrjournals.org/content/canres/62/17/4945.full.pdf · Resveratrol (3,4-trihydroxystilbene), a natural phytoalexin

[CANCER RESEARCH 62, 4945–4954, September 1, 2002]

Suppression of 7,12-Dimethylbenz(a)anthracene-induced Mammary Carcinogenesisin Rats by Resveratrol: Role of Nuclear Factor-�B, Cyclooxygenase 2, andMatrix Metalloprotease 9

Sanjeev Banerjee, Carlos Bueso-Ramos, and Bharat B. Aggarwal1

Cytokine Research Laboratory, Departments of Bioimmunotherapy [S. B., B. B. A.] and Pathology [C. B-R.], The University of Texas M. D. Anderson Cancer Center, Houston,Texas 77030

ABSTRACT

We have reported recently that resveratrol (trans-3,4�,5-trihydroxystil-bene), a polyphenolic phytoalexin found in grapes, fruits, and root extractsof the weed Polygonum cuspidatum, is a potent inhibitor of nuclear factor(NF)-�B activation. Because NF-�B suppression has been linked withchemoprevention, this prompted us to investigate the chemopreventivepotential of resveratrol by testing it against mammary carcinogenesisinduced by 7,12-dimethylbenz(a)anthracene (DMBA) in female SpragueDawley rats. Dietary administration of resveratrol (10 ppm) had no effecton body weight gain and tumor volume but produced striking reductionsin the incidence (45%; P < 0.05), multiplicity (55%; P < 0.001), andextended latency period of tumor development relative to DMBA-treatedanimals. Histopathological analysis of the tumors revealed that DMBAinduced ductal carcinomas and focal microinvasion in situ (7 of 7),whereas treatment with resveratrol suppressed DMBA-induced ductalcarcinoma. Immunohistochemistry and Western blot analysis revealedthat resveratrol suppressed the DMBA-induced cyclooxygenase-2 andmatrix metalloprotease-9 expression in the breast tumor. Gel shift analysisshowed suppression of DMBA-induced NF-�B activation by resveratrol.Treatment of human breast cancer MCF-7 cells with resveratrol alsosuppressed the NF-�B activation and inhibited proliferation at S-G2-Mphase. Overall, our results suggest that resveratrol suppresses DMBA-induced mammary carcinogenesis, which correlates with down-regulationof NF-�B, cyclooxygenase-2, and matrix metalloprotease-9 expression.

INTRODUCTION

Almost 600,000 new cases of breast cancer are identified each yearworldwide. In North America, breast cancer accounts for over onequarter of all cancers in women and is second only to lung cancer asa cause of cancer-related deaths (1). Despite abundant informationabout its etiopathogenesis and early detection, effective therapeuticmodalities for patients with advanced stages of the disease are stillneeded. Adjuvant therapy after ablative surgery is effective only whenthe tumor is detected early. Accumulating evidence derived fromlaboratory studies and study cohorts drawn from the general popula-tion have led to the search for “chemoprotection” agents to attenuatethe risk of breast cancer based on observation that most humancancers are associated with a long period of latency (2, 3). Severalnonnutritive phytochemicals found in natural products and associatedwith pharmacological attributes reveal evidence that they inhibit,delay, and/or reverse cancer evoked by either environmental insultsand/or lifestyle (4, 5). Several of these chemopreventive agents act atthe initiation, promotion, and/or progression stages conceptually as-sociated with the ontogeny of multistage carcinogenesis.

Resveratrol (3,4�-trihydroxystilbene), a natural phytoalexin presentin grapes and many other natural sources, has been suggested to play

a role in reducing the risk of coronary heart disease and cancer (6–8).In addition, resveratrol intake has been reported to have anti-inflam-matory and anti-atherosclerosis functions and to modulate hepaticalipoprotein and lipid synthesis, platelet aggregation, and productionof antiatherogenic eicosanoids by human platelets and neutrophils (7,9–11). Resveratrol has also been reported to inhibit the developmentof preneoplastic lesions in carcinogen-treated mouse mammary organcultures and the promotional stage of mouse skin carcinogenesis (7).Additionally, it mediates reduced aberrant colonic crypt foci forma-tion and inhibits of the growth of a wide variety of human-derivedtumor cells, including leukemic, prostate, breast, and endothelial cells(12–19). Other targeted cellular effects belonging to resveratrol in-volve inhibition of the enzymes protein kinase C, ribonucleotidereductase, cyclooxygenase, and nitric oxide synthase and inhibition ofaryl hydrocarbon-induced cytochrome P-450 IAI (20–25).

A close structural similarity exists between synthetic estrogen (4,4�-dihydroxy-trans-�,�-diethylstilbene) and resveratrol. It is unclear,however, whether resveratrol is an estrogen receptor agonist or an-tagonist. Estrogen agonists have been reported to exert protectiveaction against estrogen-dependent cancers, such as cancer of thebreast and endometrium; presumably, resveratrol interacts with estro-gen receptor to inhibit its activation (26). Lu and Serrano (26) reportedthat resveratrol acts as an estrogen receptor antagonist in the presenceof estrogen, leading to inhibition of growth of human breast cancercells.

In recent years, the importance of the transcription factor NF-�B2

in promoting tumorigenesis has been well recognized. NF-�B binds toconsensus elements within the promoter regions of a variety of tar-geted genes (27). Further investigations have revealed that the expres-sion of a multitude of critical genes are regulated by NF-�B, includingimmunoreceptors, transcription factor-associated proteins (c-myc andp53), cell adhesion molecules (intracellular adhesion molecule, vas-cular cell adhesion molecule 1, and endothelial leukocyte adhesionmolecule 1), and enzymes involved in tumor metastasis (COX-2,inducible nitric oxide synthase, and MMP-9). Under normal condi-tions, NF-�B is retained in the cytoplasm of cells, where it is boundby inhibitory proteins known as I�Bs. It has also been documentedthat during carcinogenesis, NF-�B has the potentiality to mediateseveral of the events associated with multistep processes includingacquisition of features such as promotion of cell survival and dys-regulation of normal control of proliferation, metastasis, and angio-genesis (27–29). The constitutive activity of NF-�B has been shownto be essential for proliferation of several cell types, e.g., smoothmuscle cells and hepatocytes during liver regeneration after partialhepatectomy or toxic damage (30, 31).

Previous reports from our laboratory and another have demon-strated the ability of resveratrol to down-regulate NF-�B expression invitro (32, 33), leading to speculation that it would in turn inhibit

Received 3/26/02; accepted 7/1/02.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Bioimmuno-therapy, The University of Texas M. D. Anderson Cancer Center, Box 143, 1515 Hol-combe Boulevard, Houston, TX 77030. Phone: (713) 792-3503, 6459; Fax: (713) 794-1613; E-mail: [email protected].

2 The abbreviations used are: NF-�B, nuclear factor �B; TNF, tumor necrosis factor;I�B, inhibitory subunit of NF-�B; EMSA, electrophoretic mobility shift assay; MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; COX, cyclooxygenase;DMBA, 7,12-dimethylbenz(a)anthracene; MMP, matrix metalloprotease.

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cellular genes regulated by NF-�B and those involved in multistagetumorigenesis. In the present study, we evaluated the effect of res-veratrol in inhibiting chemically induced mammary carcinogenesis ina rat model. This is the first report to indicate that resveratrol has achemopreventive effect on breast tumorigenesis in vivo. Given theprogressive aberrant expression of constitutive NF-�B factor withprogression of the disease (34), we hypothesize that resveratrol inter-feres with cognate signaling by inhibiting of NF-�B activity duringthe mammary tumorigenesis cascade. We therefore used immunohis-tochemical and Western blot methods to examine the expression oftwo enzymes, COX-2 and MMP-9, both of whose promoter sequencescontain binding sites for NF-�B. Additionally, we investigated theeffect of resveratrol on NF-�B activation and cell growth in MCF-7breast adenocarcinoma cells.

MATERIALS AND METHODS

Chemicals

Penicillin, streptomycin, RPMI 1640, FCS, and trypsin were obtained fromLife Technologies, Inc. (Grand Island, NY). BSA, MTT, leupeptin, aprotinin,and DMBA of highest purity were purchased from Sigma Chemical Co. (St.Louis, MO). Bacteria-derived recombinant human TNF, purified to homoge-neity with a specific activity of 5 � 107 units/mg, was kindly provided byGenentech (South San Francisco, CA). The radioisotope [5-methyl-3H]thymi-dine was obtained from Amersham Pharmacia Biotech, and [�-32P]ATP (5mCi) was purchased from ICN Radiochemicals (Costa Mesa, CA). Antibodiesused were as follows: anti-p65, against the epitope corresponding to aminoacids mapping within the NH2-terminal domain of human NF-�B p65; anti-p50, against a peptide 15 amino acids long mapping at the NLS region ofNF-�B p50; and COX-2, against the epitope corresponding to amino acids50–111 mapping near the COOH terminus of COX-2 of human origin. Thesewere purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Forimmunohistochemistry, COX-2 antibody (murine polyclonal) was purchasedfrom Cayman Laboratories (Ann Arbor, MI), whereas rabbit antirat polyclonalantibody for MMP-9 was procured from Cell Sciences, Inc. (Norwood, MA).

Resveratrol was obtained from two sources; for animal experimentation,resveratrol (�98% pure) was purchased from Alexis Cooperation, San Diego,CA). For in vitro studies, resveratrol was procured from Sigma Chemical Co.A stock solution of resveratrol was made in DMSO at a concentration of10 mM. The NF-�B oligonucleotide from the HIV long terminal repeat,5�-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCG-TGG-3� and a mutated double-stranded oligonucleotide, 5�-TTGTTACAA-CTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3� were fromLife Technologies, Inc. (Grand Island, NY; underlined regions represent aconsensus NF-�B binding sequence). All other chemicals were purchased fromauthentic sources and were of highest grade and purity.

Chemoprevention Studies

Animals. Female Sprague Dawley rats were purchased from HarlanSprague Dawley (Indianapolis, IN). The rats arrived at 40 days of age and wereplaced on a common pellet diet and quarantined for 2 days. The animals werehoused three/cage in standard rat Plexiglass cages in a room maintained atconstant temperature and humidity under 12-h light and darkness. A completehealth status was determined. None of the rats exhibited major lesions, and allwere pathogen free. Before initiating the experiment, we acclimatized all ratson pulverized diet for 3 days and then randomly assigned them by body weightto one of the three groups: group I (n � 7) received pulverized rodent diet andserved as negative control; group II (n � 12), designated as a positive control,received DMBA and pulverized diet; and group III (n � 12) received DMBAand pulverized experimental diet containing resveratrol.

Animals were allowed free access to the basal diet or diet containing thechemopreventive agent and drinking water throughout the experiment. Ourexperimental protocol was reviewed and approved by M. D. Anderson CancerCenter Animal Care and Use Committee.

Treatment with Chemopreventive Agent. Animals (groups I and II) weregiven a normal diet containing vehicle control. Starting at 45 days of age, rats

belonging to group III were treated with resveratrol (100 �g/rat) in the diet.The dose of resveratrol was computed based on average food intake, whichapproximated 12–15 g/rat/day. Resveratrol was dissolved in 70% ethanol andthen added into the diet 1 day in advance and left at room temperature for 1day. After ethanol evaporation, food was then given to the rats. Food cups werechanged two times a week.

Chemoprevention Study Design. Rats belonging to the appropriate ex-perimental group were given the chemopreventive agent beginning at day 45of age. One week later (day 0), rats belonging to groups II and III were given10 mg of DMBA by gavage in sesame oil. This dose of DMBA is suboptimalto produce sufficient tumors to allow evaluation of both reduction and increasein the end point of carcinogenicity. Rats were weighed weekly, palpated formammary tumors once/week (starting 4 weeks after DMBA treatment), andmonitored daily for signs of toxicity. The study was terminated at 120 daysafter DMBA administration. All surviving animals, including those that did notseem to develop mammary tumors as well as the control group (group I), werekilled by CO2 asphyxiation and completely necropsied to evaluate possiblesigns of toxicity. Tumors were removed and fixed in 10% buffered formalin.Thirteen DMBA-induced mammary tumors from resveratrol (group III) orcontrol group (group II) were evaluated with a blind method for histopathol-ogy. The tumors were subjectively graded either as carcinomas or fibroade-nomas. The end point for data analysis included: (a) the number of animalswith tumors (tumor incidence); (b) the number of tumors/animal (tumormultiplicity); (c) latency to tumor appearance; and (d) tumor volume. Esti-mates of tumor volume were determined using the formula V � 4/3� r3, wherer is half of the average diameter (in millimeters) measured with a verniercaliper at two different planes.

Histological Sections

Formalin-fixed tissue was paraffin-embedded, sectioned at 3–5 �m, andstained with H&E. Sections were evaluated for tumor cell cytology, mitoticrate, growth pattern, necrosis, cystic change, and associated inflammatorycellular response.

Immunohistochemistry

Immunohistochemical studies were performed using paraffin-embeddedmaterial, heat-induced antigen retrieval (citrate buffer, pH 6.0), and polyclonalantirat antibodies specific for MMP-9 (CPM601; 1:750; Cell Sciences, Nor-wood, MA) and COX-2 (1:500; Cayman Chemical Co., Ann Arbor, MI). Thedetection system used was the LSAB2 detection kit (DAKO). Negative con-trols also were run.

Preparation of Nuclear Extract from Tissue Samples

Normal mammary epithelium and tumor samples (200–250 mg) randomlyselected from untreated control and experimental groups were minced andincubated on ice for 30 min in 0.5 ml of ice-cold buffer A, composed of 10 mM

HEPES (pH 7.9), 1.5 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1% IGEPALCA-630, and 0.5 mM phenylmethylsulfonyl fluoride. The minced tissue washomogenized using a Dounce homogenizer and centrifuged at 14,000 rpm at4°C for 10 min. The nuclear pellet obtained was suspended in 0.2 ml of bufferB [20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.5mM DTT, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 4 �M

leupeptin] and incubated on ice for 2 h with intermittent mixing. The suspen-sion was then centrifuged at 14,000 rpm at 4°C for 30 min. The supernatant(nuclear extract) was collected and stored at �70°C until use. The proteinconcentration was measured by the method of Bradford (35) with BSA as thestandard. EMSA was performed by incubating 12 �g of nuclear protein extractby procedure as described (36).

Western Blot Analysis

Mammary epithelium and/or tumor specimens from each of the above-mentioned experimental groups were thawed on ice and homogenized at 4°Cusing a Dounce homogenizer in RIPA lysis buffer for extracting total cellprotein. Sixty �g of whole-cell protein was resolved on 10% SDS-PAGE gel.The protein was transferred to a nitrocellulose membrane, blocked with 5%nonfat milk, and probed with specific antibodies against MMP-9 (1:3000) orCOX-2 (1:1000), separately. The blots were washed, exposed to horseradish

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RESVERATROL BLOCKS MAMMARY CARCINOGENESIS



peroxidase-conjugated secondary antibodies for 1 h, and finally detected byECL reagent (Amersham Pharmacia Biotech, Arlington Heights, IL).

Cell Line

MCF-7 cells were a kind gift of Dr. Kapil Mehta (M. D. Anderson CancerCenter, Houston, TX). Cells were maintained in culture in RPMI 1640 sup-plemented with 10% heat-inactivated fetal bovine serum and antibiotics (100units/ml penicillin and 100 �g/ml streptomycin) in an atmosphere of 5% CO2

at 37°C.

EMSA

MCF-7 cells (1 � 106/ml) were preincubated with different concentrationsof resveratrol (0, 10, 25, and 50 �M) for 4 h and then treated with TNF (0.1 nM)for 30 min at 37°C. Nuclear extracts were then prepared according to themethod described by Chaturvedi et al. (35). The protein content was measuredby the method of Bradford (36). EMSA was performed by incubating 8 �g ofnuclear extract with 16 fmol of 32P-end-labeled, double-stranded 45-merNF-�B oligonucleotide. The incubation mixture included 2 �g of poly(deoxyi-nosinic-deoxycytidylic acid) in a binding buffer. The DNA-protein complexformed was separated from free oligonucleotide on 6.6% native polyacryl-amide gel, and then the gel was dried. The radioactive bands from dried gelswere visualized by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA)using Image Quant software.

The composition and specificity of binding was examined by competitionwith 100-fold excess of unlabeled oligonucleotide and with a mutated oligo-nucleotide. For the supershift assays, nuclear extract were incubated with theantibodies against either p50 or p65 subunits of NF-�B for 30 min at 37°Cbefore the complex was analyzed by EMSA (36).

Evaluation of Cell Viability by MTT

MCF-7 cells were plated (in triplicate) in 96-well plates (5000 cells/well),24 h before addition of resveratrol. Medium was then aspirated, and cells wereexposed to 2-fold serial dilutions of resveratrol in 0.2 ml of fresh medium andincubated at 37°C in an atmosphere of 5% CO2 for 72 h. Thereafter, cellviability was measured by the MTT method using a multiscanner autoreader(Dynatech MR 5000, Chantilly, VA; Ref. 32). To examine the antiproliferativeeffects of resveratrol, 2000 cells in 0.1 ml of medium were plated overnightand then treated with 10 or 50 �M resveratrol in 0.2 ml for 2, 4, and 6 days.Thereafter, the cell viability was determined by the MTT method.

Evaluation of Cell Viability by Thymidine Incorporation

The sensitivity of MCF-7 cells to resveratrol was also determined by[3H]thymidine incorporation. Briefly, MCF-7 cells (5000 cells/well) wereplated in 0.1 ml of medium (RPMI 1640 plus 10% fetal bovine serum) in a96-well plate. After overnight incubation in a CO2 incubator at 37°C, themedium was removed, and different concentrations of resveratrol (twofoldserial dilutions starting from 100 �M concentration) were added in 0.2 ml offresh medium for 72 h. During the last 6 h of incubation before harvesting, 0.1�Ci [3H]thymidine was added to each well. Thereafter, the medium wasaspirated, wells were washed with PBS, and cells were detached by theaddition of a solution containing trypsin (0.5%) plus EDTA (5.3 mM). The cellsuspension was harvested on a Filter Mate 196 cell harvester (Packard Instru-ments, Meriden, CT) and lysed by washing with distilled water. Radioactivitybound to the filter was measured by Direct Beta Counter (Matrix 9600;Packard Instruments, Meriden, CT).

FACS Analysis

Cells (2 � 106) in 1 ml of culture medium were plated in 6-well platesovernight and then treated with 50 �M resveratrol or medium for 6, 12, 24, or48 h. Thereafter, the cells were harvested by trypsinization, washed twice withPBS, and fixed in 5 ml of 95% ethanol. After 12 h incubation at �20°C, cellswere washed and resuspended in PBS. The cellular DNA was then stained with1 ml of propidium iodide (50 �g/ml) containing 0.1 ml of RNase (1 mg/ml).After incubation at 37°C for 30 min, cells were analyzed by FACS analysis.

Statistical Analysis

Body weight, tumor incidence, tumor multiplicity, and tumor volume weredetermined for the animals fed control diet and for those fed on the modifieddiet containing resveratrol. The data were analyzed and compared by �2 testusing Sigma-Stat software (Jandel Scientific, San Rafael, CA).

RESULTS

Resveratrol Suppresses DMBA-induced Rat Mammary Tu-morigenesis. NF-�B inhibitors have been shown to exhibit chemo-preventive properties (37–45). To determine whether resveratrol af-fects DMBA-induced rat mammary carcinogenesis, experiments weredesigned to examine chemoprevention after continuous dietary ad-ministration of resveratrol beginning 1 week before carcinogen ad-ministration and lasting until termination of the experiment (Fig. 1).The rationale for using this specific protocol relates to identifyingagents that intervene in initiation and progression of cancer. Noadverse effect on body weight gain during the observation period wasdetectable, and the average body weight gain was similar in all of thegroups throughout the experiment (267 � 2.8, 267 � 4.5, and268 � 2.7 for groups I, II, and III, respectively; P � not significant;Fig. 2). Moreover, no evidence of development of any spontaneoustumor, including tumors of the breast, occurred in animals randomizedto the negative control group (group I) during the span of the study.Administration of resveratrol and/or DMBA did not reveal any grosschanges in the liver, lung, kidneys, stomach, or intestinal tract ofanimals. In group II, 75% of the animals treated with DMBA (positivecontrol) developed palpable breast tumors at 11 weeks after DMBAtreatment (Fig. 3), and the average number of tumors/tumor-bearinganimal was 2.41 � 0.5 (mean � SE; Fig. 4). Rats of group III, whichreceived resveratrol-supplemented diet in addition to DMBA, had atumor incidence of only 41% (P � 0.05), and the average number oftumors/tumor-bearing animal was reduced to 1.08 � 0.5 (P � 0.001).Furthermore, differences between the groups (group II versus groupIII) in cancer latency was observed; in the resveratrol-treated animals,the latency was 98 days after DMBA treatment compared with 77days for DMBA-only treatment. No significant difference in the tumorvolume was noted between groups II and III (1.58 � 0.36 versus

Fig. 1. Experimental design for the evaluation of chemopreventive effect of resveratrolagainst mammary carcinogenesis. Groups of female Sprague Dawley rats were fed either0 or 10 ppm resveratrol mixed in the diet 7 days before exposure to DMBA, duringtreatment, and until termination of the study.

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1.26 � 0.70, respectively). In our study, two animals were removed,one each from group II and group III before termination schedulebecause of the presence of necrotizing tumor.

Resveratrol Suppresses DMBA-induced Ductal Carcinoma.Histopathological analysis was performed on tumor samples ran-domly selected from untreated (group I), DMBA-treated (group II),and DMBA � resveratrol-treated (group III) animals (Fig. 5). Normalmammary epithelium from control animals showed no pathologicalabnormality (Fig. 5A). Tumors obtained from DMBA-treated animalshad ductal carcinomas and focal microinvasion in situ (7 of 7; Fig. 5,

B�D). The surrounding breast tissue showed papillomatosis, atypicalductal epithelial hyperplasia, and no fibroadenomas or infarcts. Incontrast, tumors harvested from DMBA- and resveratrol-treated ani-mals exhibited evidence of fibroadenoma (2 of 4) and partially in-farcted ductal carcinoma in situ with microinvasion (4 of 4; Fig. 5, Eand F).

Resveratrol Suppresses DMBA-induced Intracellular Expres-sion of COX-2 and MMP-9 Proteins. Immunohistochemistry re-vealed the absence of COX-2 activity in normal mammary epithelium(Fig. 5G), high expression in tissue from DMBA-treated rats (7 of 7;Fig. 5H), and low expression (3 of 4; Fig. 5I) or no expression (1 of4) in DMBA � resveratrol-treated rats. When examined for MMP-9expression by immunohistochemistry, staining in the normal mam-mary epithelium (Fig. 5J) was consistently absent, the breast tissuefrom the DMBA-treated group showed uniformally positive staining(7 of 7; Fig. 5K), and the tissue from the DMBA � resveratrol-treatedgroup showed low expression (4 of 4; Fig. 5L).

To further confirm the effect of resveratrol on DMBA-inducedCOX-2 and MMP-9 expression, we examined protein expression byWestern blot analysis. Because both COX2 and MMP-9 are regulatedby NF-�B activation, we also measured the nuclear levels of NF-�Bby EMSA in the mammary tissue. As shown in Fig. 6, none of theuntreated tissues samples expressed NF-�B DNA-binding activity;tissues derived from 8 of 9 DMBA-treated animals expressed NF-�BDNA-binding activity, whereas only 3 animals of 9 from theDMBA � resveratrol-treated group expressed NF-�B. These resultssuggest that DMBA induces NF-�B DNA-binding activity in mostcases, and resveratrol suppresses it. When examined for COX-2 andMMP-9 expression, tissues from all of the DMBA-treated groupexpressed these proteins (none in the untreated group), but only lowlevels were detected in tissues from animals treated with DMBAtogether with resveratrol. These results are consistent with that ob-tained from immunohistochemistry.

Resveratrol Inhibits TNF-induced NF-�B Activation in HumanBreast Cancer Cells. Our results show that resveratrol suppressedDMBA-induced mammary carcinogenesis. NF-�B has been impli-cated in carcinogenesis. Previously, we and others have shown that

Fig. 2. Resveratrol does not affect the body weight of animals. The figure shows theaverage weekly body weights in different groups of female Sprague Dawley rats begin-ning at 45 days of age.

Fig. 3. Treatment with resveratrol suppresses the DMBA-induced incidence of mam-mary tumors in rats. The induction of mammary tumors induced by single pulse exposureto 10 mg of DMBA/rat is shown. The progressive percentage incidence of cumulativepalpable mammary tumors, as a function of time after carcinogen treatment, is shown.Values represent the mean of 8–12 rats/group.

Fig. 4. Treatment with resveratrol suppresses tumor multiplicity of DMBA-inducedmammary tumors. Rats were given resveratrol admixed in pulverized diet beginning 7days before DMBA administration and were given modified diet continuously thereafteruntil the end of the experiment. Each group was composed of 8–12 rats.

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resveratrol does suppress NF-�B activation in various cell types (32,33). Whether resveratrol inhibits NF-�B activation in breast cancercells was investigated. MCF-7 cells were pretreated for 4 h withdifferent concentrations of resveratrol and then stimulated with 0.1 nM

TNF for 30 min. As assessed by trypan blue and MTT assay, resvera-trol did not affect cell viability at this concentration and time point.Nuclear extracts were made and assayed for NF-�B by EMSA. Asshown in Fig. 7A, TNF induced DNA-binding activity of NF-�B, andresveratrol inhibited this activation in a dose-dependent manner; fullinhibition occurred at 50 �M resveratrol. Resveratrol alone at thisconcentration did not activate NF-�B.

Various combinations of Rel/NF-�B proteins can constitute an

active NF-�B heterodimer that binds to specific sequences in DNA.To show that the retarded band visualized by EMSA was indeedNF-�B, we incubated the nuclear extract from TNF-activated cellswith antibodies to either p50 (NF-�B) or the p65 (relA) subunit andthen conducted EMSA. Antibodies to either subunit of NF-�B shiftedthe bands to a higher molecular weight (Fig. 7B), thus suggesting thatthe TNF-activated complex consisted of both the p50 and p65 units.Preimmune serum had no effect on the mobility of NF-�B. Excessunlabelled NF-�B almost completely eradicated the band, indicatingthe specificity of NF-�B. Further specificity is indicated by theobservation that the oligonucleotide probe with labeled mutatedNF-�B binding site failed to bind the NF-�B protein.

Fig. 5. Effect of resveratrol on the DMBA-induced pathology and on COX-2 and MMP-9 expression. Representative H&E-stained sections (A�F) and immunohistochemical staining(G�L) for COX-2 and MMP-9 of breast lesions are shown. Breast tissue from untreated control (A, normal), from DMBA-treated animals (B, papillomatosis; C, ductal carcinoma insitu/atypical ductal hyperplasia; and D, invasive ductal carcinoma), and from DMBA- and resveratrol-treated animals (E, fibroadenoma; and F, basic carcinoma with extensive necrosis)are shown. G�I, immunohistochemical staining for COX2 protein demonstrates COX-2 negativity in breast tissue from untreated rat (G), strong positivity in ductal carcinoma fromDMBA-treated rat (H), and faint positivity in the resveratrol plus DMBA-treated tumor with necrosis (I). Scattered immunoreactivities are localized to the inflammatory cell (I). J�L,immunohistochemical staining for MMP-9. Lack of immunoreactivity in the untreated breast tissue (J), strong cytoplasmic staining in the tumor from DMBA-treated rat (K), and weakimmunopositivity in DMBA plus resveratrol-treated tumor (L) are shown.

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Resveratrol Inhibits the Growth of Human Breast CancerCells. Whether resveratrol suppresses the proliferation of humanbreast cancer cells was also investigated. MCF-7 cells were treatedwith 2-fold serial dilution of resveratrol for 72 h, either in the presenceor absence of resveratrol, and then examined for growth by MTT andcell proliferation by thymidine incorporation. Results in Fig. 8Aindicate that resveratrol inhibited the growth of human breast cancercells in a dose-dependent manner, with almost 60% suppression ofcell viability at 100 �M concentration. When examined for cell pro-liferation by DNA synthesis, 90% inhibition of thymidine incorpora-tion occurred with 100 �M resveratrol (Fig. 8B). Thus, breast cancercells were more sensitive to resveratrol in the DNA synthesis assaythan that in the assay of mitochondrial activity. We also examined theeffect of resveratrol on the proliferation of MCF-7 cells. Fifty �M

resveratrol was sufficient to completely suppress the proliferation ofbreast cancer cells (Fig. 8C).

We also examined the effect of resveratrol on the cell cycle analysisof MCF-7 cells. Results shown in Fig. 8D clearly show resveratrolinduced the accumulation of cells in S-phase of the cell cycle. In theresveratrol-treated samples at 24 h, 48% of the cells were in S-phaseas compared with 11% in the control. A complete suppression of thecells in the G2-M phase of the cycle could be noted as early as 12 hafter resveratrol treatment.

DISCUSSION

The present study demonstrates the chemopreventive action ofresveratrol in a well-established, chemically induced animal protocolof breast cancer and its relation to NF-�B expression. In recent years,“cancer chemoprevention” by biologically active dietary or nondi-etary supplements has generated immense interest in view of theirputative role in attenuating the risk of developing cancer. Against thisbackground, resveratrol is a promising agent in affording chemopro-tection against several major human epithelial and nonepithelial can-cers. Resveratrol was first evaluated in vitro and reported to inhibitmammary lesions induced by DMBA, leading to speculation of aspecific effect on the mammary gland independent of systemic drug

metabolism. Additionally, divergent beneficial modulatory effects ofresveratrol has been reported in the literature (46). We report in thisstudy that resveratrol fed to Sprague Dawley female rats inhibitedtumor formation in comparison to vehicle treatment in DMBA-initi-ated mammary carcinogenesis. Tumor incidence calculated as thepercentage of animals with one or more palpable tumors/treatmentgroup was reduced by 45% after resveratrol supplementation. Otherindices of chemopreventive response-tumor multiplicity, expressed asaverage number of tumors that developed per animal/week in eachtreatment group was reduced from 2.41 to 1.04 tumors/animal byresveratrol feeding at the termination of the experiment. Furthermore,relative to control animals, the latency to onset of tumor developmentwas prolonged by 3 weeks by resveratrol.

Precisely how resveratrol inhibits breast cancer is not certain,although several possible modes of action have been proposed andstudied at the cellular and molecular level. Resveratrol is able tomimic the activity and effects of endogenous 17�-estradiol. Accord-ing to a proposed hypothesis, estrogen has a dual affect on breastcancer risk (47). Evidence indicates that estrogens promote the growthof existing malignancies in the breast. In contrast under certain cir-cumstances such as pregnancy, during the prepubertal period andchildhood, estrogen actually reduces breast cancer risk through estro-gen-induced activation of certain tumor suppressor genes includingBRCA1 and p53 (47). It may thus be speculated that resveratrol, as aputative estrogen agonist, augments mammary tissue differentiationand maturation beyond the optimum period of carcinogen sensitivity,thus conferring a protective effect. It has been reported that completemorphological differentiation of the mammary gland protects againstmammary carcinogenesis in Wistar Furth rats. Pretreatment of ratswith estradiol and progesterone or completion of pregnancy andlactation before carcinogen exposure markedly reduces the suscepti-bility of the gland to chemical carcinogenesis (48).

In our study design, resveratrol treatment was initiated before onsetof puberty. Because resveratrol is known to induce differentiation(49), it is likely that resveratrol treatment accelerated mammary tissuedifferentiation, leading to refractory cell phenotypes during the period

Fig. 6. Effect of resveratrol on the DMBA-induced NF-�B, COX-2, and MMP-9 expression. Nuclear and whole cell extracts were prepared from mammary tissue derived fromanimals of different groups. These extracts were then analyzed for NF-�B by gel shift assay (top panel) and for MMP-9 (middle panel) and for COX-2 (bottom panel) by Western blotanalysis as described in “Materials and Methods.” �-Actin as a loading control is also depicted.

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of carcinogen sensitivity. The mammary gland of the rat undergoesextensive development after 32–35 days of age, first appearing asterminal end buds that subsequently evolve into alveolar buds andeventually into terminal ducts; this occurs at 40–60 days of age.Under normal conditions, DMBA is most effective in producingtumors during the most active transition period of terminal end budevolution into alveolar buds. The greater incidence and tumor yield inthe DMBA-treated animals occurred because transition in the mam-mary tissue was occurring in the normal window of DMBA sensitiv-ity. Such a mechanistic proposition has been put forth by Anderson etal. (50), who studied the effect of constant light on DMBA-inducedmammary tumorigenesis in rats. Neonatal genistein treatment exertsits chemopreventive action by directly enhancing maturation of theterminal ductal structures and by altering the endocrine system toreduce cell proliferation in the mammary gland (51, 52).

Several other related mechanisms relevant to the observed chemo-

preventive effects of resveratrol may be cited. As a corollary to anearlier report from this laboratory, resveratrol substantially inhibitedTNF-induced NF-�B in MCF-7 cells, further strengthening the ideathat inhibition is not cell type specific. Moreover, no general tran-scription suppression belonging to resveratrol occurs under our ex-perimental conditions (32).

How resveratrol blocks TNF-induced NF-�B in MCF-7 cells is notclear. Most inhibitors of NF-�B mediate their effect through suppres-sion of phosphorylation and degradation of I�B�. Resveratrol hasbeen shown to block the phosphorylation and the degradation of I�B�(33). We have shown that resveratrol blocks the phosphorylation ofp65 required for its transactivation function (32). Reports of anotherstudy by Kim et al. (53) revealed aberrant expression of NF-�B inmammary tumors induced by DMBA treatment of Sprague Dawleyrats compared with normal mammary gland of age-matched, vehicle-treated control animals. Moreover, they reported that NF-�B/Relactivation occurs before malignant transformation and is not presentnormally in the mammary gland, suggestive of a significant associa-tion between activation of NF-�B expression and the progression ofepithelial cells to a malignant phenotype. Moreover, aberrant expres-sion of NF-�B in human breast cancer specimens has also beenreported (34). Thus, our previous findings that resveratrol mediateddown-regulation of NF-�B factor indicate that resveratrol may atten-uate the early critical steps involved in carcinogen-driven transforma-tion of mammary epithelial cells, including dysregulation of normalcontrol of proliferation and protection from apoptosis.

Antioxidants such as N-acetylcysteine and pentoxifylline, which arealready in clinical use, repress NF-�B activity and concurrently ex-hibit significant inhibitory effects on proliferation of breast cancercells in culture (54, 55). Resveratrol has been reported recently tomediate phosphorylation of p53 at serine 15 through extracellularsignal-regulated kinase and p38 kinase activities, leading to inductionof apoptosis (56). The observed dose-dependent effect of resveratrolon the proliferation of MCF-7 cells noted in our present study impliesthat resveratrol-mediated chemoprotection may occur through theup-regulation of apoptosis. Several in vitro studies have documentedan antiproliferative effect of resveratrol on many cell types (57–63),including those derived from the human breast (14, 26). Resveratrolhas been shown to inhibit the transition of cells from the S-to-G2

phase of the cell cycle (15–18, 59, 60). Our results are in agreementwith these reports.

It is now well established that once breast cancer initiation hastaken place, estrogen promotes the growth of transformed cells, lead-ing to the development of detectable breast cancer. Resveratrol be-haves like the partial estrogen receptor agonist tamoxifen, whichblocks the action of estrogen in the breast and effectively preventsprimary and recurring breast tumor development (64). Another rele-vant important mechanism of action of many chemopreventive agentsis through their ability to modulate the xenobiotic-metabolizing en-zymes, e.g., by inhibiting metabolic activation of a procarcinogen orby increasing detoxification of reactive metabolites. In polycyclicaromatic hydrocarbon tumorigenicity, oxidative phase I biotransfor-mation results in highly reactive diolepoxides that form covalentadducts with DNA. Reports indicate that the level of polycyclicaromatic hydrocarbon-DNA adducts is related to the level of CYPIAIexpression (65). Interestingly, resveratrol has been reported to inhibitconstitutive and inducible expression of oxidative phase I biotrans-formation-related CYP1A1 in human bronchial epithelial and breastcancer cells (23, 24, 66). Furthermore, Jang et al. (8) have shown thatresveratrol induces quinone reductase activity, a phase II enzyme, incultured mouse hepatoma cells. Thus, one may also interpret theobserved chemopreventive action of resveratrol primarily at the levelof inhibition of procarcinogen activation, leading to reduced bioacti-

Fig. 7. A, resveratrol suppresses TNF-induced NF-�B activation in human breastMCF-7 cells. A total of 1 � 106 cells/ml were preincubated at 37°C for 4 h with differentconcentrations of resveratrol (0–50 �M), followed by a 30-min incubation with 0.1 nM

TNF. After these treatments, nuclear extracts were prepared and then assayed for NF-�Bas described in “Materials and Methods.” B, supershift and specificity of NF-�B activa-tion. Nuclear extracts were prepared from untreated or TNF (0.1 nM)-treated MCF-7 cells(1 � 106 cells/ml), incubated for 30 min with different antibodies as indicated, preimmunesera, unlabeled wild-type oligo or mutant oligo, and then assayed for NF-�B, as describedin “Materials and Methods.”

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vated DMBA metabolites as well as increased expression of phase IIdetoxification enzymes. Several presumptive chemopreventive agentsreveal the potential to induce and enhance detoxification activities inhost target organs. Moreover, resveratrol-mediated down-regulationof CYPIAI also implies the 2-hydroxylation of 17�-estradiol andestradiol, which may further attenuate the effect of estrogen on thedevelopment of breast cancer.

Consistent with the hypothesis that COX-2 is not expressed in

normal tissue, no evidence of its expression was detectable by immu-nohistochemistry performed on normal mammary tissue. However,resveratrol has been reported to inhibit COX-2 transcription andactivity in phorbol ester-treated human mammary epithelial cells (20).Qualitative immunohistochemical analysis for COX-2 revealed im-munoreactivity in fibroadenoma as well as in tumor specimens de-rived from resveratrol-pretreated animals. It can thus be inferred fromour findings that resveratrol-mediated down-regulation of COX-2

Fig. 8. A, resveratrol suppresses the proliferation ofhuman breast MCF-7 cells. Cells (5 � 103) in 0.1 ml ofculture medium were plated on 96-well plates and in-cubated at 37°C. After 24 h, cells were grown in either200 �l of fresh medium (control) or 200 �l of freshmedium containing 2-fold serial dilutions of resveratrol.Cells were incubated for 72 h, and viable cells at theend of incubation were determined by the MTT methodas described in “Materials and Methods.” B, resveratrolsuppresses DNA synthesis of human breast MCF-7cells. Cells (5 � 103) in 0.1 ml of medium were platedin 96-well plates and incubated overnight at 37°C. After24 h, cells were replenished with either 200 �l of freshmedium (control) or 200 �l of fresh medium containing2-fold serial dilutions of resveratrol. After 72 h ofincubation, 0.1 �Ci of [3H]thymidine was added toeach well during the last 6 h of incubation and har-vested as described in “Materials and Methods.” Pro-liferation was determined by [3H]thymidine incorpora-tion into the cells at each time point. All determinationswere made in triplicate. C, resveratrol inhibits the pro-liferation of MCF-7 cells. Cells (2 � 103) in 0.1 ml ofculture medium were plated in 96-well plates. After24 h, cells were allowed to grow in either 200 �l offresh medium (control) or 200 �l of fresh mediumcontaining 10 and 50 �M resveratrol. Cells were incu-bated for 0, 2, 4, and 6 days, and viable cells weredetermined by MTT assay as described in “Materialsand Methods.” The variations between the triplicate aretoo small to be visible at each data point. D, resveratrolinhibits MCF-7 cells at S-G2-M phase of the cell cycle.Cells (2 � 106) were plated in 6-well plates and thentreated with resveratrol for different times. Thereafter,cells were harvested, fixed in ethanol, stained withpropidium iodide, and analyzed by FACS analysis.

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may be effective in attenuating the early stages of carcinogenesis, thusreducing tumor incidence and multiplicity. COX-2 inhibitors demon-strate strong chemoprotective potential. Besides COX-2, MMP-9 isanother gene regulated by activation of NF-�B. Our results showclearly that treatment of animals with DMBA also induced MMP-9,and this induction was suppressed by treatment with resveratrol. BothCOX-2 and MMP-9 have been implicated in tumor invasion andmetastasis. Whether suppression of tumor growth by resveratrol in theanimals is attributable to inhibition of DMBA-induced NF-�B acti-vation is not clear. Because NF-�B activation was suppressed in mostof the resveratrol-treated animals, it suggests that NF-�B may play animportant role. However, NF-�B-independent mechanisms cannot beexcluded based on our studies.

In conclusion, the present study demonstrates that resveratrol in-hibits rat mammary tumor development and illustrates an emergingconcept of chemoprevention through inhibition of transcription factorNF-�B. In addition, resveratrol functions as an inhibitor of breastcancer cells in vitro mediated through modulation of cell proliferationand apoptosis. There is no study reporting on the pharmacokinetics ofresveratrol metabolism in humans subjects, and thus additional studiesare warranted to determine the optimum effective dose of this phyto-chemical compound in inhibiting cancers in humans.

ACKNOWLEDGMENTS

This research was conducted by The Clayton Foundation for Research. Wethank Walter Pagel for critical reading of the manuscript.

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2002;62:4945-4954. Cancer Res Sanjeev Banerjee, Carlos Bueso-Ramos and Bharat B. Aggarwal Metalloprotease 9

B, Cyclooxygenase 2, and MatrixκNuclear Factor-Mammary Carcinogenesis in Rats by Resveratrol: Role of

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