Anthracene-Induced Mammary Carcinogenesis in Rats by Resveratrol: Role of Nuclear Factor-␬B, Cyclooxygenase 2, and Matrix Metalloprotease 9

[CANCER RESEARCH 62, 4945–4954, September 1, 2002] Suppression of 7,12-Dimethylbenz(a)anthracene-induced Mammary Carcinogenesis in Rats by Resveratrol: Role of Nuclear Factor-␬B, Cyclooxygenase 2, and Matrix 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 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- ؅ We have reported recently that resveratrol (trans-3,4 ,5-trihydroxystil- matory and anti-atherosclerosis functions and to modulate hepatic bene), a polyphenolic phytoalexin found in grapes, fruits, and root extracts alipoprotein and lipid synthesis, platelet aggregation, and production of the weed Polygonum cuspidatum, is a potent inhibitor of nuclear factor (NF)-␬B activation. Because NF-␬B suppression has been linked with of antiatherogenic eicosanoids by human platelets and neutrophils (7, chemoprevention, this prompted us to investigate the chemopreventive 9–11). Resveratrol has also been reported to inhibit the development potential of resveratrol by testing it against mammary carcinogenesis of preneoplastic lesions in carcinogen-treated mouse mammary organ induced by 7,12-dimethylbenz(a)anthracene (DMBA) in female Sprague cultures and the promotional stage of mouse skin carcinogenesis (7). Dawley rats. Dietary administration of resveratrol (10 ppm) had no effect Additionally, it mediates reduced aberrant colonic crypt foci forma- on body weight gain and tumor volume but produced striking reductions tion and inhibits of the growth of a wide variety of human-derived in the incidence (45%; P < 0.05), multiplicity (55%; P < 0.001), and tumor cells, including leukemic, prostate, breast, and endothelial cells extended latency period of tumor development relative to DMBA-treated (12–19). Other targeted cellular effects belonging to resveratrol in- animals. Histopathological analysis of the tumors revealed that DMBA induced ductal carcinomas and focal microinvasion in situ (7 of 7), volve inhibition of the enzymes protein kinase C, ribonucleotide whereas treatment with resveratrol suppressed DMBA-induced ductal reductase, cyclooxygenase, and nitric oxide synthase and inhibition of carcinoma. Immunohistochemistry and Western blot analysis revealed aryl hydrocarbon-induced cytochrome P-450 IAI (20–25). that resveratrol suppressed the DMBA-induced cyclooxygenase-2 and A close structural similarity exists between synthetic estrogen (4,4Ј- matrix metalloprotease-9 expression in the breast tumor. Gel shift analysis dihydroxy-trans-␣,␤-diethylstilbene) and resveratrol. It is unclear, showed suppression of DMBA-induced NF-␬B activation by resveratrol. however, whether resveratrol is an estrogen receptor agonist or an- Treatment of human breast cancer MCF-7 cells with resveratrol also tagonist. Estrogen agonists have been reported to exert protective suppressed the NF-␬B activation and inhibited proliferation at S-G -M 2 action against estrogen-dependent cancers, such as cancer of the phase. Overall, our results suggest that resveratrol suppresses DMBA- breast and endometrium; presumably, resveratrol interacts with estro- induced mammary carcinogenesis, which correlates with down-regulation of NF-␬B, cyclooxygenase-2, and matrix metalloprotease-9 expression. gen receptor to inhibit its activation (26). Lu and Serrano (26) reported that resveratrol acts as an estrogen receptor antagonist in the presence of estrogen, leading to inhibition of growth of human breast cancer INTRODUCTION cells. ␬ 2 Almost 600,000 new cases of breast cancer are identified each year In recent years, the importance of the transcription factor NF- B ␬ worldwide. In North America, breast cancer accounts for over one in promoting tumorigenesis has been well recognized. NF- B binds to quarter of all cancers in women and is second only to lung cancer as consensus elements within the promoter regions of a variety of tar- a cause of cancer-related deaths (1). Despite abundant information geted genes (27). Further investigations have revealed that the expres- ␬ about its etiopathogenesis and early detection, effective therapeutic sion of a multitude of critical genes are regulated by NF- B, including modalities for patients with advanced stages of the disease are still immunoreceptors, transcription factor-associated proteins (c-myc and needed. Adjuvant therapy after ablative surgery is effective only when p53), cell adhesion molecules (intracellular adhesion molecule, vas- the tumor is detected early. Accumulating evidence derived from cular cell adhesion molecule 1, and endothelial leukocyte adhesion laboratory studies and study cohorts drawn from the general popula- molecule 1), and enzymes involved in tumor metastasis (COX-2, tion have led to the search for “chemoprotection” agents to attenuate inducible nitric oxide synthase, and MMP-9). Under normal condi- ␬ the risk of breast cancer based on observation that most human tions, NF- B is retained in the cytoplasm of cells, where it is bound ␬ cancers are associated with a long period of latency (2, 3). Several by inhibitory proteins known as I Bs. It has also been documented nonnutritive phytochemicals found in natural products and associated that during carcinogenesis, NF-␬B has the potentiality to mediate with pharmacological attributes reveal evidence that they inhibit, several of the events associated with multistep processes including delay, and/or reverse cancer evoked by either environmental insults acquisition of features such as promotion of cell survival and dys- and/or lifestyle (4, 5). Several of these chemopreventive agents act at regulation of normal control of proliferation, metastasis, and angio- the initiation, promotion, and/or progression stages conceptually as- genesis (27–29). The constitutive activity of NF-␬B has been shown sociated with the ontogeny of multistage carcinogenesis. to be essential for proliferation of several cell types, e.g., smooth Resveratrol (3,4Ј-trihydroxystilbene), a natural phytoalexin present muscle cells and hepatocytes during liver regeneration after partial in grapes and many other natural sources, has been suggested to play hepatectomy or toxic damage (30, 31). Previous reports from our laboratory and another have demon- ␬ Received 3/26/02; accepted 7/1/02. strated the ability of resveratrol to down-regulate NF- B expression in The costs of publication of this article were defrayed in part by the payment of page vitro (32, 33), leading to speculation that it would in turn inhibit charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Bioimmuno- 2 The abbreviations used are: NF-␬B, nuclear factor ␬B; TNF, tumor necrosis factor; therapy, The University of Texas M. D. Anderson Cancer Center, Box 143, 1515 Hol- I␬B, inhibitory subunit of NF-␬B; EMSA, electrophoretic mobility shift assay; MTT, combe Boulevard, Houston, TX 77030. Phone: (713) 792-3503, 6459; Fax: (713) 794- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; COX, cyclooxygenase; 1613; E-mail: [email protected]. DMBA, 7,12-dimethylbenz(a)anthracene; MMP, matrix metalloprotease. 4945

cellular genes regulated by NF-␬B and those involved in multistage belonging to group III were treated with resveratrol (100 ␮g/rat) in the diet. tumorigenesis. In the present study, we evaluated the effect of res- The dose of resveratrol was computed based on average food intake, which veratrol in inhibiting chemically induced mammary carcinogenesis in approximated 12–15 g/rat/day. Resveratrol was dissolved in 70% ethanol and a rat model. This is the first report to indicate that resveratrol has a then added into the diet 1 day in advance and left at room temperature for 1 chemopreventive effect on breast tumorigenesis in vivo. Given the day. After ethanol evaporation, food was then given to the rats. Food cups were changed two times a week. progressive aberrant expression of constitutive NF-␬B factor with Chemoprevention Study Design. Rats belonging to the appropriate ex- progression of the disease (34), we hypothesize that resveratrol inter- perimental group were given the chemopreventive agent beginning at day 45 ␬ feres with cognate signaling by inhibiting of NF- B activity during of age. One week later (day 0), rats belonging to groups II and III were given the mammary tumorigenesis cascade. We therefore used immunohis- 10 mg of DMBA by gavage in sesame oil. This dose of DMBA is suboptimal tochemical and Western blot methods to examine the expression of to produce sufficient tumors to allow evaluation of both reduction and increase two enzymes, COX-2 and MMP-9, both of whose promoter sequences in the end point of carcinogenicity. Rats were weighed weekly, palpated for contain binding sites for NF-␬B. Additionally, we investigated the mammary tumors once/week (starting 4 weeks after DMBA treatment), and effect of resveratrol on NF-␬B activation and cell growth in MCF-7 monitored daily for signs of toxicity. The study was terminated at 120 days breast adenocarcinoma cells. after DMBA administration. All surviving animals, including those that did not seem to develop mammary tumors as well as the control group (group I), were

killed by CO2 asphyxiation and completely necropsied to evaluate possible MATERIALS AND METHODS signs of toxicity. Tumors were removed and fixed in 10% buffered formalin. Thirteen DMBA-induced mammary tumors from resveratrol (group III) or Chemicals control group (group II) were evaluated with a blind method for histopathol- ogy. The tumors were subjectively graded either as carcinomas or fibroade- Penicillin, streptomycin, RPMI 1640, FCS, and trypsin were obtained from nomas. The end point for data analysis included: (a) the number of animals Life Technologies, Inc. (Grand Island, NY). BSA, MTT, leupeptin, aprotinin, with tumors (tumor incidence); (b) the number of tumors/animal (tumor and DMBA of highest purity were purchased from Sigma Chemical Co. (St. multiplicity); (c) latency to tumor appearance; and (d) tumor volume. Esti- Louis, MO). Bacteria-derived recombinant human TNF, purified to homoge- mates of tumor volume were determined using the formula V ϭ 4/3␲ r3, where neity with a specific activity of 5 ϫ 107 units/mg, was kindly provided by r is half of the average diameter (in millimeters) measured with a vernier Genentech (South San Francisco, CA). The radioisotope [5-methyl-3H]thymi- caliper at two different planes. dine was obtained from Amersham Pharmacia Biotech, and [␥-32P]ATP (5 mCi) was purchased from ICN Radiochemicals (Costa Mesa, CA). Antibodies Histological Sections used were as follows: anti-p65, against the epitope corresponding to amino ␬ acids mapping within the NH2-terminal domain of human NF- B p65; anti- Formalin-fixed tissue was paraffin-embedded, sectioned at 3–5 ␮m, and p50, against a peptide 15 amino acids long mapping at the NLS region of stained with H&E. Sections were evaluated for tumor cell cytology, mitotic NF-␬B p50; and COX-2, against the epitope corresponding to amino acids rate, growth pattern, necrosis, cystic change, and associated inflammatory 50–111 mapping near the COOH terminus of COX-2 of human origin. These cellular response. were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For immunohistochemistry, COX-2 antibody (murine polyclonal) was purchased Immunohistochemistry from Cayman Laboratories (Ann Arbor, MI), whereas rabbit antirat polyclonal antibody for MMP-9 was procured from Cell Sciences, Inc. (Norwood, MA). Immunohistochemical studies were performed using paraffin-embedded Resveratrol was obtained from two sources; for animal experimentation, material, heat-induced antigen retrieval (citrate buffer, pH 6.0), and polyclonal resveratrol (Ն98% pure) was purchased from Alexis Cooperation, San Diego, antirat antibodies specific for MMP-9 (CPM601; 1:750; Cell Sciences, Nor- CA). For in vitro studies, resveratrol was procured from Sigma Chemical Co. wood, MA) and COX-2 (1:500; Cayman Chemical Co., Ann Arbor, MI). The A stock solution of resveratrol was made in DMSO at a concentration of detection system used was the LSAB2 detection kit (DAKO). Negative con- trols also were run. 10 mM. The NF-␬B oligonucleotide from the HIV long terminal repeat, 5Ј-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCG- Preparation of Nuclear Extract from Tissue Samples TGG-3Ј and a mutated double-stranded oligonucleotide, 5Ј-TTGTTACAA- CTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3Ј were from Normal mammary epithelium and tumor samples (200–250 mg) randomly Life Technologies, Inc. (Grand Island, NY; underlined regions represent a selected from untreated control and experimental groups were minced and ␬ consensus NF- B binding sequence). All other chemicals were purchased from incubated on ice for 30 min in 0.5 ml of ice-cold buffer A, composed of 10 mM authentic sources and were of highest grade and purity. HEPES (pH 7.9), 1.5 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1% IGEPAL CA-630, and 0.5 mM phenylmethylsulfonyl fluoride. The minced tissue was Chemoprevention Studies homogenized using a Dounce homogenizer and centrifuged at 14,000 rpm at 4°C for 10 min. The nuclear pellet obtained was suspended in 0.2 ml of buffer Animals. Female Sprague Dawley rats were purchased from Harlan B [20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl , 420 mM NaCl, 0.5 Sprague Dawley (Indianapolis, IN). The rats arrived at 40 days of age and were 2 mM DTT, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 4 ␮M placed on a common pellet diet and quarantined for 2 days. The animals were leupeptin] and incubated on ice for 2 h with intermittent mixing. The suspen- housed three/cage in standard rat Plexiglass cages in a room maintained at sion was then centrifuged at 14,000 rpm at 4°C for 30 min. The supernatant constant temperature and humidity under 12-h light and darkness. A complete (nuclear extract) was collected and stored at Ϫ70°C until use. The protein health status was determined. None of the rats exhibited major lesions, and all concentration was measured by the method of Bradford (35) with BSA as the were pathogen free. Before initiating the experiment, we acclimatized all rats standard. EMSA was performed by incubating 12 ␮g of nuclear protein extract on pulverized diet for 3 days and then randomly assigned them by body weight by procedure as described (36). to one of the three groups: group I (n ϭ 7) received pulverized rodent diet and ϭ served as negative control; group II (n 12), designated as a positive control, Western Blot Analysis received DMBA and pulverized diet; and group III (n ϭ 12) received DMBA and pulverized experimental diet containing resveratrol. Mammary epithelium and/or tumor specimens from each of the above- Animals were allowed free access to the basal diet or diet containing the mentioned experimental groups were thawed on ice and homogenized at 4°C chemopreventive agent and drinking water throughout the experiment. Our using a Dounce homogenizer in RIPA lysis buffer for extracting total cell experimental protocol was reviewed and approved by M. D. Anderson Cancer protein. Sixty ␮g of whole-cell protein was resolved on 10% SDS-PAGE gel. Center Animal Care and Use Committee. The protein was transferred to a nitrocellulose membrane, blocked with 5% Treatment with Chemopreventive Agent. Animals (groups I and II) were nonfat milk, and probed with specific antibodies against MMP-9 (1:3000) or given a normal diet containing vehicle control. Starting at 45 days of age, rats COX-2 (1:1000), separately. The blots were washed, exposed to horseradish 4946

peroxidase-conjugated secondary antibodies for 1 h, and finally detected by Statistical Analysis ECL reagent (Amersham Pharmacia Biotech, Arlington Heights, IL). Body weight, tumor incidence, tumor multiplicity, and tumor volume were determined for the animals fed control diet and for those fed on the modified Cell Line diet containing resveratrol. The data were analyzed and compared by ␹2 test MCF-7 cells were a kind gift of Dr. Kapil Mehta (M. D. Anderson Cancer using Sigma-Stat software (Jandel Scientific, San Rafael, CA). Center, Houston, TX). Cells were maintained in culture in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 RESULTS ␮ units/ml penicillin and 100 g/ml streptomycin) in an atmosphere of 5% CO2 at 37°C. Resveratrol Suppresses DMBA-induced Rat Mammary Tu- morigenesis. NF-␬B inhibitors have been shown to exhibit chemo- EMSA preventive properties (37–45). To determine whether resveratrol af- fects DMBA-induced rat mammary carcinogenesis, experiments were ϫ 6 MCF-7 cells (1 10 /ml) were preincubated with different concentrations designed to examine chemoprevention after continuous dietary ad- ␮ of resveratrol (0, 10, 25, and 50 M) for 4 h and then treated with TNF (0.1 nM) ministration of resveratrol beginning 1 week before carcinogen ad- for 30 min at 37°C. Nuclear extracts were then prepared according to the ministration and lasting until termination of the experiment (Fig. 1). method described by Chaturvedi et al. (35). The protein content was measured by the method of Bradford (36). EMSA was performed by incubating 8 ␮gof The rationale for using this specific protocol relates to identifying nuclear extract with 16 fmol of 32P-end-labeled, double-stranded 45-mer agents that intervene in initiation and progression of cancer. No NF-␬B oligonucleotide. The incubation mixture included 2 ␮g of poly(deoxyi- adverse effect on body weight gain during the observation period was nosinic-deoxycytidylic acid) in a binding buffer. The DNA-protein complex detectable, and the average body weight gain was similar in all of the formed was separated from free oligonucleotide on 6.6% native polyacryl- groups throughout the experiment (267 Ϯ 2.8, 267 Ϯ 4.5, and amide gel, and then the gel was dried. The radioactive bands from dried gels 268 Ϯ 2.7 for groups I, II, and III, respectively; P ϭ not significant; were visualized by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) Fig. 2). Moreover, no evidence of development of any spontaneous using Image Quant software. tumor, including tumors of the breast, occurred in animals randomized The composition and specificity of binding was examined by competition to the negative control group (group I) during the span of the study. with 100-fold excess of unlabeled oligonucleotide and with a mutated oligo- Administration of resveratrol and/or DMBA did not reveal any gross nucleotide. For the supershift assays, nuclear extract were incubated with the changes in the liver, lung, kidneys, stomach, or intestinal tract of antibodies against either p50 or p65 subunits of NF-␬B for 30 min at 37°C before the complex was analyzed by EMSA (36). animals. In group II, 75% of the animals treated with DMBA (positive control) developed palpable breast tumors at 11 weeks after DMBA treatment (Fig. 3), and the average number of tumors/tumor-bearing Evaluation of Cell Viability by MTT animal was 2.41 Ϯ 0.5 (mean Ϯ SE; Fig. 4). Rats of group III, which MCF-7 cells were plated (in triplicate) in 96-well plates (5000 cells/well), received resveratrol-supplemented diet in addition to DMBA, had a 24 h before addition of resveratrol. Medium was then aspirated, and cells were tumor incidence of only 41% (P Ͻ 0.05), and the average number of exposed to 2-fold serial dilutions of resveratrol in 0.2 ml of fresh medium and tumors/tumor-bearing animal was reduced to 1.08 Ϯ 0.5 (P Ͻ 0.001). incubated at 37°C in an atmosphere of 5% CO2 for 72 h. Thereafter, cell Furthermore, differences between the groups (group II versus group viability was measured by the MTT method using a multiscanner autoreader III) in cancer latency was observed; in the resveratrol-treated animals, (Dynatech MR 5000, Chantilly, VA; Ref. 32). To examine the antiproliferative the latency was 98 days after DMBA treatment compared with 77 effects of resveratrol, 2000 cells in 0.1 ml of medium were plated overnight days for DMBA-only treatment. No significant difference in the tumor and then treated with 10 or 50 ␮M resveratrol in 0.2 ml for 2, 4, and 6 days. Ϯ Thereafter, the cell viability was determined by the MTT method. volume was noted between groups II and III (1.58 0.36 versus

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) were plated in 0.1 ml of medium (RPMI 1640 plus 10% fetal bovine serum) in a

96-well plate. After overnight incubation in a CO2 incubator at 37°C, the medium was removed, and different concentrations of resveratrol (twofold serial dilutions starting from 100 ␮M concentration) were added in 0.2 ml of fresh medium for 72 h. During the last6hofincubation before harvesting, 0.1 ␮Ci [3H]thymidine was added to each well. Thereafter, the medium was aspirated, wells were washed with PBS, and cells were detached by the addition of a solution containing trypsin (0.5%) plus EDTA (5.3 mM). The cell suspension was harvested on a Filter Mate 196 cell harvester (Packard Instru- ments, Meriden, CT) and lysed by washing with distilled water. Radioactivity bound to the filter was measured by Direct Beta Counter (Matrix 9600; Packard Instruments, Meriden, CT).

FACS Analysis

Cells (2 ϫ 106) in 1 ml of culture medium were plated in 6-well plates overnight and then treated with 50 ␮M resveratrol or medium for 6, 12, 24, or 48 h. Thereafter, the cells were harvested by trypsinization, washed twice with PBS, and fixed in 5 ml of 95% ethanol. After 12 h incubation at Ϫ20°C, cells were washed and resuspended in PBS. The cellular DNA was then stained with Fig. 1. Experimental design for the evaluation of chemopreventive effect of resveratrol ␮ against mammary carcinogenesis. Groups of female Sprague Dawley rats were fed either 1 ml of propidium iodide (50 g/ml) containing 0.1 ml of RNase (1 mg/ml). 0 or 10 ppm resveratrol mixed in the diet 7 days before exposure to DMBA, during After incubation at 37°C for 30 min, cells were analyzed by FACS analysis. treatment, and until termination of the study. 4947

1.26 Ϯ 0.70, respectively). In our study, two animals were removed, one each from group II and group III before termination schedule because of the presence of necrotizing tumor. Resveratrol Suppresses DMBA-induced Ductal Carcinoma. Histopathological analysis was performed on tumor samples randomly selected from untreated (group I), DMBA-treated (group II), and DMBA ϩ resveratrol-treated (group III) animals (Fig. 5). Normal mammary epithelium from control animals showed no pathological abnormality (Fig. 5A). Tumors obtained from DMBA-treated animals had ductal carcinomas and focal microinvasion in situ (7 of 7; Fig. 5,

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

BϪD). The surrounding breast tissue showed papillomatosis, atypical ductal epithelial hyperplasia, and no fibroadenomas or infarcts. In contrast, tumors harvested from DMBA- and resveratrol-treated animals exhibited evidence of fibroadenoma (2 of 4) and partially in- farcted ductal carcinoma in situ with microinvasion (4 of 4; Fig. 5, E and F). Resveratrol Suppresses DMBA-induced Intracellular Expres- sion of COX-2 and MMP-9 Proteins. Immunohistochemistry re- Fig. 2. Resveratrol does not affect the body weight of animals. The figure shows the average weekly body weights in different groups of female Sprague Dawley rats begin- vealed the absence of COX-2 activity in normal mammary epithelium ning at 45 days of age. (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 of 4) in DMBA ϩ resveratrol-treated rats. When examined for MMP-9 expression by immunohistochemistry, staining in the normal mammary epithelium (Fig. 5J) was consistently absent, the breast tissue from the DMBA-treated group showed uniformally positive staining (7 of 7; Fig. 5K), and the tissue from the DMBA ϩ resveratrol-treated group showed low expression (4 of 4; Fig. 5L). To further confirm the effect of resveratrol on DMBA-induced COX-2 and MMP-9 expression, we examined protein expression by Western blot analysis. Because both COX2 and MMP-9 are regulated by NF-␬B activation, we also measured the nuclear levels of NF-␬B by EMSA in the mammary tissue. As shown in Fig. 6, none of the untreated tissues samples expressed NF-␬B DNA-binding activity; tissues derived from 8 of 9 DMBA-treated animals expressed NF-␬B DNA-binding activity, whereas only 3 animals of 9 from the DMBA ϩ resveratrol-treated group expressed NF-␬B. These results suggest that DMBA induces NF-␬B DNA-binding activity in most cases, and resveratrol suppresses it. When examined for COX-2 and MMP-9 expression, tissues from all of the DMBA-treated group expressed these proteins (none in the untreated group), but only low levels were detected in tissues from animals treated with DMBA together with resveratrol. These results are consistent with that obtained from immunohistochemistry. Fig. 3. Treatment with resveratrol suppresses the DMBA-induced incidence of mam- Resveratrol Inhibits TNF-induced NF-␬B Activation in Human mary tumors in rats. The induction of mammary tumors induced by single pulse exposure Breast Cancer Cells. Our results show that resveratrol suppressed to 10 mg of DMBA/rat is shown. The progressive percentage incidence of cumulative ␬ palpable mammary tumors, as a function of time after carcinogen treatment, is shown. DMBA-induced mammary carcinogenesis. NF- B has been impli- Values represent the mean of 8–12 rats/group. cated in carcinogenesis. Previously, we and others have shown that 4948

Fig. 5. Effect of resveratrol on the DMBA-induced pathology and on COX-2 and MMP-9 expression. Representative H&E-stained sections (AϪF) and immunohistochemical staining (GϪL) 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 in situ/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. GϪI, immunohistochemical staining for COX2 protein demonstrates COX-2 negativity in breast tissue from untreated rat (G), strong positivity in ductal carcinoma from DMBA-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). JϪL, 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 weak immunopositivity in DMBA plus resveratrol-treated tumor (L) are shown.

resveratrol does suppress NF-␬B activation in various cell types (32, active NF-␬B heterodimer that binds to specific sequences in DNA. 33). Whether resveratrol inhibits NF-␬B activation in breast cancer To show that the retarded band visualized by EMSA was indeed cells was investigated. MCF-7 cells were pretreated for 4 h with NF-␬B, we incubated the nuclear extract from TNF-activated cells different concentrations of resveratrol and then stimulated with 0.1 nM with antibodies to either p50 (NF-␬B) or the p65 (relA) subunit and TNF for 30 min. As assessed by trypan blue and MTT assay, resvera- then conducted EMSA. Antibodies to either subunit of NF-␬B shifted trol did not affect cell viability at this concentration and time point. the bands to a higher molecular weight (Fig. 7B), thus suggesting that Nuclear extracts were made and assayed for NF-␬B by EMSA. As the TNF-activated complex consisted of both the p50 and p65 units. shown in Fig. 7A, TNF induced DNA-binding activity of NF-␬B, and Preimmune serum had no effect on the mobility of NF-␬B. Excess resveratrol inhibited this activation in a dose-dependent manner; full unlabelled NF-␬B almost completely eradicated the band, indicating inhibition occurred at 50 ␮M resveratrol. Resveratrol alone at this the specificity of NF-␬B. Further specificity is indicated by the concentration did not activate NF-␬B. observation that the oligonucleotide probe with labeled mutated Various combinations of Rel/NF-␬B proteins can constitute an NF-␬B binding site failed to bind the NF-␬B protein. 4949

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 from animals 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 blot analysis as described in “Materials and Methods.” ␤-Actin as a loading control is also depicted.

Resveratrol Inhibits the Growth of Human Breast Cancer metabolism. Additionally, divergent beneficial modulatory effects of Cells. Whether resveratrol suppresses the proliferation of human resveratrol has been reported in the literature (46). We report in this breast cancer cells was also investigated. MCF-7 cells were treated study that resveratrol fed to Sprague Dawley female rats inhibited with 2-fold serial dilution of resveratrol for 72 h, either in the presence tumor formation in comparison to vehicle treatment in DMBA-initi- or absence of resveratrol, and then examined for growth by MTT and ated mammary carcinogenesis. Tumor incidence calculated as the cell proliferation by thymidine incorporation. Results in Fig. 8A percentage of animals with one or more palpable tumors/treatment indicate that resveratrol inhibited the growth of human breast cancer group was reduced by 45% after resveratrol supplementation. Other cells in a dose-dependent manner, with almost 60% suppression of indices of chemopreventive response-tumor multiplicity, expressed as cell viability at 100 ␮M concentration. When examined for cell pro- average number of tumors that developed per animal/week in each liferation by DNA synthesis, 90% inhibition of thymidine incorpora- treatment group was reduced from 2.41 to 1.04 tumors/animal by ␮ tion occurred with 100 M resveratrol (Fig. 8B). Thus, breast cancer resveratrol feeding at the termination of the experiment. Furthermore, cells were more sensitive to resveratrol in the DNA synthesis assay relative to control animals, the latency to onset of tumor development than that in the assay of mitochondrial activity. We also examined the was prolonged by 3 weeks by resveratrol. ␮ effect of resveratrol on the proliferation of MCF-7 cells. Fifty M Precisely how resveratrol inhibits breast cancer is not certain, resveratrol was sufficient to completely suppress the proliferation of although several possible modes of action have been proposed and breast cancer cells (Fig. 8C). studied at the cellular and molecular level. Resveratrol is able to We also examined the effect of resveratrol on the cell cycle analysis mimic the activity and effects of endogenous 17␤-estradiol. Accord- of MCF-7 cells. Results shown in Fig. 8D clearly show resveratrol ing to a proposed hypothesis, estrogen has a dual affect on breast induced the accumulation of cells in S-phase of the cell cycle. In the cancer risk (47). Evidence indicates that estrogens promote the growth resveratrol-treated samples at 24 h, 48% of the cells were in S-phase of existing malignancies in the breast. In contrast under certain cir- as compared with 11% in the control. A complete suppression of the cumstances such as pregnancy, during the prepubertal period and cells in the G -M phase of the cycle could be noted as early as 12 h 2 childhood, estrogen actually reduces breast cancer risk through estro- after resveratrol treatment. gen-induced activation of certain tumor suppressor genes including BRCA1 and p53 (47). It may thus be speculated that resveratrol, as a DISCUSSION putative estrogen agonist, augments mammary tissue differentiation The present study demonstrates the chemopreventive action of and maturation beyond the optimum period of carcinogen sensitivity, resveratrol in a well-established, chemically induced animal protocol thus conferring a protective effect. It has been reported that complete of breast cancer and its relation to NF-␬B expression. In recent years, morphological differentiation of the mammary gland protects against “cancer chemoprevention” by biologically active dietary or nondi- mammary carcinogenesis in Wistar Furth rats. Pretreatment of rats etary supplements has generated immense interest in view of their with estradiol and progesterone or completion of pregnancy and putative role in attenuating the risk of developing cancer. Against this lactation before carcinogen exposure markedly reduces the suscepti- background, resveratrol is a promising agent in affording chemopro- bility of the gland to chemical carcinogenesis (48). tection against several major human epithelial and nonepithelial can- In our study design, resveratrol treatment was initiated before onset cers. Resveratrol was first evaluated in vitro and reported to inhibit of puberty. Because resveratrol is known to induce differentiation mammary lesions induced by DMBA, leading to speculation of a (49), it is likely that resveratrol treatment accelerated mammary tissue specific effect on the mammary gland independent of systemic drug differentiation, leading to refractory cell phenotypes during the period 4950

preventive effects of resveratrol may be cited. As a corollary to an earlier report from this laboratory, resveratrol substantially inhibited TNF-induced NF-␬B in MCF-7 cells, further strengthening the idea that inhibition is not cell type specific. Moreover, no general transcription suppression belonging to resveratrol occurs under our experimental conditions (32). How resveratrol blocks TNF-induced NF-␬B in MCF-7 cells is not clear. Most inhibitors of NF-␬B mediate their effect through suppression of phosphorylation and degradation of I␬B␣. Resveratrol has been shown to block the phosphorylation and the degradation of I␬B␣ (33). We have shown that resveratrol blocks the phosphorylation of p65 required for its transactivation function (32). Reports of another study by Kim et al. (53) revealed aberrant expression of NF-␬Bin mammary tumors induced by DMBA treatment of Sprague Dawley rats compared with normal mammary gland of age-matched, vehicle- treated control animals. Moreover, they reported that NF-␬B/Rel activation occurs before malignant transformation and is not present normally in the mammary gland, suggestive of a significant association between activation of NF-␬B expression and the progression of epithelial cells to a malignant phenotype. Moreover, aberrant expression of NF-␬B in human breast cancer specimens has also been reported (34). Thus, our previous findings that resveratrol mediated down-regulation of NF-␬B factor indicate that resveratrol may attenuate the early critical steps involved in carcinogen-driven transformation of mammary epithelial cells, including dysregulation of normal control of proliferation and protection from apoptosis. Antioxidants such as N-acetylcysteine and pentoxifylline, which are already in clinical use, repress NF-␬B activity and concurrently exhibit significant inhibitory effects on proliferation of breast cancer cells in culture (54, 55). Resveratrol has been reported recently to mediate phosphorylation of p53 at serine 15 through extracellular signal-regulated kinase and p38 kinase activities, leading to induction of apoptosis (56). The observed dose-dependent effect of resveratrol on the proliferation of MCF-7 cells noted in our present study implies that resveratrol-mediated chemoprotection may occur through the up-regulation of apoptosis. Several in vitro studies have documented an antiproliferative effect of resveratrol on many cell types (57–63), including those derived from the human breast (14, 26). Resveratrol

Fig. 7. A, resveratrol suppresses TNF-induced NF-␬B activation in human breast has been shown to inhibit the transition of cells from the S-to-G2 MCF-7 cells. A total of 1 ϫ 106 cells/ml were preincubated at 37°C for 4 h with different phase of the cell cycle (15–18, 59, 60). Our results are in agreement concentrations 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-␬B with these reports. as described in “Materials and Methods.” B, supershift and specificity of NF-␬B activa- It is now well established that once breast cancer initiation has tion. Nuclear extracts were prepared from untreated or TNF (0.1 nM)-treated MCF-7 cells taken place, estrogen promotes the growth of transformed cells, lead- (1 ϫ 106 cells/ml), incubated for 30 min with different antibodies as indicated, preimmune sera, unlabeled wild-type oligo or mutant oligo, and then assayed for NF-␬B, as described ing to the development of detectable breast cancer. Resveratrol be- in “Materials and Methods.” haves like the partial estrogen receptor agonist tamoxifen, which blocks the action of estrogen in the breast and effectively prevents primary and recurring breast tumor development (64). Another rele- of carcinogen sensitivity. The mammary gland of the rat undergoes vant important mechanism of action of many chemopreventive agents extensive development after 32–35 days of age, first appearing as is through their ability to modulate the xenobiotic-metabolizing en- terminal end buds that subsequently evolve into alveolar buds and zymes, e.g., by inhibiting metabolic activation of a procarcinogen or eventually into terminal ducts; this occurs at 40–60 days of age. by increasing detoxification of reactive metabolites. In polycyclic Under normal conditions, DMBA is most effective in producing aromatic hydrocarbon tumorigenicity, oxidative phase I biotransfor- tumors during the most active transition period of terminal end bud mation results in highly reactive diolepoxides that form covalent evolution into alveolar buds. The greater incidence and tumor yield in adducts with DNA. Reports indicate that the level of polycyclic the DMBA-treated animals occurred because transition in the mam- aromatic hydrocarbon-DNA adducts is related to the level of CYPIAI mary tissue was occurring in the normal window of DMBA sensitiv- expression (65). Interestingly, resveratrol has been reported to inhibit ity. Such a mechanistic proposition has been put forth by Anderson et constitutive and inducible expression of oxidative phase I biotrans- al. (50), who studied the effect of constant light on DMBA-induced formation-related CYP1A1 in human bronchial epithelial and breast mammary tumorigenesis in rats. Neonatal genistein treatment exerts cancer cells (23, 24, 66). Furthermore, Jang et al. (8) have shown that its chemopreventive action by directly enhancing maturation of the resveratrol induces quinone reductase activity, a phase II enzyme, in terminal ductal structures and by altering the endocrine system to cultured mouse hepatoma cells. Thus, one may also interpret the reduce cell proliferation in the mammary gland (51, 52). observed chemopreventive action of resveratrol primarily at the level Several other related mechanisms relevant to the observed chemo- of inhibition of procarcinogen activation, leading to reduced bioacti- 4951

Fig. 8. A, resveratrol suppresses the proliferation of human breast MCF-7 cells. Cells (5 ϫ 103) in 0.1 ml of culture medium were plated on 96-well plates and incubated at 37°C. After 24 h, cells were grown in either 200 ␮l of fresh medium (control) or 200 ␮l of fresh medium containing 2-fold serial dilutions of resveratrol. Cells were incubated for 72 h, and viable cells at the end of incubation were determined by the MTT method as described in “Materials and Methods.” B, resveratrol suppresses DNA synthesis of human breast MCF-7 cells. Cells (5 ϫ 103) in 0.1 ml of medium were plated in 96-well plates and incubated overnight at 37°C. After 24 h, cells were replenished with either 200 ␮l of fresh medium (control) or 200 ␮l of fresh medium containing 2-fold serial dilutions of resveratrol. After 72 h of incubation, 0.1 ␮Ci of [3H]thymidine was added to each well during the last6hofincubation and harvested as described in “Materials and Methods.” Pro- liferation was determined by [3H]thymidine incorporation into the cells at each time point. All determinations were made in triplicate. C, resveratrol inhibits the proliferation of MCF-7 cells. Cells (2 ϫ 103) in 0.1 ml of culture medium were plated in 96-well plates. After 24 h, cells were allowed to grow in either 200 ␮lof fresh medium (control) or 200 ␮l of fresh medium containing 10 and 50 ␮M resveratrol. Cells were incubated for 0, 2, 4, and 6 days, and viable cells were determined by MTT assay as described in “Materials and Methods.” The variations between the triplicate are too small to be visible at each data point. D, resveratrol inhibits MCF-7 cells at S-G2-M phase of the cell cycle. Cells (2 ϫ 106) were plated in 6-well plates and then treated with resveratrol for different times. Thereafter, cells were harvested, fixed in ethanol, stained with propidium iodide, and analyzed by FACS analysis.

vated DMBA metabolites as well as increased expression of phase II normal tissue, no evidence of its expression was detectable by immu- detoxification enzymes. Several presumptive chemopreventive agents nohistochemistry performed on normal mammary tissue. However, reveal the potential to induce and enhance detoxification activities in resveratrol has been reported to inhibit COX-2 transcription and host target organs. Moreover, resveratrol-mediated down-regulation activity in phorbol ester-treated human mammary epithelial cells (20). of CYPIAI also implies the 2-hydroxylation of 17␤-estradiol and Qualitative immunohistochemical analysis for COX-2 revealed im- estradiol, which may further attenuate the effect of estrogen on the munoreactivity in fibroadenoma as well as in tumor specimens de- development of breast cancer. rived from resveratrol-pretreated animals. It can thus be inferred from Consistent with the hypothesis that COX-2 is not expressed in our findings that resveratrol-mediated down-regulation of COX-2 4952

may be effective in attenuating the early stages of carcinogenesis, thus 17. Hsieh, T. C., Juan, G., Darzynkiewicz, Z., and Wu, J. M. Resveratrol increases nitric WAF1/CIP1 reducing tumor incidence and multiplicity. COX-2 inhibitors demon- oxide synthase, induces accumulation of p53 and p21 , and suppresses cultured bovine pulmonary artery endothelial cell proliferation by perturbing progres-

strate strong chemoprotective potential. Besides COX-2, MMP-9 is sion through S and G2. Cancer Res., 59: 2596–2601, 1999. another gene regulated by activation of NF-␬B. Our results show 18. Hsieh, T. C., and Wu, J. M. Differential effects on growth, cell cycle arrest, and clearly that treatment of animals with DMBA also induced MMP-9, induction of apoptosis by resveratrol in human prostate cancer cell lines. Exp. Cell Res., 249: 109–115, 1999. and this induction was suppressed by treatment with resveratrol. Both 19. Surh, Y. J., Hurh, Y. J., Kang, J. Y., Lee, E., Kong, G., and Lee, S. J. Resveratrol, an COX-2 and MMP-9 have been implicated in tumor invasion and antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia metastasis. Whether suppression of tumor growth by resveratrol in the (HL-60) cells. Cancer Lett., 140: 1–10, 1999. 20. Subbaramaiah, K., Michaluart, P., Chung, W. J., Tanabe, T., Telang, N., and animals is attributable to inhibition of DMBA-induced NF-␬B acti- Dannenberg, A. J. Resveratrol inhibits cyclooxygenase-2 transcription in human vation is not clear. Because NF-␬B activation was suppressed in most mammary epithelial cells. Ann. N. Y. Acad. Sci., 889: 214–223, 1999. of the resveratrol-treated animals, it suggests that NF-␬B may play an 21. Garcia-Garcia, J., Micol, V., de Godos, A., and Gomez-Fernandez, J. C. The cancer chemopreventive agent resveratrol is incorporated into model membranes and inhibits important role. However, NF-␬B-independent mechanisms cannot be protein kinase C␣ activity. Arch. Biochem. Biophys., 372: 382–388, 1999. excluded based on our studies. 22. Fontecave, M., Lepoivre, M., Elleingand, E., Gerez, C., and Guittet, O. Resveratrol, In conclusion, the present study demonstrates that resveratrol in- a remarkable inhibitor of ribonucleotide reductase. FEBS Lett., 421: 277–279, 1998. 23. Ciolino, H. P., and Yeh, G. C. Inhibition of aryl hydrocarbon-induced cytochrome hibits rat mammary tumor development and illustrates an emerging P-450 1A1 enzyme activity and CYP1A1 expression by resveratrol. Mol. Pharmacol., concept of chemoprevention through inhibition of transcription factor 56: 760–767, 1999. NF-␬B. In addition, resveratrol functions as an inhibitor of breast 24. Ciolino, H. P., Daschner, P. J., and Yeh, G. C. Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor. Cancer cancer cells in vitro mediated through modulation of cell proliferation Res., 58: 5707–5712, 1998. and apoptosis. There is no study reporting on the pharmacokinetics of 25. Tsai, S. H., Lin-Shiau, S. Y., and Lin, J. K. Suppression of nitric oxide synthase and ␬ resveratrol metabolism in humans subjects, and thus additional studies the down-regulation of the activation of NF- B in macrophages by resveratrol. Br. J. Pharmacol., 126: 673–680, 1999. are warranted to determine the optimum effective dose of this phyto- 26. Lu, R., and Serrero, G. Resveratrol, a natural product derived from grape, exhibits chemical compound in inhibiting cancers in humans. antiestrogenic activity and inhibits the growth of human breast cancer cells. J. Cell. Physiol. 179: 297–304, 1999. 27. de Martin, R., Schmid, J. A., and Hofer-Warbinek, R. The NF-␬B/Rel family of transcription factors in oncogenic transformation and apoptosis. Mutat. Res., 437: ACKNOWLEDGMENTS 231–243, 1999. 28. Gilmore, T. D., Koedood, M., Piffat, K. A., and White, D. W. Rel/NF-␬B/I␬B This research was conducted by The Clayton Foundation for Research. We proteins and cancer. Oncogene, 13: 1367–1378, 1996. 29. Jang, M., and Pezzuto, J. M. Effects of resveratrol on 12-O-tetradecanoylphorbol- thank Walter Pagel for critical reading of the manuscript. 13-acetate-induced oxidative events and gene expression in mouse skin. Cancer Lett., 134: 81–89, 1998. 30. Bellas, R. E., Lee, J. S., and Sonenshein, G. E. Expression of a constitutive NF-␬B- REFERENCES like activity is essential for proliferation of cultured bovine vascular smooth muscle cells. J. Clin. Investig., 96: 2521–2527, 1995. 1. Parker, S. L., Tong, T., Bolden, S., and Wingo, P. A. Cancer statistics, 1997. CA 31. Taub, R. Liver regeneration. IV. Transcriptional control of liver regeneration. FASEB Cancer J. Clin., 47: 5–27, 1977. J., 10: 413–427, 1996. 2. Food, Nutrition and the Prevention of Cancer: A Global Perspective. Washington, 32. Manna, S. K., Mukhopadhyay, A., and Aggarwal, B. B. Resveratrol suppresses DC: World Cancer Research Fund/American Institute for Cancer Research, 1997. TNF-induced activation of nuclear transcription factors NF-␬B, activator protein-1, 3. Benner, S. E., and Hong, W. K. Clinical chemoprevention: developing a cancer and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. prevention strategy. J. Natl. Cancer Inst. (Bethesda), 85: 1446–1447, 1993. J. Immunol., 164: 6509–6519, 2000. 4. Waladkhani, A. R., and Clemens, M. R. Effect of dietary phytochemicals on cancer 33. Holmes-McNary, M., and Baldwin, A. S., Jr. Chemopreventive properties of trans- development. Bioorg. Med. Chem. Lett., 1: 747–753, 1998. resveratrol are associated with inhibition of activation of the I␬B kinase. Cancer Res., 5. Kelloff, G. J., Crowell, J. A., Steele, V. E., Lubet, R. A., Malone, W. A., Boone, 60: 3477–3483, 2000. C. W., Kopelovich, L., Hawk, E. T., Lieberman, R., Lawrence, J. A., Ali, I., Viner, 34. Sovak, M. A., Bellas, R. E., Kim, D. W., Zanieski, G. J., Rogers, A. E., Traish, A. M., J. L., and Sigman, C. C. Progress in cancer chemoprevention: development of and Sonenshein, G. E. Aberrant nuclear factor-␬B/Rel expression and the pathogen- diet-derived chemopreventive agents. J. Nutr., 13(Suppl. 2S): 467S–471S, 2000. esis of breast cancer. J. Clin. Investig., 100: 2952–2960, 1997. 6. Kopp, P. Resveratrol, a phytoestrogen found in red wine. A possible explanation for 35. Bradford, M. M. A rapid and sensitive method for the quantitation of micrograms of the conundrum of the “French paradox”? Eur. J. Endocrinol., 138: 619–620, 1998. protein utilizing the principle of protein dye binding. Anal. Biochem., 72: 248–254, 7. Jang, M., and Pezzuto, J. M. Cancer chemopreventive activity of resveratrol. Drugs 1976. Exp. Clin. Res., 25: 65–77, 1999. 36. Chaturvedi, M. M., Mukhopadhyay, A., and Aggarwal, B. B. Assay for redox- 8. Jang, M., Cai, L., Udeani, G. O., Slowing, K. V., Thomas, C. F., Beecher, C. W., sensitive transcription factor. Methods Enzymol., 319: 585–602, 2000. Fong, H. H., Farnsworth, N. R., Kinghorn, A. D., Mehta, R. G., Moon, R. C., and 37. Singh, S., and Aggarwal, B. B. Activation of transcription factor NF-␬B is suppressed Pezzuto, J. M. Cancer chemopreventive activity of resveratrol, a natural product by curcumin (diferuloylmethane). J. Biol. Chem., 270: 24995–5000, 1995. derived from grapes. Science (Wash. DC), 275: 218–220, 1997. 38. Manna, S. K., Mukhopadhyay, A., Van, N. T., and Aggarwal, B. B. Silymarin 9. Frankel, E. N., Waterhouse, A. L., and Kinsella, J. E. Inhibition of human LDL suppresses TNF-induced activation of NF-␬B, c-Jun N-terminal kinase, and apopto- oxidation by resveratrol. Lancet, 341: 1103–1104, 1993. sis. J. Immunol., 163: 6800–6809, 1999. 10. Fauconneau, B., Waffo-Teguo, P., Huguet, F., Barrier, L., Decendit, A., and Merillon, 39. Natarajan, K., Singh, S., Burke, T. R., Jr., Grunberger, D., and Aggarwal, B. B. J. M. Comparative study of radical scavenger and antioxidant properties of phenolic Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear compounds from Vitis vinifera cell cultures using in vitro tests. Life Sci., 61: transcription factor NF-␬B. Proc. Natl. Acad. Sci. USA, 93: 9090–9095, 1996. 2103–2110, 1997. 40. Yamamoto, Y., Yin, M. J., Lin, K. M., and Gaynor, R. B. Sulindac inhibits activation 11. Pace-Asciak, C. R., Rounova, O., Hahn, S. E., Diamandis, E. P., and Goldberg, D. M. of the NF-␬B pathway. J. Biol. Chem., 274: 27307–27314, 1999. Wines and grape juices as modulators of platelet aggregation in healthy human subjects. Clin. Chim. Acta, 246: 163–182, 1996. 41. Kelloff, G. J. Perspectives on cancer chemoprevention research and drug develop- 12. Tessitore, L., Davit, A., Sarotto, I., and Caderni, G. Resveratrol depresses the growth ment. Adv. Cancer Res., 78: 199–334, 2000. of colorectal aberrant crypt foci by affecting bax and p21 (CIP) expression. Carcino- 42. Chainy, G. B., Manna,. S. K., Chaturvedi, M. M., and Aggarwal, B. B. Anethole genesis (Lond.), 21: 1619–1622, 2000. blocks both early and late cellular responses transduced by tumor necrosis factor: ␬ 13. Carbo, N., Costelli, P., Baccino, F. M., Lopez-Soriano, F. J., and Argiles, J. M. effect on NF- B, AP-1, JNK, MAPKK and apoptosis. Oncogene, 19: 2943–2950, Resveratrol, a natural product present in wine, decreases tumour growth in a rat tumor 2000. model. Biochem. Biophys. Res. Commun., 254: 739–743, 1999. 43. Manna, S. K., Gad, Y. P., Mukhopadhyay, A., and Aggarwal, B. B. Suppression of 14. Mgbonyebi, O. P., Russo, J., and Russo, I. H. Antiproliferative effect of synthetic tumor necrosis factor-activated nuclear transcription factor-␬B, activator protein-1, resveratrol on human breast epithelial cells. Int. J. Oncol., 12: 865–869, 1998. c-Jun N-terminal kinase, and apoptosis by ␤-lapachone. Biochem. Pharmacol., 57: 15. Ragione, F. D., Cucciolla, V., Borriello, A., Pietra, V. D., Racioppi, L., Soldati, G., 763–764, 1999. Manna, C., Galletti, P., and Zappia, V. Resveratrol arrests the cell division cycle at 44. Ahmad, N., Gupta, S., and Mukhtar, H. Green tea polyphenol epigallocatechin-3- ␬ S/G2 phase transition. Biochem. Biophys. Res. Commun., 250: 53–58, 1998. gallate differentially modulates nuclear factor B in cancer cells versus normal cells. 16. Hsieh, T. C., Burfeind, P., Laud, K., Backer, J. M., Traganos, F., Darzynkiewicz, Z., Arch. Biochem. Biophys., 376: 338–346, 2000. and Wu, J. M. Cell cycle effects and control of gene expression by resveratrol in 45. Sharma, R. A., Manson, M. M., Gescher, A., and Steward, W. P. Colorectal cancer human breast carcinoma cell lines with different metastatic potentials. Int. J. Oncol., chemoprevention: biochemical targets and clinical development of promising agents. 15: 245–252, 1999. Eur. J. Cancer, 37: 12–22, 2001. 4953

46. Gusman, J., Malonne, H., and Atassi, G. A reappraisal of the potential chemopre- 57. Holian, O., Wahid, S., Atten, M. J., and Attar, B. M. Inhibition of gastric cancer cell ventive and chemotherapeutic properties of resveratrol. Carcinogenesis (Lond.), 22: proliferation by resveratrol: role of nitric oxide. Am. J. Physiol. Gastrointest. Liver 1111–1117, 2001. Physiol., 82: G809ϪG816, 2002. 47. Hilakivi-Clarke, L. Estrogens, BRCA1, and breast cancer. Cancer Res., 60: 4993– 58. Holian, O., and Walter, R. J. Resveratrol inhibits the proliferation of normal human 5001, 2000. keratinocytes in vitro. J. Cell. Biochem., 36 (Suppl.): 55–62, 2001. 48. Kimura, J., Hasegawa, R., Yaono, M., Kato, T., Wakabayashi, K., Sugimura, T., Ito, 59. Kuwajerwala, N., Cifuentes, E., Gautam, S., Menon, M., Barrack, E. R., and Reddy, N., and Shirai, T. Inhibitory potential of pregnancy and lactation on mammary G. P. Resveratrol induces prostate cancer cell entry into S phase and inhibits DNA carcinogenesis induced by a food carcinogen, 2-amino-1-methyl-6-phenylimi- synthesis. Cancer Res., 62: 2488–2492, 2002. dazo[4,5-b]pyridine, in Sprague Dawley rats. Cancer Lett., 101: 73–78, 1996. 60. Park, J. W., Choi, Y. J., Jang, M. A., Lee, Y. S., Jun, D. Y., Suh, S. I., Baek, W. K., 49. Mizutani, K., Ikeda, K., Kawai, Y., and Yamamori, Y. Resveratrol stimulates the Suh, M. H., Jin, I. N., and Kwon, T. K. Chemopreventive agent resveratrol, a natural proliferation and differentiation of osteoblastic MC3T3–E1 cells. Biochem. Biophys. product derived from grapes, reversibly inhibits progression through S and G2 phases Res. Commun., 253: 859–863, 1998. of the cell cycle in U937 cells. Cancer Lett., 163: 43–49, 2001. 50. Anderson, L. E., Morris, J. E., Sasser, L. B., and Stevens, R. G. Effect of constant 61. Moreno, J. J. Resveratrol modulates arachidonic acid release, prostaglandin synthesis, light on DMBA mammary tumorigenesis in rats. Cancer Lett., 148: 121–126, 2000. and 3T6 fibroblast growth. J. Pharmacol. Exp. Ther., 294: 333–338, 2000. 51. Lamartiniere, C. A., Moore, J. B., Brown, N. M., Thompson, R., Hardin, M. J., and 62. Adhami, V. M., Afaq, F., and Ahmad, N. Involvement of the retinoblastoma (pRb)- Barnes, S. Genistein suppresses mammary cancer in rats. Carcinogenesis (Lond.), 16: E2F/DP pathway during antiproliferative effects of resveratrol in human epidermoid 2833–2840, 1995. carcinoma (A431) cells. Biochem. Biophys. Res. Commun., 288: 579–585, 2001. 52. Lamartiniere, C. A., Moore, J., Holland, M., and Barnes, S. Neonatal genistein chemoprevents mammary cancer. Exp. Biol. Med., 208: 120–123, 1995. 63. Bhat, K. P., and Pezzuto, J. M. Resveratrol exhibits cytostatic and antiestrogenic 53. Kim, D. W., Sovak, M. A., Anieski, G., Nonet, G., Romieu-Mourez, R., Lau, A. W., properties with human endometrial adenocarcinoma (Ishikawa) cells. Cancer Res., Hafer, L. J., Yaswen, P., Stampfer, M., Rogers, A. E., Russo, J., and Sonenshein, 61: 6137–6144, 2001. G. E. Activation of NF-B/Rel occurs early during neoplastic transformation of 64. Gelber, R. D., Cole, B. F., Goldhirsch, A., Rose, C., Fisher, B., Osborne, C. K., mammary cells. Carcinogenesis (Lond.), 21: 871–879, 2000. Boccardo, F., Gray, R., Gordon, N. H., Bengtsson, N. O., and Sevelda, P. Adjuvant 54. Biswas, D. K., Dezube, B. J., Ahlers, C. M., and Pardee, A. B. Pentoxifylline inhibits chemotherapy plus tamoxifen compared with tamoxifen alone for postmenopausal HIV-1 LTR-driven gene expression by blocking NF-␬B action. J. Acquired Immune breast cancer: meta-analysis of quality-adjusted survival. Lancet, 347: 1066–1071, Defic. Syndr., 6: 778–786, 1993. 1996. 55. Staal, J., Roederer, M., Herzenberg, L. A., and Herzenberg, L. A. Intracellular thiols 65. Mollerup, S., Ryberg, D., Hewer, A., Phillips, D. H., and Haugen, A. Sex differences regulate activation of nuclear factor ␬B and transcription of human immunodeficiency in lung CYP1A1 expression and DNA adduct levels among lung cancer patients. virus. Proc. Natl. Acad. Sci. USA, 87: 9943–9947, 1990. Cancer Res., 59: 3317–3320, 1999. 56. Qing-Bai, S., Bode, A. M., Ma, W. Y., Chen, N. Y., and Dong, Z. Resveratrol- 66. Mollerup, S., Ovrebo, S., and Haugen, A. Lung carcinogenesis: resveratrol modulates induced activation of p53 and apoptosis is mediated by extracellular signal regulated the expression of genes involved in the metabolism of PAH in human bronchial protein kinases and p38 kinase. Cancer Res., 61: 1604–1610, 2001. epithelial cells. Int. J. Cancer, 92: 18–25, 2001.

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

Cancer Res 2002;62:4945-4954.

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