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1 City of Hope Graduate School of Biological Science and
2 Department of Surgical Research, Beckman Research Institute of the City of Hope, Duarte, California, and
3 Department of Medicine, Cedars-Sinai Medical Center-University of California at Los Angeles School of Medicine, Los Angeles, California
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ABSTRACT |
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INTRODUCTION |
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Many epidemiological studies have shown that a diet high in fruits and vegetables can reduce cancer incidence, including that of breast cancer (10, 11, 12) . Previous studies from our laboratory have found that grape juice and red wine contain chemicals that suppress estrogen biosynthesis in vitro as well as in vivo (13 , 14) . Grapes and red wine are a rich source of flavonoids known for their strong antioxidant properties. Over the past several years, multiple studies have looked at the nutritional and pharmacological benefits of wine and grape products. Moderate red wine consumption has also been linked to a decreased risk in cardiovascular disease (15 , 16) and to a reduction in cancer risk (17) . In the latter report, after adjusting for other sources of alcohol, wine intake among postmenopausal women was found to significantly affect the percentage of breast density, where white wine showed a positive association, and red wine showed an inverse association. Women with very dense breast tissue as determined by mammography have a risk of breast cancer that is 1.8–6 times that of women of the same age with normal density (18) . Additional studies have demonstrated that specific components isolated from wine are responsible for its chemopreventive activities (19 , 20) .
The association of alcohol consumption as a breast cancer risk factor has been established in numerous studies (21) . Our results on white wine suggest a weak inductive effect of alcohol on aromatase activity (22) . However, the inductive effect of alcohol was not seen when aromatase activity was suppressed by chemicals in red wine. The opposite action of alcohol and phytochemicals in red wine on aromatase activity may complicate the evaluation of the effect of wine drinking on breast cancer. Therefore, we have set our goals to separate the effect of alcohol from the action of phytochemicals in red wine and to identify the chemicals in red wine that have the ability to suppress estrogen biosynthesis. The antiaromatase action of alcohol-free red wine extract has been examined using a transgenic mouse model in which aromatase is overexpressed in the mammary tissues (14) . It was found that, similar to the treatment with the selective and potent aromatase inhibitor letrozole, the intake of the extract by gavage completely abrogated aromatase-induced hyperplasia as well as other changes in the mammary tissue. This was a direct demonstration of the chemopreventive effect of red wine chemicals against breast cancer by suppressing in situ estrogen formation.
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MATERIALS AND METHODS |
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The HPLC4 system used was a Beckman Gold System Programmable Solvent Module 126 with a Shimadzsu SPD-6A UV spectrophotometric detector set at 214 nm. The active wine extract was separated on a Discovery C18 column (5 µM, 25 cm x 4.6 mm; Supelco) using a shallow gradient at a 1 ml/min flow rate with solvent A (0.1% trifluoroacetic acid; Pierce) and solvent B [90% acetonitrile (Burdick and Jackson), 0.1% trifluoroacetic acid]: 5–10% B (5 min), 10–30% B (60 min), and 30–98% B (15 min).
MS.
The accurate mass-mass spectral analysis was performed using a Mariner time-of-flight mass spectrometer (PerSeptive Biosystems, Framingham, MA). Tandem MS of isolated HPLC peaks was performed on a LCQ quadropole ion trap mass spectrometer (Thermo-Finnigan, San Jose, CA) through ESI-MS.
In Vitro Aromatase Assay.
Aromatase activity was determined using the tritiated water release method developed by Thompson and Siiteri (23)
. The assay was performed using placental microsomes or microsomal fractions prepared from aromatase-expressing CHO cells. A detailed procedure was described previously (14)
. Briefly, the assay reaction mixture (500 µl) contained the substrate [1ß-3H(N)]androst-4-ene-3,17-dione (specific activity, 24.7 Ci/mmol; 100 nM), microsomal preparations (20 µg), progesterone (10 µM), BSA (0.1%), and potassium phosphate [67 mM (pH 7.4)]. Progesterone was required to suppress endogenous 5
-reductase in the cell homogenates that also consume the androgen substrate. After incubation for 10 min at room temperature, 50 µl of NADPH (12 mM) were added to the mixture and incubated in a 37°C water bath for 10 min. At the end of 10 min, the reaction was stopped by the addition of 500 µl of 5% trichloroacetic acid. After a 10-min centrifugation at 1000 x g, supernatants were removed to new glass tubes and mixed with an equal volume of chloroform to remove unreacted substrate. For the second extraction, the upper aqueous phase was transferred to microcentrifuge tubes containing a dextran-charcoal pellet. Charcoal mixtures were vortexed and subsequently pelleted by centrifugation at 15,000 x g for 5 min. For each sample, a 300-µl aliquot of the supernatant containing the tritiated water product was mixed with 3 ml of Scintisafe (Fisher’s Scientific, Tustin, CA) 30% liquid scintillation mixture and counted in a Tri-Carb Liquid Scintillation Analyzer 1600CA (Packard, Downers Grove, IL).
To determine the aromatase suppression activity of wine chemicals, the assay was performed in the presence of wine fractions at the indicated amounts. Inhibition kinetic analysis on wine chemicals was performed with various concentrations of the substrate androstenedione at 20, 40, 60, 100, and 200 nM.
In Vivo Experiments.
In the experiments using intact animals, 5–6-week-old female BALB/c-nu-nu, athymic, nonovariectomized mice were purchased (Charles River Laboratories). At about 8 weeks of age, mice received s.c. implant with 5 mg/60 day time-release androstenedione pellets (Innovative Research of America, Sarosota, FL). A week later, mice were individually gavage fed (p.o. force-fed) with 100 µl of water control or 25, 50, or 100 µl of a 1x concentrated wine polyamide extract (in water). Each animal received daily gavage treatment for 42 consecutive days. At 10 weeks old, mice were given two s.c. injections of MCF-7aro cells. These cells were grown in MEM with nonessential amino acids, sodium pyruvate, and Earle’s salts in 10% FCS. The MCF-7aro cells were harvested and resuspended in an equal volume of Matrigel (BD Biosciences) to a final concentration of 1 x 107 cells/ml. Body weights were monitored weekly as an indicator of the overall health of the animals. At the end of 6 weeks of gavage treatment, mice were euthanized, blood samples were collected, and tumors and ovaries-uteri were removed, weighed, and sent for H&E histological staining through the City of Hope Pathology Department Core Facility.
Estrogen concentrations were determined from mouse sera. Mouse blood was obtained through cardiac puncture and immediately combined with heparin. Whole blood was separated to serum and plasma in a tabletop centrifuge. Sera were frozen until later evaluation by radioimmunoassay for estrogen and estrone levels.
For the animal experiments using ovariectomized animals, athymic nude ovariectomized female mice were used (Charles River Laboratories). Mice also received the same s.c. androstenedione 60 day time-release pellet implants as described in the previous experiment. Control animals were given androstenedione pellet and gavage fed 100 µl of sterile water. Treatment groups were given androstenedione pellets and either 1x or 3x concentrate of the Pinot Noir polyamide 70% methanol fraction in a 100-µl volume. All other parameters (i.e., number of cells injected, duration of experiment, and experimental analyses) were identical to the animal experiment using intact mice described previously. For the animal experiments using MCF-7 cells (the cells without aromatase), MCF-7 cells and estrogen pellets, instead of androgen pellets, were used. Other conditions were identical to those described for the experiments using MCF-7aro cells.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. The most effective inhibition curve was observed with HPLC peak 1, the IC50 of which was between 15 and 20 µM (calculated using Mr 579; discussed further below). The reconstituted mixture was significantly more effective than any single chemical (see Fig. 1
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. For HPLC peak 1 chemical, the elemental analysis gave the most likely chemical formula as C30H27O12 with an 89.1% isotope match score. In practice, the mass of a compound measured with an error of <10 ppm from the theoretical mass is considered an exact mass. Therefore, HPLC peak 1 exhibited a mass (M+) of 579 and an elemental formula of C30H27O12, which confirmed that HPLC peak 1 is a procyanidin B dimer.
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The HPLC fractions were also analyzed by ESI-MS with collision-induced dissociation. Tandem fragmentation and ionization of the parent ion can help elucidate chemical and structural features of the molecule. As expected, HPLC peaks 2, 3, and 6 had identical tandem MS profiles up to MS (3)
as did procyanidin B1, B3, and B2. HPLC peaks 1 and 5 had a different fragmentation pattern from HPLC peaks 2, 3, and 6, as seen by their MS (2)
fragment ion of 301 compared with MS (2)
fragment ion of 427. Perhaps there is no noticeable difference among dimers B1, B2, or B3 because they all belong to the same subgroup of procyanidin dimers with the 4-8 linkage. Thus, HPLC peaks 1 and 5 could be procyanidin B dimers with the 4-6 linkage. It would be ideal to analyze the subgroup of authentic procyanidin B dimers with the 4-6 linkage, which could confirm the nature of HPLC peak 1. However, no compounds from the 4-6 linkage subgroup are commercially available. Thus, using mass spectral analyses, we have determined that the major chemicals in the 60–80% methanol-eluted polyamide fraction are isomers of procyanidin B dimer, and HPLC peak 1 is probably a procyanidin B dimer with 4-6 linkage (see Fig. 3
). Finally, NMR analysis was carried out and confirmed that peak 1 is a procyanidin B dimer, but it was not able to determine which isomer.
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. A Ki value of 6 µM for HPLC peak 1 was determined from the secondary plot (1/v versus [I]). Therefore, this procyanidin B dimer inhibits aromatase/estrogen biosynthesis in a specific manner with a Ki value similar to the known aromatase inhibitor, aminoglutethimide (24)
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. Mutant D309A produced the most dramatic effect by completely eliminating the ability of HPLC peak 1 dimer to inhibit aromatase. Two other mutants, S478T and H480Q, also proved to be significant in reversing the inhibitory action of HPLC peak 1. Interestingly, the T310S mutant produced a more effective dose response after treatment with HPLC peak 1 procyanidin B dimer when compared with the wild-type aromatase. These results support the finding from enzyme kinetic analysis that this compound binds to the active site of aromatase, and they indicate that Asp-309, Thr-310, Ser-478, and His-480 are involved in the interaction with the peak 1 procyanidin B dimer.
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Serum from each mouse was also collected and analyzed for estradiol and estrone concentrations. Mice treated with increasing concentrations of wine extract showed a decreasing trend in the levels of estradiol and estrone compared with the control. The average estrogen concentration values for wine extract-treated mice did show a clear difference when compared with mice treated with water. Unfortunately, the SD values were too large to determine any statistical significance. The large SD values may be because of the intact ovaries in the mice that contribute to the variation in circulating estrogen levels. Over the course of the 2-month experiment, the weights of animals in each group were recorded where all groups had similar body weight.
To eliminate the effect of estrogens generated endogenously by the ovary, in the second type of animal experiments, athymic nude-ovariectomized female mice were used. Additionally, a more concentrated polyamide wine extract was used in these experiments; i.e., at a 100-µl dose each, a 1x or 3x concentrated extract was used as compared with just a 1x concentrate used at different volumes (25, 50, and 100 µl). All mice received a 5 mg/60-day release androstenedione pellet that was implanted s.c. For 6 weeks, mice were gavage fed daily with either 100 µl of 1x or 3x concentrated 70M wine extract or water. After the first week of gavage treatment, each mouse was given a 0.2-ml s.c. injection into each hind flank containing MCF-7aro cells suspended in equal volume Matrigel to a final concentration of 1 x 107 cells/ml. At the completion of 6 weeks of gavage treatment, mice were sacrificed, and blood, tumor, and uteri were removed, weighed, and sent out for histological evaluation. Mice treated with 1x and 3x concentrated 70M wine extract showed a significant reduction (P < 0.01) in tumor size compared with the androstenedione control mice fed with water (15.7 ± 16.2 and 9.9 ± 20.2 versus 35.4 ± 25.2 mg, respectively; Fig. 6
). A more detailed analysis of individual mice per treatment group is summarized in Table 1
. The positive control group And+/H2O was the group that had the highest percentage (62.5%) of mice with tumor growth on each flank. Fifty percent of the 1x concentrate-treated group had growth of two tumors. Whereas the group with the lowest number of mice having two tumors was the mice treated with the 3x concentrate, the 3x concentrate-treated mice had the most dramatic suppression of tumor growth, as indicated by the high percentage (62.5%) of animals that had no MCF-7aro growth at either injection site. Interestingly, only 12.5% of the 1x concentrate-treated mice had complete tumor suppression, whereas 37.5% of the mice had just one tumor with an average tumor weight of 22.7 mg. To evaluate whether these treatments caused any deleterious effects on other endocrine glands, the uteri were removed and weighed. The wet uterine weights of all wine extract-treated mice were not found to be statistically different from the uteri of androstenedione control mice. Blood samples were taken, and serum was analyzed for the circulating estrogen. The concentrations of circulating estrogen in ovariectomized mice were found to be significantly lower than those found in intact animals (with ovary). Furthermore, the results suggested only a slight trend in reduced blood estrogen concentrations after treatment with increasing concentrations of wine extract. The results from uterine weight measurements and circulating estrogen levels support the hypothesis that in situ-produced estrogen plays a larger role in aromatase expressing and ER-positive breast tumor growth than circulating estrogen.
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These mice tumors were further evaluated using immunohistochemistry with M30 CytoDEATH mouse monoclonal antibodies that bind to an early apoptotic marker, the caspase-cleaved product of human CK18 cytoskeletal protein. The M30 CytoDEATH antibody recognizes a specific caspase-cleavage site in CK18 that is not detected in native CK18 of normal cells. The tumors of mice treated with wine extract showed a slight increase in the number of apoptotic cells when compared with tumors of control mice; however, the difference did not appear to be statistically significant. These results suggest that the observed reduction in tumor growth in the wine extract-treated mice was attributable to the inhibition of aromatase and not because of a nonspecific cytotoxic effect.
We have also examined the effect of polyamide wine extract in a MCF-7 breast cancer xenograft model. In these in vivo studies, athymic nude-ovariectomized female mice were given two s.c. injections of MCF-7 instead of MCF-7aro cells, and the tumor formation was induced using 5 mg/60-day release 17ß-estradiol pellet (implanted s.c.). Other experimental conditions were identical to those described for the experiments using MCF-7aro cells. MCF-7 cells have minimal aromatase activity. We found that 3x concentrated 70M wine extract fed daily at 100 µl was not able to suppress the growth of MCF-7 tumors (P = 0.286). The average tumor weights for the estrogen control and 3x concentrated extract-treated mice were 44.2 ± 60.3 versus 52.4 ± 45.9 mg, respectively. The weights of the tumors from mice without estrogen pellet were 21.0 ± 15.4 mg. These results are very crucial for confirming the antiaromatase activity of procyanidin B dimers in red wine.
Although our studies have demonstrated a highly specific inhibitory activity of procyanidin B dimers against aromatase/estrogen biosynthesis, these chemicals are thought to exhibit additional activities in cells. For example, it has been shown by Agarwal et al. (26) that a GSP largely composed of procyanidins could irreversibly inhibit breast carcinoma MDA-MB468 cells by reducing constitutive extracellular signal-regulated kinase 1/2 and p38 activation. Additionally, GSP arrested MDA-MB468 cells at G1 as seen by the increase of Cip1/p21 levels and the decrease of CDK4 and cyclin D1 G1-phase regulators. Similarly, this same group evaluated the GSP on human prostate cancer DU145 cells. This fraction caused a decrease in extracellular signal-regulated kinase 1, an increase in Cip1/p21, and a decrease in CDK2, CDK4, and cyclin E that also led to G1 cell cycle arrest (27) . Previously, this group examined the anti-tumor-promoting activity of the same GSP in a 12-O-tetradecanoylphorbol-13-acetate-promoted SENCAR mouse skin two-stage initiation-promotion model. The grape seed fraction reduced tumor occurrence and tumor volume (28) . Others have also studied grape seed proanthocyanidin extract and shown that it inhibited growth of MCF-7 human breast cancer, A-427 human lung cancer, and CRL 1730 human gastric adenocarcinoma cells in a concentration- and dose-dependent manner (29) . The GSP contains procyanidin B dimers (28) . These published results suggest that grape seed extract may modulate cell proliferation, but it has not yet been determined whether procyanidin B dimers are directly responsible for the reported cell growth suppression activity of grape seed extract. Because our procyanidin B preparation did not suppress the growth of MCF-7 tumors in nude mice, the cytotoxic effect of the grape seed extract may result from different phytochemicals in the extract.
In summary, previous studies from this laboratory have shown that red wine contains phytochemicals that inhibit aromatase activity in vitro and suppress aromatase-mediated breast tumor formation in vivo. In this study, we have isolated a bioactive fraction from red wine. The chemicals in this fraction were identified to be procyanidin B dimers that were shown to be aromatase inhibitors. The most active procyanidin B dimer was found to inhibit aromatase with a Ki value of 6 µM, and the preparation of procyanidin B dimer mixture was found to be much more potent than any one of the individual procyanidin B dimers. Using a MCF-7aro tumor induction model, oral intake of the procyanidin B dimer mixture was found to be effective in suppressing aromatase-mediated tumor formation in vivo. On the other hand, the procyanidin B dimer mixture was not able to suppress the formation of MCF-7 tumors. These in vitro and in vivo studies demonstrated that procyanidin B dimers in red wine could be used as chemopreventive agents against breast cancer by suppressing in situ estrogen biosynthesis. Five ml of red wine can produce 100 µl of a 1x fraction. A typical conversion factor for determining mouse to human dosage of chemotherapeutic agents is 25, calculated based on body surface area (30) . Therefore, consumption of 125 ml of red wine/day would provide adequate amounts of procyanidin B dimers to suppress in situ aromatase in an average postmenopausal woman.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dudley Williams, Allergan Inc., 2525 Dupont Drive, Mail Stop: TL-2B, Irvine, California 92612.
Requests for reprints: Shiuan Chen, Department of Surgical Science, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010. Phone: (626) 359-8111, ext. 63454; Fax: (626) 301-8972; E-mail: schen{at}coh.org
4 The abbreviations used are: HPLC, high-performance liquid chromatography; CHO, Chinese hamster ovary; absmax, maximal absorbance; ppm, parts/million; ESI, electrospray; MS, mass spectrometry; NMR, nuclear magnetic resonance; ER, estrogen receptor; 70M, 70% methanol-water fraction; CK18, cytokeratin 18; GSP, grape seed polyphenolic fraction; CDK, cyclin-dependent kinase. 
Received 7/16/03. Revised 9/ 8/03. Accepted 9/22/03.
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; 5 µM HPLC peak 1,
; 15 µM HPLC peak 1,
. The inset graph is the secondary plot (1/v versus [I]) used to determine the Ki value for HPLC peak 1 procyandin dimer.
