Breast Cancer Prevention with Phytoestrogens in Grape Juice

Institution: Beckman Research Institute of the City of Hope
Investigator(s): Shiuan  Chen , Ph.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4PB-0115 Award: $539,331
Award Type: Request for Applications
Research Priorities
Prevention & Risk Reduction>Other searches for the causes

Initial Award Abstract (1998)
Grape juice has been found to suppress breast cancer cell growth by preventing the synthesis of the female hormone estrogen, which plays a major role in the development of breast cancer. Approximately 60% of premenopausal and 75% of postmenopausal patients have estrogen-dependent tumors. In estrogen-dependent breast tumors, estrogen stimulates the formation of growth factors that are essential for breast cancer growth. In cells, a protein called aromatase produces estrogen, and in breast cancer patients, some tumors contain an abnormally high level of aromatase, which generates a large amount of estrogen. Therefore, an abnormal expression of aromatase in breast is considered to be a risk factor for breast cancer.

During the previous year, it was found that grape juice stopped the estrogen production in cells in a test tube. Recent experiments using mice demonstrated that the tumors implanted in mice fed with a very small amount of grape juice daily for 5 weeks were one-third the size of those in similarly implanted mice not given grape juice. These results suggest the possibility that drinking grape juice may prevent breast cancer.

In this study, more extensive animal studies using mice and rats will be performed to critically evaluate these preliminary findings. In addition, experiments will be designed to identify the active components in grape juice that are responsible for the suppression of breast tumor formation. The latter research will provide us with a scientific basis as to why grape juice is capable of suppressing breast tumor formation in mice. Such information could be very important for designing more selective and effective breast cancer prevention strategies given the ready availability of grape juice.

Final Report (2003)
Identification of the active components in grape juice.
Separation of the components of grape juice were performed using high performance liquid chromatography (HPLC) and electronic chemical detection (ECD). However, we found that the sugar in grape juice caused problems in a large scale separation. We therefore used these separation techniques to extract components from red wine, and tested the effect of these wine-extracted components on aromatase activity. Our study has found that red wine extracts, but not white wine extracts, suppress aromatase (the enzyme that produces estrogen in tumor cells). A wine extract fraction was recently isolated from whole red wine that exhibited a potent inhibitory action on aromatase activity. Through UV absorbance analysis, HPLC profiling, accurate mass-mass spectrometry and nanospray tandem mass spectrometry, most of the compounds in our wine fraction were identified as compounds called procyanidin B dimers that were shown to be aromatase inhibitors. Grape seeds are known to contain high levels of procyanidin B dimers.

An analysis, using MCF-7aro breast cancer cell lines (and a technique called inhibition kinetic analysis), of one of the most potent procyanidin B dimer compounds, showed that it competes with the binding of one of the compounds in the aromatase activity pathway (the androgen substrate).

Animal experiments
The efficacy of these procyanidin B dimer compounds was then evaluated in an aromatase transfected MCF-7 breast cancer mouse model. The B dimer mixture was able to reduce androgen dependent tumor growth, indicating that procyanidin B dimers suppress in situ estrogen formation. This was done in collaboration with Dr. Rajeshwar Rao Tekmal at Emory University using a transgenic mouse model that over-expresses aromatase in the mammary tissues. Like the known specific aromatase inhibitor, Letrozole, the active B dimer fraction from red wine (administered by gavage) is a potent blocker of estrogen biosynthesis. When feed to the mice by gavage, it led not only to complete reduction and elimination of breast hyperplasia and other preneoplastic and neoplastic changes, but also affected other normal endocrine functions that are typical for estrogen deprivation.

These in vitro and in vivo studies demonstrated that procyanidin B dimers in red wine and grape seeds could be used as chemopreventive agents against breast cancer by suppressing in situ estrogen biosynthesis. We are very excited with our results. A manuscript is being prepared for publication.

Symposium Abstract (2005)
Suppression of aromatase/estrogen biosynthesis is thought to have potential for chemoprevention of breast cancer. Aromatase is the enzyme that converts androgen to estrogen. An abnormally high expression of aromatase in breast tissue is considered to be a risk factor for breast cancer. In our laboratory, we found that of seven fruit juices tested, grape juice was the most effective in inhibiting the activity of human placental aromatase activity.

During the last several years, we have learned that: (1) the methanol extract of grape juice and red wine suppresses aromatase in a dose-dependent manner; (2) the extract suppresses the proliferation of an aromatase over-expressing and estrogen receptor-positive breast cancer cell line MCF-7aro; (3) oral administration of the extract completely stops aromatase-induced hyperplasia and other changes in the mammary tissue of aromatase transgenic mice; (4) procyanidin B dimers, the major phytochemicals in the seeds and skins of grapes, are the chemicals responsible for the anti-aromatase activity; and (5) oral feeding of procyanidin B dimers reduces androgen-dependent MCF-7aro tumor growth in nude mice.

The tumors of mice treated with an extract enriched with procyanidin B dimers showed a minimal increase in apoptotic cells compared with tumors of control mice, suggesting that the reduction in tumor growth in the treated mice was due to inhibition of aromatase and not due to a non-specific cytotoxic effect. Further, results from the feeding experiments indicate that these procyanidin B dimers are orally active and maintain their activity after ingestion.

Recently, we have examined the content of procyanidin B dimers in commercially available grape seed extract formulations and determined that grape seed extract may be a useful chemopreventive agent against breast cancer. The mass spectral analysis has revealed that grape seed extract contains high levels of procyanidin B dimers, oligomers and gallate esters of procyanidins. Further testing in the nude mouse model revealed suppression of MCF-7aro tumor shrinkage when they were fed 700mg/day for 6 weeks. Based on this preclinical data, we hypothesize that postmenopausal women could reduce their breast cancer risk with a grape seed extract dietary supplement, containing phytochemicals that have been shown to be effective aromatase inhibitors. A Phase I chemoprevention trial to evaluate the anti-aromatase activity of grape seed extract in postmenopausal women has been initiated at the City of Hope. The design and progress of this clinical trial will be discussed.

Symposium Abstract (2007)
Aromatase is the enzyme that converts androgen to estrogen. It is expressed at higher levels in breast cancer tissues than normal breast tissues. Grape seed extract (GSE) contains high levels of procyanidin dimers that have been shown in our laboratory to be potent inhibitors of aromatase. In this study, GSE was found to inhibit aromatase activity in a dose-dependent manner and reduce androgen-dependent tumor growth in an aromatase-transfected MCF-7 (MCF-7aro) breast cancer xenograft model, agreeing with our previous findings.

We have also examined the effect of GSE on aromatase expression. RT-PCR experiments showed that treatment with 60 g/ml of GSE suppressed the levels of exons I.3-, PII-, and I.6-containing aromatase mRNA in MCF-7 and SK-BR-3 cells. The levels of exon I.1- containing mRNA, however, did not change with GSE treatment. Transient transfection experiments with luciferase-aromatase promoter I.3/II or I.4 reporter vectors showed the suppression of the promoter activity in a dose-dependent manner. The GSE treatment also led to the down-regulation of two transcription factors, cAMP-responsive element binding protein-1 (CREB-1) and glucocorticoid receptor (GR). CREB-1 and GR are known to up-regulate aromatase gene expression through promoters I.3/II and I.4, respectively. We believe that these results are exciting in that they demonstrate GSE to be potentially useful in the prevention/treatment of hormone-dependent breast cancer through the inhibition of aromatase activity as well as its expression.

Suppression of Estrogen Biosynthesis by Procyanidin Dimers in Red Wine and Grape Seeds
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Eng ET, Ye J, Williams D, Phung, S, et al
Yr: 2003 Vol: 63 Nbr: Abs: Pg:8516-8522

Prevention and treatment of breast cancer by suppressing aromatase activity and expression
Periodical:Annals of the New York Academy of Sciences
Index Medicus: Ann N Y Acad Sci
Authors: Chen S, Zhou D, Okubo T, Kao YC, Eng ET, Grube B, Kwon A, Yang C, Yu B
Yr: 2002 Vol: 963 Nbr: Abs: Pg:229-38

Suppression of aromatase (estrogen synthetase) by red wine phytochemicals.
Periodical:Breast Cancer Research and Treatment
Index Medicus: Breast Cancer Res Treat
Authors: ET Eng, D Williams, U Mandava, N Kirma, RR Tekmal, and S Chen.
Yr: 2001 Vol: 67 Nbr: 2 Abs: Pg:133-46

Grape seed extract is an aromatase inhibitor and a suppressor of aromatase expression.
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Kijima, I., Phung, S., Hur, G., Kwok, S.-L. and Chen, S.
Yr: 2006 Vol: 66 Nbr: Abs: Pg:5960-7

Resveratrol suppresses aromatase at the levels of enzyme activity and mRNA expression.
Periodical:Toxicological Sciences
Index Medicus: Toxicol Sci
Authors: Wang, Y., Chan, F. L., Chen, S. and Leung, L. K.
Yr: 2006 Vol: 92 Nbr: Abs: Pg:71-7