Abstract
2265
Aromatase is the enzyme responsible for the conversion of androgen to estrogen. It is expressed at higher levels in breast cancer tissues than normal breast tissues. The in situ produced estrogen, due to the over-expressed aromatase in breast cancer cells, is thought to play a critical role in stimulating cancer cell growth. The human aromatase gene contains nine translated exons (II-X) and at least seven untranslated exons I (I.1, I.2, I.3, I.4, I.5, I.6 and PII). Studies conducted in this and two other laboratories have revealed that exons I.3 and PII are the major exon-I’s in aromatase mRNA isolated from breast cancer, indicating that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer. Promoter I.4 is the major promoter used in breast stromal cells. Our group previously reported that procyanidin B dimers present in red wine/grape seed extract (GSE) are potent inhibitors of aromatase. Furthermore, procyanidin B dimers were found to reduce androgen-dependent tumor growth in an aromatase-transfected MCF-7 breast cancer xenograft model, suggesting that procyanidin B dimers suppress in situ estrogen formation (Cancer Res 63: 8516-8522, 2003). In this study, we have found that GSE suppresses not only aromatase activity, but also aromatase promoter activities and its expression. RT-PCR experiments showed that treatment with 60μg/ml of GSE suppressed the levels of exons I.3-, PII-, and I.6-containing mRNA in MCF-7 and SK-BR-3 cells. The levels of exon I.1- or exon I.5-containing mRNA, however, did not change upon GSE treatment. Transient transfection experiments with luciferase-aromatase promoters I.3/II or I.4 reporter vectors showed the suppression of the promoter activities 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 promoter I.3/II and I.4, respectively. The decreased levels of mRNA and protein of CREB-1 and GR were confirmed by real-time PCR and Western blotting experiments in MCF-7 and SK-BR-3 cells. Our results indicate that GSE can suppress estrogen production in breast cancer through two mechanisms. It inhibits the activity of aromatase. More interestingly, through suppression of the expression of CREB-1 and GR, GSE will selectively decrease the expression of aromatase expression in breast cancer tissue by reducing the activity of promoters I.3, II and I.4. Animal studies have been performed to demonstrate that GSE intake suppresses aromatase-expressing breast tumor growth in vivo. Based on these preclinical studies, a phase I/II clinical trial involving GSE has been initiated at our Institution (Sponsored by NIH grant CA44735 and California Breast Cancer Research Program CBCRP-10GB-0088).
[Proc Amer Assoc Cancer Res, Volume 47, 2006]