Influence of Genistein and Daidzein on Brca1 Protein Levels in Human Breast Cell Lines

Phyto-estrogens potentially have anticarcinogenic biologic activities and could play a role in the dietary etiology of breast cancer. They have estrogenic as well as antiestrogenic properties and can also inhibit specific enzyme activities. In vitro and in vivo studies have shown that phyto-estrogens are tumor inhibitory. Moreover, Asian populations who consume large amounts of phytoestrogens derived from a soy-rich diet have a lower frequency of breast tumors than western populations who consume much lower quantities of these compounds (1). The BRCA1 gene is involved in breast cancer families with a pattern of autosomal-dominant inheritance of the disease (2). Comparison of BRCA1 messenger RNA levels in a series of normal breast tissues and sporadic breast cancer tissues demonstrated an apparent decrease in BRCA1 gene expression by tumor cells (3). Moreover, there is some evidence that BRCA1 gene transcription is directly and indirectly regulated by estrogen (4). We investigated whether two selected phyto-estrogens—genistein and daidzein—modulate the expression of Brca1 protein in human breast cells. The effects of these phyto-estrogens were compared in three human mammary cell lines, MCF7 cells (estrogen receptorpositive cells), MDA-MB231 cells (estrogen receptor-negative adenocarcinomaderived cells), and MCF10a cells (estrogen receptor-positive epithelial cells). Each cell line was treated for 96 hours at the IC50 of each phytoestrogen—genistein (5 mg) or daidzein (20 mg). (P = .05). Brca1 protein determinations following either genistein or daidzein treatment, total cell extracts were prepared from cells metabolically labeled with [35S]methionine. Levels of Brca1 protein were determined by use of a magnetic protein purification protocol (Dynabeads M-450 coated with Brca1 antibodies D-20; Santa Cruz Biotechnologies, Santa Cruz, CA). The beadswere incubated with the radiolabeled cell extracts, collected by use of a magnet, and washed three times with lysing solution. The Brca1 proteins were eluted with 0.5 M acetate buffer (pH 2.4). Brca1-specific immunoselected protein was quantified by liquid scintillation counting. The amount of Brca1 protein was expressed as a percentage, calculated as follows: 100 × (disintegrations per minute [dpm] Brca1 immunoselected proteintotal cell extract). The results of the Brca1 protein determination are shown in . Genistein had no effect on Brca1 protein expression in MCF7 cells (P 4 .35; P values are two-sided), MDA-MB231 cells (P 4 .20) or MCF10a cells (P 4 .35) when compared with untreated cells. Daidzein had no effect on Brca1 protein expression in MCF7 cells (P 4 .20), whereas Brca1 protein levels were increased in MDA-MB231 cells (P 4 .05). Brca1 protein levels were decreased in MCF10a cells (P 4 .05). Because daidzein increased Brca1 protein levels in estrogen receptornegative MDA-MB231 cells, the response was not mediated via the estrogen receptor. Isoflavones have biologic propertiesthat are quite separate from classic estrogen action and may influence carcinogenesis (5). Our results suggest that the lack of an effect by genistein on Brca1 protein expression may be explained by nonestrogenic biologic properties of isoflavones. Genistein apparently signals via a separate mechanism. For example, genistein augments signaling mechanisms mediated by transforming growth factor b, which inhibits progression of cells through the G1phase of the cell cycle (6). It is of interest that the BRCA1 gene is expressed in late G1phase (7). Further research on dietary phyto-estrogens should be undertaken by use of such approaches as a quantitative reverse transcriptasecoupled polymerase chain reaction assay to determine whether regulation of the BRCA1 gene at the level of messenger RNA is influenced by phytoestrogens. Fig. 1. Relative amount of Brca1 proteins expressed by human mammary cell lines (MCF7, MDAMB231, and MCF10a) following genistein (5 mg) and daidzein (20 mg) treatments. Each value corresponds to mean ± standard deviation of three assays for each group (two-sided P values by Wilcoxon rank-sum tests). * P 4 .05. Fig. 1. Relative amount of Brca1 proteins expressed by human mammary cell lines (MCF7, MDAMB231, and MCF10a) following genistein (5 mg) and daidzein (20 mg) treatments. Each value corresponds to mean ± standard deviation of three assays for each group (two-sided P values by Wilcoxon rank-sum tests). * P 4 .05.