Preferential induction of cytochrome P450 1A1 over cytochrome P450 1B1 in human breast epithelial cells following exposure to quercetin

Abstract

Estrogen metabolism is suggested to play an important role in estrogen-induced breast carcinogenesis. Epidemiologic studies suggest that diets rich in phytoestrogens are associated with a reduced risk of breast cancer. Phytoestrogens are biologically active plant compounds that structurally mimic 17beta-estradiol (E(2)). We hypothesize that phytoestrogens, may provide protection against breast carcinogenesis by altering the expression of estrogen-metabolizing enzymes cytochrome P450 1A1 (Cyp1A1) and 1B1 (Cyp1B1). Cyp1A1 and Cyp1B1 are responsible for the metabolism of E(2) to generate 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)), respectively. Studies suggest that 2-OHE(2) and 2-methoxyestradiol may protect against breast carcinogenesis, while 4-OHE(2) is carcinogenic in rodent models. Thus, agents that increase the metabolism of E(2) by Cyp1A1 to produce 2-OHE(2) may have chemoprotective properties. The human immortalized non-neoplastic breast cell line MCF10F was treated with quercetin at 10 and 50muM concentrations for time points ranging from 3 to 48h. Total RNA and protein were isolated. Real-time PCR was used to measure the expression of Cyp1A1 and Cyp1B1 mRNA. Quercetin treatment produced differential regulation of Cyp1A1 and Cyp1B1 mRNA expression in a time- and dose-dependent manner. Treatment with 10 and 50 microM doses of quercetin produced 6- and 11-times greater inductions of Cyp1A1 mRNA over Cyp1B1 mRNA, respectively. Furthermore, quercetin dramatically increased Cyp1A1 protein levels and only slightly increased Cyp1B1 protein levels in MCF10F cells. Thus, our data suggest that phytoestrogens may provide protection against breast cancer by modulating expression of estrogen-metabolizing genes such that production of the highly carcinogenic estrogen metabolite 4-OHE(2) by Cyp1B1 is reduced and the production of the less genotoxic 2-OHE(2) by Cyp1A1 is increased.

Figure 1. Regulation of Cyp1A1 mRNA expression by quercetin

MCF10F human breast epithelial cells were treated with either vehicle (ethanol) or 10 and 50 µM concentrations of quercetin for 3 to 48 hours. Total RNA was isolated and reverse transcribed to generate cDNA. Real-time quantitative PCR was performed to analyze the expression of cyclophilin and Cyp1A1 mRNA. Expression of Cyp1A1 was determined by dividing the number of cDNA molecules of Cyp1A1 by the number of cDNA molecules of cyclophilin, a housekeeping gene. Fold change was determined by dividing the expression of Cyp1A1 in the quercetin-treated MCF10F cells by the expression of Cyp1A1 in the vehicle-treated cells. Each experiment was performed in quadruplicate and the data are expressed as fold change +/− standard deviation relative to control. A * indicates that Cyp1A1 expression is significantly different than Cyp1A1 expression in vehicle-treated cells, with a p value of less than 0.05.

Figure 2. Regulation of Cyp1B1 mRNA expression by quercetin

MCF10F human breast epithelial cells were treated with either vehicle or 10 and 50 µM concentrations of quercetin for 3 to 48 hours. Total RNA was isolated and reverse transcribed to generate cDNA. Real-time quantitative PCR was performed to analyze the expression of cyclophilin and Cyp1B1 mRNA. Expression of Cyp1B1 was determined by dividing the number of cDNA molecules of Cyp1B1 by the number of cDNA molecules of cyclophilin, a housekeeping gene. Fold change was determined by dividing the expression of Cyp1B1 in the quercetin-treated MCF10F cells by the expression of Cyp1B1 in the vehicle-treated cells. Each experiment was performed in quadruplicate and the data are expressed as fold change +/− standard deviation relative to control. A * indicates that Cyp1B1 expression is significantly different than Cyp1B1 expression in vehicle-treated cells, with a p value of less than 0.05.

Figure 3. Western blot shows quercetin increases protein levels of Cyp1A1 more than Cyp1B1

MCF10F human breast epithelial cells were treated with either vehicle or 10 and 50 µM concentrations of quercetin for 3 to 48 hours. Protein was isolated and subjected to Western blotting analysis. PVDF membranes were probed using antibodies to Cyp1A1, Cyp1B1 and GAPDH. Equal amounts of protein were loaded into each lane.