Development of an analytical method to measure 17<font face="symbol">b</font>-estradiol metabolite concentrations in MCF-7 and MCF-12A cell lines

Abstract

Breast cancer is one of the most common cancers affecting women. It remains the leading cause of death in American women from 30 – 70 years of age and approximately 10% of the women living in western countries will develop breast cancer during their life time. Estrogens are a family of female hormones involved in the reproductive function of the human body. Estradiol is the most abundant estrogen in premenopausal woman. Initially it was thought that estradiol itself was responsible for tumourigenesis, but it has since been discovered that the catechol metabolites of estradiol and other estrogens cause carcinogenesis. 4-Hydroxyestradiol (4-HE2) is a potent cell proliferating estrogen whereas 2-methoxyestradiol (2-ME2) is a potent inhibitor of cell proliferation through the activation of apoptosis. 2-Hydroxyestradiol (2-HE2) also causes increased cell proliferation but it is not as potent as 4-HE2 and it is <font face="symbol">O</font>-methylated rapidly to 2-ME2. Catechol metabolites of estradiol are also involved in producing reactive oxygen species through redox cycling. The reactive oxygen species cause DNA damage and mutations to occur which can lead to carcinogenesis. A significant ratio to consider as a biomarker for breast cancer risk therefore is the 4-HE2/2-ME2 ratio. In this study, an analytical method was developed to measure the concentration levels of E2, 2-ME2, 2-HE2 and 4-HE2 in cell culture medium. The analytical method made use of gas chromatographymass spectrometry (GCMS) analysis, since the expected physiological concentrations of these metabolites were very low. Various extraction and derivatisation techniques were applied during the development of the method. The final method made use of protein precipitation with concentrated hydrochloric acid, liquid-liquid extraction using diethyl ether and derivatisation with trimethylsilylimidazole (TMSI). During the validation of this method, it was found that the method did not produce accurate measurements and that it could only be used to determine trends. Since the precise in vitro concentration levels of the metabolites were still unknown, it was decided to proceed with experiments using this method, to provide preliminary results with which further course of action could be planned. Equal numbers (1x106 cells/flask) of MCF-7 and MCF-12A cells were provided with 11 ml medium containing E2/2-HE2/4-HE2/2-ME2 (10-6 M) and medium containing E2/2-HE2/4-HE2/2-ME2 (10-8 M). Of each metabolite and each concentration two flasks were prepared. Each flask represented a specific time interval. At the appropriate time 10 ml of the medium was extracted. The time intervals used for each experiment were 0 hours, 8 hours and 24 hours for incubation with E2, 0 hours, 1 hour and 8 hours for incubation with 2-ME2, 0 hours, 10 minutes and 1 hour for incubation with 2-HE2 and 4-HE2. The time intervals used were according to the expected rate of metabolism. Each experiment was repeated three times. Differences in the metabolism of breast tumour cells and normal cells were found and the concentration of the metabolites present in the cell incubation medium had an influence on the metabolism of the cells. The need to investigate the intracellular concentrations of the metabolites has also been accentuated through the results obtained.