The Mechanism Of Hydroxylation Metabolism Of Estradiol In MCF-7 And MDA-MB-231 Cells

Objective:In this study,we researched characteristics of estradiol metabolites and metabolic enzymes of hydroxyl in MCF-7 and MDA-MB-231 cells to explore the role of key metabolic enzymes and key metabolites in the development of the two cancer cells,and provide new ideas for the prevention and treatment of breast cancer,especially the study of triple negative breast cancer Meanwhile,it further clarifies the role of estradiol metabolic pathway in the development of breast cancer and enriches the mechanism of estrogen carcinogenesis.Methods:(1)UPLC-MS/MS method was established for the determination of estradiol and its hydroxy metabolites in cell samples.(2)The concentration and time of estradiol were screened by MTT.Then the UPLC-MS/MS method was used to study the pharmacokinetics of estradiol and its hydroxyl metabolites in MCF-7 and MDA-MB-231 cells,including uptake and disposition.(3)Finally,RT-PCR and western-blotting were used to detect the effects of estradiol on the gene and protein expression of CYP1A1 and CYP1B1 in MCF-7 and MDA-MB-231 cells,and to explore the carcinogenesis mechanism of estrogen.Results:In this study,the UPLC-MS/MS method for the determination of estradiol and its hydroxy metabolites in biological samples was successfully established for the disposition study of estradiol and its hydroxyl metabolites in MCF-7 and MDA-MB-231cells.Multiple reaction monitoring,positive ion scan mode,ethinylestradiol as internal standard,were m/z 414.2/350.2,571.2/365.2,571.2/365.2,438.2/374.2 for estradiol,2-hydroxyestradiol,4-hydroxyestradiol and ethinyl estradiol,respectively.We chose the ion mode of[M+H]~+and successfully set up a reproducible,sensitive and facile UPLC-MS/MS method for the determination of E2,2-OH E2,4-OH E2 and EE2.Cell toxicity test was carried out by MTT colorimetry.The results showed that cytotoxicity was not observed when estradiol concentration was no more than 1?M.Considering the following experiment and cell status,we finally chose estradiol concentration gradient of 0.4?M,0.8?M and 1.0?M.The disposition of estradiol in MCF-7 and MDA-MB-231 cells is not the same,the intake of estradiol was dose-dependent at 0.4?M,0.8?M and 1.0?M,and the drug concentration reached saturation at 12 h in both cells.The amount of drugs in MCF-7 cells was much higher than that of MDA-MB-231cells.The metabolites that are generated are different in the two cells,estradiol were mainly metabolized into 2-OH estradiol in MCF-7 cells and we failed to detect 4-OH estradiol,whereas MDA-MB-231 cells are contrary.The results of the drug scavenging experiment showed that two cells were able to remove estradiol and its hydroxyl metabolites within 12 hours.RT-PCR and western blotting were used to detect the effects of the different concentrations of estradiol on CYP1A1 and CYP1B1 gene and protein expression in MCF-7 and MDA-MB-231 cells.The results showed that in MCF-7 cells,different concentrations(0.4?M,0.8?M and 1.0?M)of estradiol could significantly increase the expression of gene and protein of CYP1A1,and the gene and protein expression of CYP1B1 did not change,whereas MDA-MB-231 cells are contrary.Conclusion:The hydroxylation process of estradiol in estrogen receptor negative and positive breast cancer cells is different.The main metabolite of estradiol in MCF-7 cells is 2-OH estradiol,and in MDA-MB-231 cells is 4-OH estradiol.Low,medium and high concentrations of estradiol can upregulate the expression of CYP1A1 in MCF-7 cells.In addition,the expression of CYP1B1 in MDA-MB-231 cells increased significantly,which may be related to the carcinogenic effect of estradiol.The results of this study may provide a theoretical basis for the study of estrogen carcinogenesis.