MicroRNA-181a suppresses norethisterone-promoted tumorigenesis of breast epithelial MCF10A cells through the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway

BackgroundResearch suggests that hormone replacement therapy may increase the risk of breast cancer, and progestins such as norethisterone (NET) play a key role in this phenomenon. We have demonstrated that microRNA-181a (miR-181a) suppresses NET-promoted breast cancer cell survival. Nonetheless, the effects of NET and miR-181a on the tumorigenesis of human breast epithelial cells have not yet been elaborated.MethodsAssays of cell viability, proliferation, migration, apoptosis, and colony formation were performed to investigate the pro-tumorigenesis effect of NET and the effects of miR-181a on human breast epithelial MCF10A cells. The expressions of cell-proliferation-related genes and apoptotic factors were analyzed by quantitative RT-PCR and Western blot in MCF10A cells treated with NET and miR-181a.ResultsNET significantly increased MCF10A cell viability, proliferation, migration, and colony formation, but reduced cellular apoptosis. In addition, NET increased the expression of progesterone receptor membrane component 1 (PGRMC1), EGFR, B-cell lymphoma 2, cyclin D1, and proliferating cell nuclear antigen, but decreased the expression of pro-apoptosis factors, such as Bax, caspase-7, and caspase-9. Overexpression of miR-181a strongly inhibited the effects of NET on MCF10A cells and abrogated NET-stimulated PGRMC1, EGFR, and mTOR expression.ConclusionsActivation of the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway is the primary mechanism underlying the pro-tumorigenesis effects of NET on human breast epithelial MCF10A cells. Additionally, miR-181a can suppress the effects of NET on these cells. These data suggest a therapeutic potential for miR-181a in reducing or preventing the risk of breast cancer in hormone replacement therapy using NET.     

Doping in sport: 3. Metabolic conversion of oral norethisterone to urinary 19-norandrosterone

The detection of 19 norandrosterone (19-NA) in a competitor's urine sample is taken as prima facie evidence of administration of nandrolone or other 19-norsteroid but a potential problem is that administration of norethisterone, a progestogen used for menstrual disorders and for hormonal contraception, also results in the excretion of 19-NA that can exceed the laboratory reporting threshold of 2 ng/mL. The contribution of norethisterone to urinary 19-NA with and without 19-norandrostenedione, a known norethisterone tablet impurity, requires evaluation. Preparations containing, either <2 ng or 1 μg 19-norandrostenedione impurity per 5 mg of norethisterone, administered to female volunteers (n = 10) in doses comparable to those used for menstrual disorders (5 mg three times daily for 10 days), resulted in maximal 19-NA concentrations of 51 and 63 ng/mL, respectively. The maximal concentration of 19-NA, 2 h post-administration of a single 1 μg dose of 19-norandrostenedione, was 2.4 ng/mL. These results prove unequivocally that norethisterone is metabolized to 19-NA and that there is only a minor contribution from the impurity 19-norandrostenedione. Administration to women (n = 30) of a single contraceptive tablet containing norethisterone (1 mg) with one of the highest proportions of the impurity 19-norandrostenedione (∼0.5 μg, 0.05%, w/w) resulted in a urinary 19-NA concentration of 9.1 ng/mL, with a maximum concentration ratio of 19-NA to the norethisterone metabolite 3α,5β-tetrahydronorethisterone of 0.36. We provide data that should remove the need for time-consuming follow-up investigations to consider whether doping with 19-norandrogens has occurred.     

Doping in sport—2. Quantification of the impurity 19-norandrostenedione in pharmaceutical preparations of norethisterone

The finding of measurable amounts of 19-norandrostenedione in norethisterone tablets prompted us to develop an assay to quantify this steroid. 19-Norandrostenedione is an anabolic steroid whose use in sport is prohibited by the World Anti-Doping Agency (WADA). The assay was developed using isotope dilution and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 19-norandrostenedione in norethisterone formulations, with [3,4-¹³C₂]-19-norandrostenedione as the internal standard. The results showed amounts up to 1.01 ± 0.01 μg (mean ± S.E.M.) per tablet in those containing 5 mg of norethisterone or norethisterone acetate (0.02%, w/w) and up to 0.5 ± 0.01 μg (mean ± S.E.M.) per tablet (0.05%, w/w) in oral contraceptive tablets containing 0.35-1.5 mg of norethisterone or norethisterone acetate. No tablet tested exceeded the British Pharmacopoeia limit of 0.1% for this impurity.     

Simultaneous determination of norethisterone and six metabolites in human plasma by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of norethisterone (NET) and six metabolites in human plasma by capillary gas chromatography-mass-selective detection (GC-MS) is described. The compounds are determined in plasma after enzymatic hydrolysis. After addition of norgestrel as the internal standard, the compounds are extracted from plasma at pH 5 using an Extrelut column and elution with dichloromethane. After evaporation, the compounds are converted into bistrimethylsilyl derivatives which are determined by gas chromatography using a mass-selective detector at m/z 429 for the two dihydro-NET (5β-NET and 5α-NET), m/z 431 for the four tetrahydro-NET (3α,5α-NET, 3α,5β-NET, 3β,5β-NET and 3β,5α-NET), m/z 442 for NET and m/z 456 for the internal standard. The reproducibility and accuracy of the method were found suitable over the range of concentrations between 0.50 and 8 ng/ml for NET, and metabolites except for 5α-dihydro-NET (between 1 and 8 ng/ml). The method was applied to clinical samples.     

Doping in sport—1. Excretion of 19-norandrosterone by healthy women, including those using contraceptives containing norethisterone

19-Norandrosterone (19-NA) is the principal urinary metabolite of the anabolic steroid nandrolone and its prohormones. The administration of these 19-nor androgens is prohibited in sport by the World Anti-Doping Agency (WADA) but, even so, adverse findings for 19-NA continue to be commonly reported. Little is known about the urinary concentrations of 19-NA that can occur in women who are not using anabolic steroids, including those using oral contraceptives containing the 19-nor progestogen norethisterone. In 2004, WADA lowered the reporting threshold for 19-NA for females from 5 to 2 ng/mL. The lack of any substantial data on 19-NA excretion in women prompted this large-scale investigation. In this investigation, single untimed urines collected from 1202 female volunteers, 38 of whom were taking norethisterone containing contraceptives, were analysed for 19-NA. None of the women was a competitive athlete and pregnancy had been excluded by a urinary test for human chorionic gonadotropin (hCG). Only one sample exceeded the 19-NA reporting threshold having a concentration of 4.1 ng/mL. This sample was from a user of a norethisterone-containing contraceptive.     

Irreversible protein binding of norethisterone (norethindrone) epoxide.

14,15-3H-Norethisterone-4 beta, 5 beta-epoxide, a metabolite of norethisterone, was incubated with several proteins and nucleic acids. After 30 min incubation 0.19 nmol of the epoxide were irreversibly bound per mg albumin which contains free sulfhydryl groups; proteins without SH-groups, such as concanavalin A, gamma-globulin, DNA and RNA, did not irreversibly bind norethisterone epoxide. A superoxide (O2) generating enzyme system comprised of xanthine oxidase and hypoxanthine was capable of catalyzing the irreversible binding of the parent compound, norethisterone, to albumin, indicating that an oxidation product was formed which reacted with the protein. When norethisterone epoxide was incubated for 60 min with hepatic microsomes of rats in absence of NADPH, about 2.0 nmol of the epoxide were irreversibly incorporated per mg microsomal protein. This binding was increased to 5.2 nmol by addition of a NADPH regenerating system. Addition of glutathione and cytosol decreased only the NADPH-dependent protein binding; phenobarbital pretreatment of rats induced this NADPH-dependent binding of norethisterone epoxide to microsomal protein by a factor of 2. In presence of NADPH, binding of the epoxide to microsomal protein depended on substrate concentration used. The results indicate that norethisterone epoxide is able to chemically react with proteins. In addition, hepatic microsomal enzymes convert the epoxide to another metabolite which also can react with proteins.     

11β-Hydroxylation of norethisterone acetate by Curvularia lunata

Abstract: The biotransformation of norethisterone acetate by Curvularia lunata was studied. The 11β-hydroxy-norethisterone acetate produced was identified by mass spectrometry, and ¹H and ¹³C nuclear magnetic resonance after extractive sample preparation.