Breast cancer is the most prevalent cancer in women and the second leading cause of cancer-related deaths among females worldwide (Ferlay et al., 2015). Chemoprevention in combination with anticancer treatment is therefore crucial to reduce rates of morbidity and mortality. Evidence from epidemiological and experimental studies indicates that several natural products may act as chemopreventive agents and inhibit mammary carcinogenesis (Pan et al., 2015). Among these products is soy, which contains variable amounts of genistein and daidzein as the major isoflavones (approximately 47 and 44%, respectively) and minor amounts of glycitein (approximately 9% of the total isoflavones in soybeans). The genistein and daidzein content is therefore also predomit in soy-derived foods and dietary supplements (Wiseman et al., 2002 Clarke et al., 2008). Epidemiological studies have indicated that soy intake post-diagnosis not only improves prognosis but is also associated with statistically si... See full list on Materials 16α-hydroxy-17β-estradiol (E3), E2, 17β-estradiol-3-(β-D-glucuronide) sodium salt (E2-G) and E1, as well as acetic acid, acetonitrile, ammonium acetate, DMSO, genistein and daidzein, were purchased from Sigma–Aldrich Chemical Co. (Munich, Germany). 17β-estradiol-3-sulfate sodium salt (E2-S) and estrone-3-sulfate sodium salt (E1-S) w (Newport, RI, United States). 16α-hydroxy-17β-estradiol-2,4,17-d3 (E3-d3), 17β-estradiol-2,4,16,16-d4 (E2-d4), 17β-estradiol-16,... Cell Proliferation Studies MCF-7 breast cancer cells were purchased from the American Type Culture Collection (ATCC Rockville, MD, United States). All experiments were performed during the exponential growth phase of the cell line. MCF-7 cells were routinely cultivated at 37°C (95% humidity and 5% CO2) in phenol red-free Dulbecco’s Modified Eagle Medium F-12 (DMEM/F-12 Invitrogen, Karlsruhe, Germany), fortified with 1% PenStrep®-solution and 10% fetal bovine serum (Invitrogen). For experimental conditions, cells were... Inhibition Studies MCF-7 breast cancer cells were cultivated in the presence of HyClone® heat-inactivated charcoal-stripped fetal bovine serum as describedeasing concentrations of E1 (10, 25, 50, 75, and 100 nM) in the presence of 1, 5, and 10 μM genistein or daidzein, respectively. After 24 and 48 h, 2000 μl media aliquots were mixed with 20 μl deuterated internal standard solution and pre-cleaned by SPE on Oasis HLB 1 cc SPE cartridges (30 mg Waters Corporation, Milford, MA,... See full list on Influence of Estrone and Soy Isoflavones on MCF-7 Cell Proliferation To assess the influence of E1 on MCF-7 ceasing concentrations of E1 for 48 h, detached by application of TrypLe® solution and counted using a Coulter® Z1 cell counter. Compared with control samples (containing DMSO only), the number of viable eased in the presence of E1 (2.31 ± 0.33 × 106 vs. 3.99 ± 0.31 × 106 cells) (Figure 1A), confirming the hormone-dependency of the MCF-7 cell line. The observed proliferati... Formation of Estrogen Metabolites by MCF-7 Cells In preliminary experiments, MCF-7 breast cancer cells were treated with 100 nM E1 as a hormone precursor and cell media aliquots were analyzed for E1 and its metabolites after 24 and 48 h. By using a highly specific and sensitive LC-HRMS assay, five biotransformation products could be quantified besides the precursor E1 in the cellular medium (Figure 2). As metabolite formation showed a lineabations in all further experiments were finalized after this time-sp... Inhibition of Estrogen Conjugations by Genistein To assess the possible inhibitory effect of genistein on estrogen metabolism, MCF-7 cells were first treated with E1 (100 nM) for 48 h ieasing genistein concentrations (1, 5, and 10 μM). As shown in Table 1, a marked inhibition of E1 and E2 conjugation by this isoflavone was observed (Supplementary Figure S1A). Even in the presence of 1 μM genistein, the formation rates of E1-S, E2-S and E2-G decreased by approximately 25–35% compared to control. At 10 μM gen... See full list on To the best of our knowledge, the present study was the first to investigate the concentration-dependent impacts of the soy isoflavones genistein and daidzein on the formation of estrogen conjugates in human ERα+ breast cancer cells (MCF-7). When cells were exposed solely to the hormoneased up to 1.6-fold. A stimulatory effect on cell proliferation was bated with genistein or daidzein (1–10 μM) in the absence or in the presence of low E1 concentrations (1 and 2.5 nM). Our findings are in line with previous studies that have also reported a stimulatory effect of these isoflavone concentrations on MCF-7 cell growth (Chen et al., 2015 Wei et al., 2015). Our data also correlate with the in vitro study by Kuiper et al. (1998), which shows that genistein presents a 20- to 30-fold higher binding affinity for ERβ than for ERα whileeased affinity for ERβ, explaining the observed slightly in... See full list on The present work identified a non-competitive inhibition of E1 and E2 conjugation by low micromolar concentrations of soy isoflavones in the human breast cancer cell line MCF-7, which leads to a minor ease in unconjugated E2 of approximately 20%. As the content of genistein and daidzein in seased risk of breast cancer development and progression in women might only be observed after the continuous consumption of high-dose isoflavone supplements. Further long-term human studies monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the efficacy and safety of high-dose genistein and daidzein supplementation, especially in patients diagnosed with ERα+ breast cancer. See full list on SP performed all the cell culture experiments, the LC-HRMS analysis and the data analysis, and contributed to the manuscript. AM-S analyzed the data and contributed to the manuscript. MZ, JW, and DD performed the LC-HRMS analysis. BP and KS cultivated the MCF-7 cells and performed inhibition experiments and WJ planned the experiments, analyzed the data and wrote the final version of the manuscript. See full list on The authors declare that the research was conducted in the absence of any commercial or ficial relationships that could be construed as a potential conflict of interest. See full list on This research was supported by a grant from the Austrian Science Fund (FWF) [I 3417-B31] awarded to WJ. See full list on The Supplementary Material for this article can be found online at: https:///articles/10.3389/fphar.2017.00699/full#supplementary-material See full list on CYP, cytochrome P450 enzyme DMEM, Dulbecco’s modified Eagle’s medium DMSO, dimethyl sulfoxide DPBS, Dulbecco’s phosphate-buffered saline E1, estrone E1-S, estrone-3-sulfate E2, 17β-estradiol E2-G, 17β-estradiol-3-(β-D-glucuronide) E2-S, 17β-estradiol-3-sulfate E3, estriol (16α-OH-17β-estradiol) EIC, extracted ion chromatogram ERα, estrogen receptor alpha ERβ, estrogen receptor beta ESI, electro-spray ionization 17β-HSD, 17β-hydroxysteroid dehydrogenase Ki, inhibition constant Km, Michaelis constant LC-HRMS, liquid chromatography-high resolution mass spectrometry LLOQ, lower limit of quantification SD, standard deviation SPE, solid phase extraction SULT, sulfotransferase UGT, UDP-glucuronosyl transferase Vmax, maximum reaction velo. See full list on
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