Oestrogenic activity of benzyl salicylate,benzyl benzoate and butylphenylmethylpropional (Lilial) in MCF7 human breast cancer cells in vitro

Benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) are added to bodycare cosmetics used around the human breast. We report here that all three compounds possess oestrogenic activity in assays using the oestrogen‐responsive MCF7 human breast cancer cell line. At 3 000 000‐fold molar excess, they were able to partially displace [³H]oestradiol from recombinant human oestrogen receptors ERα and ERβ, and from cytosolic ER of MCF7 cells. At concentrations in the range of 5 × 10^?5 to 5 × 10^?4 m , they were able to increase the expression of a stably integrated oestrogen‐responsive reporter gene (ERE‐CAT) and of the endogenous oestrogen‐responsive pS2 gene in MCF7 cells, albeit to a lesser extent than with 10^?8 m 17β‐oestradiol. They increased the proliferation of oestrogen‐dependent MCF7 cells over 7 days, which could be inhibited by the antioestrogen fulvestrant, suggesting an ER‐mediated mechanism. Although the extent of stimulation of proliferation over 7 days was lower with these compounds than with 10^?8 m 17β‐oestradiol, given a longer time period of 35 days the extent of proliferation with 10^?4 m benzyl salicylate, benzyl benzoate or butylphenylmethylpropional increased to the same magnitude as observed with 10^?8 m 17β‐oestradiol over 14 days. This demonstrates that benzyl salicylate, benzyl benzoate and butylphenylmethylpropional are further chemical components of cosmetic products which give oestrogenic responses in a human breast cancer cell line in culture. Further research is now needed to investigate whether oestrogenic responses are detectable using in vivo models and the extent to which these compounds might be absorbed through human skin and might enter human breast tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

N‐substituted Piperazinopyridylsteroid Derivatives as Abiraterone Analogues Inhibit Growth and Induce Pro‐apoptosis in Human Hormone‐independent Prostate Cancer Cell Lines

Nine new 17‐(piperazin‐1‐yl)pyridin‐5‐yl)steroids as abiraterone analogues were synthesized. Compounds 5d and 5g showed selective activities against 17α‐hydroxylase/C17,20‐lyase (CYP17A1) and aromatase (CYP19), respectively. IC₅₀ values of 5d were 5.09 and >50? μm , whereas these values for 5g were >50 ?μm and 7.40 μm , respectively, for CYP17A1 and CYP19. Molecular modelling highlighted that the inhibitor designed to bind cytochrome P450 haem iron is a necessary condition but not the only rationale to explain inhibitory activity. These abiraterone analogues were then evaluated on hormone‐independent prostate cancer cell lines DU‐145 and PC‐3 and on hormone‐dependent breast and prostate cancer cell lines MCF‐7 and LNCaP, respectively. Compounds 5e , 5g and 5i have showed potent activities only on hormone‐independent prostate cancer cell lines DU‐145 and PC‐3 with 60–85% inhibition of both cell viability and growth at 10 nm with pro‐apoptotic mechanism as illustrated in PC‐3 cells by DNA ladder assay and Western blotting of Bax, Casp‐3 and its substrate, the poly (ADP–ribose) polymerase. We conclude that hybrid heterocycle steroids could be good lead compounds in the drug design especially against hormone‐independent prostate cancer.     

Effects of exposure to six chemical ultraviolet filters commonly used in personal care products on motility of MCF‐7 and MDA‐MB‐231 human breast cancer cells in vitro

Benzophenone (BP)‐1, BP‐2, BP‐3, octylmethoxycinnamate (OMC), 4‐methylbenzilidenecamphor and homosalate are added to personal care products to absorb ultraviolet light. Their presence in human milk and their oestrogenic activity suggests a potential to influence breast cancer development. As metastatic tumour spread is the main cause of breast cancer mortality, we have investigated the effects of these compounds on migration and invasion of human breast cancer cell lines. Increased motility of oestrogen‐responsive MCF‐7 human breast cancer cells was observed after long‐term exposure (>20 weeks) to each of the six compounds at ≥10^?7 m concentrations using three independent assay systems (scratch assay, live cell imaging, xCELLigence technology) and increased invasive activity was observed through matrigel using the xCELLigence system. Increased motility of oestrogen‐unresponsive MDA‐MB‐231 human breast cancer cells was observed after 15 weeks of exposure to each of the six compounds by live cell imaging and xCELLigence technology, implying the increased migratory activity was not confined to oestrogen‐responsive cells. Molecular mechanisms varied between compounds and cell lines. Using MCF‐7 cells, reduction in E‐cadherin was observed following 24 weeks' exposure to 10^?5 m BP‐1 and 10^?5 m homosalate, and reduction in β‐catenin was noted following 24 weeks' exposure to 10^?5 m OMC. Using MDA‐MB‐231 cells, increased levels of matrix metalloproteinase 2 were observed after 15 weeks exposure to 10^?7 m OMC and 10^?7 m 4‐methylbenzilidenecamphor. Although molecular mechanisms differ, these results demonstrate that exposure to any of these six compounds can increase migration and invasion of human breast cancer cells.     

In-vitro antiproliferative activity of benzopyranone derivatives in comparison with standard chemotherapeutic drugs

The cytotoxic activities of five new benzopyranone derivatives containing basic amino side chain are described. Their cytotoxicities against ER(+) MCF‐7 and ER(–) MDA‐MB‐231 human breast cancer cell lines, and Ishikawa human endometrial cell line were determined after 72 h drug exposure employing CellTiter‐Glo assay at concentrations ranging from 0.01–1.0 × 10⁵ nM. The antiproliferative activities of these compounds were compared to tamoxifen (TAM), 4‐hydroxytamoxifen (4‐OHT, active metabolite of tamoxifen), and raloxifene (RAL). In‐vitro results indicated that compounds 9 , 10 , 12 , and 13 were more potent than TAM against the human breast cancer cell lines with IC₅₀ < 20 µM. The in‐silico structure–activity relationships of these compounds and their binding mode within the estrogen receptor (ER) binding site using AutoDock vina are discussed.     

Design,synthesis, and biological evaluation of pyrimidine derivatives as potential inhibitors of human calcium/calmodulin‐dependent protein kinase IV

Calcium/calmodulin‐dependent protein kinase IV (CAMKIV ) is a multifunctional Ser/Thr kinase, associated with cerebral hypoxia, cancer, and neurodegenerative diseases. Here, we report design, synthesis, and biological evaluation of seven pyrimidine‐substituted novel inhibitors of CAMKIV . We successfully synthesized and extensively characterized (ESI ‐MS , ¹H NMR , and ¹³C NMR studies) seven compounds that are showing appreciable binding affinity to the CAMKIV . Molecular docking and fluorescence binding studies revealed that compound 1 is showing very high binding free energy (ΔG  = ?11.52 kcal/mol) and binding affinity (K  =  9.2 × 10¹⁰ m ^?1) to the CAMKIV . We further performed MTT assay to check the cytotoxicity and anticancer activity of these compounds. An appreciable IC ₅₀ (39 μm ) value of compound 1 was observed on human hepatoma cell line and nontoxic till the 400 μm on human embryonic kidney cells. To ensure anticancer activity of all these compounds, we further performed propidium iodide assay to evaluate cell viability and DNA content during the cell cycle. We found that compound 1 is again showing a better anticancer activity on both human hepatoma and human embryonic kidney cell lines.     

Design,synthesis, and cytotoxic evaluation of novel furo[2,3‐b]quinoline derivatives

A number of novel furo[2,3‐b]quinoline derivatives were designed and synthesized by introducing benzyl ether, benzoate, and benzenesulfonate to 6‐position of furo[2,3‐b]quinoline and their chemical structures were confirmed by ESI‐MS, ¹H NMR, and ¹³C NMR spectra. All target compounds were evaluated in vitro against four human cancer cell lines (HCT‐116, MCF‐7, U2OS, and A549) by MTT method. Cytotoxic assay showed that compounds 8a , 8e , 10a , 10b, and 10c exhibited more potent cytotoxicities compared to 2‐bromine‐6‐hydroxy‐furo‐[2,3‐b]quinoline ( 7 ). Compound 10c exhibited higher antiproliferative activity (IC₅₀ values ranging from 4.32 to 24.96 μm ) against four human cancer cell lines (HCT‐116, MCF‐7, U2OS, and A549) and weak cytotoxicity on normal cell HL‐7702, which suggested that 10c might be an ideal anticancer candidate. Compounds 8a , 10a , 10b showed good selectivities to MCF‐7 and HCT‐116, which could be considered as ideal selective candidates for further study. The SARs showed that the introduction of the benzyl ether and benzenesulfonate could significantly improve cytotoxicities, while the benzoate failed to enhance potency obviously.     

Fludioxonil induced the cancer growth and metastasis via altering epithelial–mesenchymal transition via an estrogen receptor‐dependent pathway in cellular and xenografted breast cancer models

Fludioxonil is an antifungal agent used in agricultural applications that is present at measurable amounts in fruits and vegetables. In this study, the effects of fludioxonil on cancer cell viability, epithelial–mesenchymal transition (EMT), and metastasis were examined in MCF‐7 clonal variant breast cancer cell (MCF‐7 CV cells) with estrogen receptors (ERs). MCF‐7 CV cells were cultured with 0.1% DMSO (control), 17β‐estradiol (E2; 1 ×10^?9 M, positive control), or fludioxonil (10^?5?10^?8 M). MTT assay revealed that fludioxonil increased MCF‐7 CV cell proliferation 1.2 to 1.5 times compared to the control, while E2 markedly increased the cell proliferation by about 3.5 times. When the samples were co‐treated with ICI 182,780 (10^?8 M), an ER antagonist, fludioxonil‐induced cell proliferation was reversed to the level of the control. Protein levels of cyclin E1, cyclin D1, Snail, and N‐cadherin increased in response to fludioxonil as the reaction to E2, but these increases were not observed when fludioxonil was administered with ICI 182,780. Moreover, the protein level of p21 and E‐cadherin decreased in response to treatment with fludioxonil, but remained at the control level when co‐treated with ICI 182,780. In xenografted mouse models transplanted with MCF‐7 CV cells, fludioxonil significantly increased the tumor mass formation by about 2.5 times as E2 did when compared to vehicle (0.1% DMSO) during the experimental period (80 days). Immunohistochemistry revealed that the protein level of proliferating cell nuclear antigen (PCNA), Snail, and cathepsin D increased in response to fludioxonil as the reaction to E2. These results imply that fludioxonil may have a potential to induce growth or metastatic behaviors of breast cancer by regulation of the expression of cell cycle‐, EMT‐, and metastasis‐related genes via the ER‐dependent pathway. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1439–1454, 2017.     

DIRECT EFFECT OF DEXMEDETOMIDINE ON RAT ISOLATED AORTA INVOLVES ENDOTHELIAL NITRIC OXIDE SYNTHESIS AND ACTIVATION OF THE LIPOXYGENASE PATHWAY

• 1 The aims of the present in vitro study were to examine the roles of pathways associated with arachidonic acid metabolism in dexmedetomidine‐induced contraction and to determine which endothelium‐derived vasodilators are involved in the endothelium‐dependent attenuation of vasoconstriction elicited by dexmedetomidine.
• 2 Dexmedetomidine (10^?9–10^?6 mol/L) concentration–response curves were constructed in: (i) aortic rings with no drug pretreatment; (ii) endothelium‐denuded aortic rings pretreated with either 2 × 10^?5 mol/L quinacrine dihydrochloride, 10^?5 mol/L nordihydroguaiaretic acid (NDGA), 3 × 10^?5 mol/L indomethacin or 10^?5 mol/L fluconazole; and (iii) endothelium‐intact aortic rings pretreated with either 5 × 10^?5 mol/L N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME), 10^?5 mol/L fluconazole, 10^?5 mol/L indomethacin, 10^?5 mol/L glibenclamide, 5 × 10^?3 mol/L tetraethylammonium or 5 × 10^?5 mol/L l ‐NAME plus rauwolscine (10^?5, 10^?6 mol/L). The production of nitric oxide (NO) metabolites was determined in human umbilical vein endothelial cells treated with dexmedetomidine.
• 3 Quinacrine dihydrochloride, NDGA and indomethacin attenuated the dexmedetomidine‐induced contraction of endothelium‐denuded rings. Dexmedetomidine (10^?7–10^?6 mol/L)‐induced contractions of endothelium‐denuded rings were enhanced compared with those of endothelium‐intact rings, as were dexmedetomidine‐induced contractions of endothelium‐intact rings pretreated with l ‐NAME or tetraethylammonium. Rauwolscine attenuated dexmedetomidine‐induced contractions in endothelium‐intact rings pretreated with l ‐NAME. Dexmedetomidine (10^?6 mol/L) was found to activate NO production.
• 4 Taken together, the results indicate that dexmedetomidine‐induced contraction of aortic rings involves activation of the lipoxygenase and cyclo‐oxygenase pathways and is attenuated by increased NO production following stimulation of endothelial α₂‐adrenoceptors by dexmedetomidine.

     

Interactions of an acidic cyclooctapeptide with metal ions: microcalorimetric and fluorescence analyses

Abstract: The metal‐ion binding preferences of an acidic amphipathic cyclopeptide, cyclo[d ‐Leu‐Leu‐d ‐Leu‐Trp‐(d ‐Glu‐Glu)₂] (CP), was studied by isothermal titration calorimetry and steady‐state fluorescence spectroscopy. CP adopted a partial beta structure, and variable temperature circular dichroism showed small secondary structural changes over the temperature range from 5 °C to 95 °C. The peptide did not bind alkali or alkaline earth metal ions but exhibited selectivity for some divalent transition metal ions (with association constants K_Cu2+ 4.5 × 10⁴ m ^?1, K_Zn2+ 1.6 × 10⁵ m ^?1, K_Cd2+ 1.3 × 10⁴ m ^?1, K_{Hg2+ (1)} 2.2 × 10⁶ m ^?1, and K_{Hg2+ (2)} 6.5 × 10³ m ^?1), for Pb²⁺ (2.0 × 10⁵ m ^?1), and a trivalent Group III metal, Al³⁺ (1.6 × 10⁵ m ^?1). The thermodynamic data show that the interaction between CP and these metal ions are spontaneous and entropically driven. A large range of binding enthalpies coupled with a smaller range of binding free energies of CP for these metal ions indicate an entropy–enthalpy compensation dependent on the ionic size of participating metal ions. The interaction of Pb²⁺, Hg²⁺, and Cu²⁺ with CP in aqueous solution specifically modulates the fluorescence emission properties of CP. The results of this study show that CP exhibits selectivity in metal‐ion binding, which is reflected in its fluorescence spectra. The observed trends can be useful for the design of heavy‐metal sensors based on fluorophore‐tagged acidic cyclopeptides.     

Novel menadione hybrids: Synthesis,anticancer activity,and cell‐based studies

A series of novel menadione‐based triazole hybrids were designed and synthesized by employing copper‐catalyzed azide‐alkyne cycloaddition (CuAAC). All the synthesized hybrids were characterized by their spectral data (¹H NMR, ¹³C NMR, IR, and HRMS). The synthesized compounds were evaluated for their anticancer activity against five selected cancer cell lines including lung (A549), prostate (DU‐145), cervical (Hela), breast (MCF‐7), and mouse melanoma (B‐16) using MTT assay. The screening results showed that majority of the synthesized compounds displayed significant anticancer activity. Among the tested compounds, the triazoles 5 and 6 exhibited potent activity against all cell lines. In particular, compound 6 showed higher potency than the standard tamoxifen and parent menadione against MCF‐7 cell line. Flow cytometric analysis revealed that compound 6 arrested cell cycle at G0/G1 phase and induced apoptotic cell death which was further confirmed by Hoechst staining, measurement of mitochondrial membrane potential (ΔΨm) and Annexin‐V‐FITC assay. Thus, compound 6 can be considered as lead molecule for further development as potent anticancer therapeutic agent.     

Comparison of in vitro cytotoxicity,estrogenicity and anti‐estrogenicity of triclosan,perfluorooctane sulfonate and perfluorooctanoic acid

Concern with increasing levels of emerging contaminants exists on a global scale. Three commonly observed emerging environmental contaminants: triclosan (2,4,4‐trichloro‐2′‐hydroxydiphenyl ether), a synthetic, broad‐spectrum antibacterial agent, and perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), used in stain‐ and water‐resistant treatments, have become distributed ubiquitously across ecosystems and have been detected in wildlife and humans. MCF‐7 BOS human breast cancer cells were used to investigate the potential for cytotoxicity, estrogenicity and anti‐estrogenicity of these three compounds at environmentally relevant concentrations using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt assay (MTS) and the E‐SCREEN bioassay. The doses used were 0.002–200 µg ml^?1 for triclosan and 0.03–30 µg ml^?1 for PFOS and PFOA. Quantitative results from the MTS assay revealed no significant cytotoxicity at lower concentrations for any of the test compounds; however, both triclosan and PFOA were cytotoxic at the highest concentrations examined (100–200 and 30 µg ml^?1, respectively), while PFOS showed no significant cytotoxicity at any of the concentrations tested. Positive estrogenic responses (P < 0.05) were elicited from the E‐SCREEN at all concentrations examined for triclosan and PFOA and at 30 µg ml^?1 for PFOS. Further, significant anti‐estrogenic activity (P < 0.05) was detected for all compounds tested at all concentrations when cells were co‐exposed with 10^?9 m 17‐β estradiol (E₂). The overall results demonstrated that triclosan, PFOS and PFOA have estrogenic activities and that co‐exposure to contaminants and E₂ produced anti‐estrogenic effects. Each of these compounds could provide a source of xenoestrogens to humans and wildlife in the environment. Published 2011. This article is a US Government work and is in the public domain in the USA.     

Anticoagulant, anti-aggregation and antithrombotic effects of a novel hexapeptide

Objectives Hexapeptide is a novel synthetic oligopeptide with a structure similar to that of eptifibatide. This study was designed to investigate the anticoagulant, anti‐aggregation, disaggregation and anti‐thrombogenesis effects of hexapeptide. Methods The effects of antiplatelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin, and the effect of disaggregation of platelet aggregation induced by ADP were determined. The anticoagulation indexes were determined by different kits. Key findings Hexapeptide 1 × 10^?5–1 × 10^?4m could significantly prolong rabbit blood clotting time, thrombin time, prothrombin time and activated partial thromboplastin enzyme time, and reduce the length, wet weight, dry weight and the index of thrombus in a concentration‐dependent manner. Hexapeptide 1 × 10^?4m decreased platelet adhesion rate by 40.2%. The platelet aggregation inhibition of hexapeptide in dogs and humans was more obvious than in rabbits and rats. The aggregation inhibition rate of 1 × 10^?5m hexapeptide in dogs, rabbits, rats and humans induced by ADP was 93.9 ± 1.3%, 66.2 ± 1.4%, 76.1 ± 3.2% and 99.8 ± 0.2%, respectively; the 50% inhibitory concentration (IC50) of hexapeptide was 7.24 × 10^?8, 3.24 × 10^?6, 6.61 × 10^?6 and 8.91 × 10^?8m , respectively. For the aggregation inhibition rate of hexapeptide in dogs, rabbits and humans induced by AA, the IC50 was 1.29 × 10^?9, 1.32 × 10^?6 and 9.33 × 10^?8m , respectively; the IC50 of aggregation inhibition rates induced by thrombin was 2.88 × 10^?6, >1 × 10^?5 and 4.17 × 10^?6m , respectively. The disaggregation rate of 1 × 10^?4m hexapeptide in dog induced by ADP was 68.8 ± 7.4%. Conclusions Hexapeptide has anticoagulant, antiplatelet aggregation, disaggregation and antithrombotic effects in vitro.     

Determination of pethidine hydrochloride using potentiometric coated graphite and carbon paste electrodes

A new approach for lowering the detection limit of a pethidine ion‐selective electrode is presented. A coated graphite (CGE) and carbon paste (CPE) electrodes for pethidine ions based on pethidine‐phosphotungstate (PD‐PT) as ion‐pair complex are described. The sensors exhibit a Nernstian slope of 58.1 and 54.2 mVdecade^?1 for pethidine ion over a wide concentration range from 2.6 × 10^?7 to 1.0 × 10^?2 M and 2.1 × 10^?6 to 1.0 × 10^?2 M with a detection limit of 1.8 × 10^?7 M and 7.3 × 10^?7 M for pethidine coated graphite (PD‐CGE) and pethidine carbon paste electrode (PD‐CPE), respectively. These sensors exhibited a fast response time (about 5–8 s) and good stability. The standard electrode potentials, E^o, were determined at different temperatures and used to calculate the isothermal temperature coefficient (dE^o/dT) of the PD‐CGE and PD‐CPE, which was 0.0062 and 0.0071 V/ °C, respectively. Selectivity coefficients, determined by matched potential method (MPM) and separate solution method (SSM), showed high selectivity for pethidine hydrochloride (PDCl) over a large number of inorganic cations, organic cations, sugars, urine components, and some common drug excipients. The sensors were applied for determination of PDCl in ampoule and in spiked urine samples using potentiometric determination, standard addition and the calibration curve methods. The results obtained were satisfactory with excellent percentage recovery comparable and sometimes better than those obtained by other routine methods for the assay. Copyright © 2011 John Wiley & Sons, Ltd.     

Syntheses and In Vitro Anticancer Properties of Novel Radiosensitizers

Series of 4‐(ethylsulfonyl)‐1‐halogen‐2‐nitrobenzene ( 3a – e ) and 1‐(4‐halogen‐3‐nitrophenyl) propan‐1‐one ( 5a – d ) analogs designed as novel radiosensitizers using bromonitropropiophenone and bromonitrobenzonitrile as lead compounds were synthesized. The anticancer activities of the compounds were evaluated in vitro using human prostate cancer (DU‐145) and breast cancer (MCF‐7) cell lines and the MTT assay. From the series, six compounds ( 3b – e , 5b – c ) exhibited potent growth inhibitory effects against both cell lines. The most active, compound 3d, is an iodosulfone and is significantly more potent than the lead compound 5c at 10 μm . Compounds were then compared with doxorubicin, a clinically used anticancer compound for breast and prostate cancers. Our most active compound 3d is more effective than doxorubicin at the dose level of 10 μm at 3 days after radiation, cell viabilities of 18%, 13% compared to 87%, 94% against MCF‐7, and 15%, 20% compared to 60%, 75% against DU‐145 without and with radiation, respectively. At 10 μm , compound 5c had no effects as compared to control, whereas compound 3d reduced DU‐145 cell viability to 16% and that of MCF‐7 cells to 9% even at 5 days after radiation. These results are very encouraging. Future studies include testing the compounds in vivo with and without radiation.     

Actions of dopamine and apomorphine on the vasoconstrictor responses of perfused mesenteric arteries of mouse,rat and rabbit

Dopamine (10^?7-10^?6 m) and apomorphine (5 × 10^?7 ? 5 × 10^?6 m) inhibited the vasoconstrictor responses of the perfused mesenteric artery preparations of rat, rabbit and mouse to adrenergic nerve stimulation but did not affect responses to added noradrenaline. The inhibitory effects of both dopamine and apomorphine were prevented by haloperidol (3 × 10^?7 m) but not by yohimbine (3 × 10^?8 m) in rat and rabbit mesenteric artery preparations. In contrast, yohimbine (3 × 10^?8 m), but not haloperidol, antagonized the inhibitory effect of dopamine and apomorphine in mouse mesenteric artery preparations. In higher concentrations, dopamine (10^?6 ? 10^?4 m) produced a direct vascoconstrictor effect, which involved post-junctional α-adrenoceptors in all three species. However, in preparations contracted with 10^?7 m 5-hydroxytryptamine and in the presence of phentolamine (3 × 10^?7 m) and propranolol (10^?6 m), dopamine (10^?6 ? 10^?4 m) produced a direct relaxant effect in rabbit mesenteric artery preparations but not in those of rat and mouse. It is suggested that inhibition of neurogenic vasoconstrictor responses, by dopamine and apomorphine, may be mediated through a specific prejunctional inhibitory dopamine receptor in the mesenteric artery of rat and rabbit whereas in the mouse they involve activation of α-adrenoceptors.     

Methylparaben stimulates tumor initiating cells in ER+ breast cancer models

A body of epidemiological evidence implicates exposure to endocrine disrupting chemicals (EDCs) with increased susceptibility to breast cancer. To evaluate the physiological effects of a suspected EDC in vivo, we exposed MCF‐7 breast cancer cells and a patient‐derived xenograft (PDX, estrogen receptor positive) to physiological levels of methylparaben (mePB), which is commonly used in personal care products as a preservative. mePB pellets (4.4 μg per day) led to increased tumor size of MCF‐7 xenografts and ER⁺ PDX tumors. mePB has been thought to be a xenoestrogen; however, in vitro exposure of 10 nM mePB failed to increase MCF‐7 cell proliferation or induction of canonical estrogen‐responsive genes (pS2 and progesterone receptor), in contrast to 17β‐estradiol (E2) treatment. MCF‐7 and PDX‐derived mammospheres exhibited increased size and up‐regulation of canonical stem cell markers ALDH1, NANOG, OCT4 and SOX2 when exposed to mePB; these effects were not observed for MDA‐MB‐231 (ER⁻) mammospheres. As tumor‐initiating cells (TICs) are also believed to be responsible for chemoresistance, mammospheres were treated with either tamoxifen or the pure anti‐estrogen fulvestrant in the presence of mePB. Blocking the estrogenic response was not sufficient to block NANOG expression in mammospheres, pointing to a non‐classic estrogen response or an ER‐independent mechanism of mePB promotion of mammosphere activity. Overall, these results suggest that mePB increases breast cancer tumor proliferation through enhanced TIC activity, in part via regulation of NANOG, and that mePB may play a direct role in chemoresistance by modulating stem cell activity. Copyright © 2016 John Wiley & Sons, Ltd.     

A novel ERα-mediated reporter gene assay for screening estrogenic/antiestrogenic chemicals based on LLC-MK2 cells

Low concentration of endocrine-disrupting chemicals (EDCs) may lead to serious consequences in animals and human, so it is essential to develop an effective assay for EDCs detection. In this study, we developed a novel ERα-mediated reporter gene assay based on the LLC-MK2 cells by co-transfecting pERE-sv40-Luc, hERα-pcDNA3.1, and pRL-tk. Then we determined 17β-estradiol (E2) and some estrogenic/antiestrogenic chemicals to verify the validity of this assay. Data showed that the assay possesses a concentration-dependent responses to E2 and diethylstilbestrol (DES) from 10^?12?M to 10^?8?M with EC₅₀ 3.4?×?10^?10?M and 5.9?×?10^?10?M, and ICI 182,780 completely blocks the luciferase activity induced by 10^?9?M E2. Bisphenol A (BPA), nonylphenol (NP), genistein (GS), and tamoxifen (TAM) also showed corresponding estrogenic or antiestrogenic activity at test concentrations. All evidences proved that the LLC-MK2 reporter gene assay was specific and sensitive to estrogen receptor (ER) agonistic and antagonistic chemicals.     

The in Vitro estrogenic activities of triclosan and triclocarban

Triclosan (TCS) and triclocarban (TCC), as broad spectrum antibacterial agents, are distributed widely in the environment and humans. Most studies have focused on their distribution and biodegradation, but the endocrine‐disrupting effects of these chemicals, especially their estrogenic effects, are still unclear. In the present study, we investigated the estrogenic effects of TCS and TCC using a series of in vitro assays, including the ER reporter gene assay in the CV‐1 cells, E‐screen assay and evaluation of estrogen‐responsive genes in the MCF‐7 cells. The tested concentrations of TCS and TCC were both from 1 × 10^–9 to 1 × 10^–6 M. Results showed that TCS and TCC exerted estrogenic activities by inducing luciferase activities in an ER reporter gene assay, promoting the proliferation of the MCF‐7 cells, up‐regulating the expression of pS2 and down‐regulating ERα expression at both the mRNA and protein levels in the MCF‐7 cells. We further found that TCS and TCC could alter the expression of multiple microRNAs (mir‐22, mir‐206 and mir‐193b) in the MCF‐7 cells, which would help understand the mechanisms of their estrogenic effects on regulating the expression of ERα. In brief, our results demonstrated the potential estrogenic effects and profiled in vitro data for further risk assessment of TCS and TCC. Copyright © 2014 John Wiley & Sons, Ltd.