RESUMEN
Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17ß-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals.
Asunto(s)
Aromatasa/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Receptores de Estrógenos/efectos de los fármacos , Secuencia de Bases , Cartilla de ADN , Humanos , Células MCF-7RESUMEN
Aromatase inhibitors (AI) are currently the first line therapy for estrogen receptor (ER)-positive postmenopausal women. De novo AI resistance is when a patient intrinsically does not respond to an AI therapy as well as other targeted endocrine therapy. To characterize this type of resistance and to examine potential therapies for treatment, we have generated two cell models for de novo resistance. These models derive from MCF-7 cells that stably overexpress aromatase and Akt (AKT-aro) or HER2 (HER2-aro). Evaluation of these cell lines revealed that the activities of aromatase and ER were inhibited by AI and ICI 187280 (ICI) treatment, respectively; however, cell growth was resistant to therapy. Proliferation in the presence of the pure anti-estrogen ICI, indicates that these cells do not require ER for cell growth and distinguishes these cells from the acquired AI resistant cells. We further determined that the HSP90 inhibitor 17-DMAG suppressed the growth of the AI-resistant cell lines studied. Our analysis revealed 17-DMAG-mediated decreased expression of growth promoting signaling proteins. It was found that de novo AI resistant AKT-aro and HER2-aro cells could not be resensitized to letrozole or ICI by treatment with 17-DMAG. In summary, we have generated two cell lines which display the characteristics of de novo AI resistance. Together, these data indicate the possibility that HSP90 inhibitors may be a viable therapy for endocrine therapy resistance although additional clinical evaluation is needed.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de la Aromatasa/farmacología , Aromatasa/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos/uso terapéutico , Inhibidores de la Aromatasa/uso terapéutico , Benzoquinonas/farmacología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia sin Enfermedad , Sinergismo Farmacológico , Estradiol/análogos & derivados , Estradiol/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Fulvestrant , Regulación Neoplásica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Lactamas Macrocíclicas/farmacología , Letrozol , Nitrilos/farmacología , Receptores de Estrógenos/metabolismo , Activación Transcripcional , Triazoles/farmacologíaRESUMEN
Estrogen plays important roles in hormone receptor-positive breast cancer. Endocrine therapies, such as the antiestrogen tamoxifen, antagonize the binding of estrogen to estrogen receptor (ER), whereas aromatase inhibitors (AIs) directly inhibit the production of estrogen. Understanding the mechanisms of endocrine resistance and the ways in which we may better treat these types of resistance has been aided by the development of cellular models for resistant breast cancers. In this review, we will discuss what is known thus far regarding both de novo and acquired resistance to tamoxifen or AIs. Our laboratory has generated a collection of AI- and tamoxifen-resistant cell lines in order to comprehensively study the individual types of resistance mechanisms. Through the use of microarray analysis, we have determined that our cell lines resistant to a particular AI (anastrozole, letrozole, or exemestane) or tamoxifen are distinct from each other, indicating that these mechanisms can be quite complex. Furthermore, we will describe two novel de novo AI-resistant cell lines that were generated from our laboratory. Initial characterization of these cells reveals that they are distinct from our acquired AI-resistant cell models. In addition, we will review potential therapies which may be useful for overcoming resistant breast cancers through studies using endocrine resistant cell lines. Finally, we will discuss the benefits and shortcomings of cell models. Together, the information presented in this review will provide us a better understanding of acquired and de novo resistance to tamoxifen and AI therapies, the use of appropriate cell models to better study these types of breast cancer, which are valuable for identifying novel treatments and strategies for overcoming both tamoxifen and AI-resistant breast cancers.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Investigación Biomédica Traslacional/métodos , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , HumanosRESUMEN
It has been demonstrated that growth factors produced by breast cancer cells stimulate aromatase expression in both breast cancer and adjacent adipose fibroblasts and stromal cells. However, whether these growth factors affect aromatase activity by other mechanisms still remain unclear. In the current study, MCF-7aro and T47Daro aromatase transfected breast carcinoma cells were used to explore the mechanisms of post-transcriptional regulation of aromatase activity by growth factor pathways. Our study reveals that PI3K/Akt and MAPK inhibitors suppressed aromatase activity in MCF-7aro cells. However, PI3K/Akt pathway inhibitors stimulated aromatase activity in T47Daro cells. This is due to enhanced MAPK phosphorylation as compensation after the PI3K/Akt pathway has been blocked. IGF-1 treatment increased aromatase activity in both breast cancer cell lines. In addition, LTEDaro cells (long-term estrogen deprived MCF-7aro cells) which have enhanced MAPK activity, show higher aromatase activity compared to parental MCF-7aro cells, but the aromatase protein level remains the same. These results suggest that aromatase activity could be enhanced by growth factor signaling pathways via post-transcriptional mechanisms.
Asunto(s)
Aromatasa/metabolismo , Neoplasias de la Mama/enzimología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Transducción de Señal , Aromatasa/genética , Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Aromatase inhibitors are important drugs to treat estrogen receptor alpha (ERalpha)-positive postmenopausal breast cancer patients. However, development of resistance to aromatase inhibitors has been observed. We examined whether the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) can inhibit the growth of aromatase inhibitor-resistant breast cancers and the mechanisms by which 17-DMAG affects proliferation. Aromatase inhibitor-responsive MCF-7aro and aromatase inhibitor-resistant LTEDaro breast epithelial cells were used in this study. We observed that 17-DMAG inhibited proliferation in both MCF-7aro and LTEDaro cells in a dose-dependent manner. 17-DMAG induced apoptosis and G(2) cell cycle arrest in both cell lines. Although inhibition of HSP90 decreased the levels of ERalpha, the ERalpha transcriptional activity was not affected when cells were treated with 17-DMAG together with estradiol. Moreover, detailed mechanistic studies suggested that 17-DMAG inhibits cell growth via degradation of HSP90 client proteins AKT and HER2. Collectively, results from this study provide data to support that HSP90 inhibitors may be an effective therapy to treat aromatase inhibitor-resistant breast cancers and that improved efficacy can be achieved by combined use of a HSP90 inhibitor and an AKT inhibitor.
Asunto(s)
Antineoplásicos/farmacología , Benzoquinonas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de la Aromatasa/uso terapéutico , Western Blotting , Neoplasias de la Mama , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/efectos de los fármacos , Femenino , Proteínas HSP90 de Choque Térmico/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Receptor ErbB-2/efectos de los fármacosRESUMEN
OBJECTIVE: The purpose of this study was to evaluate the role of coactivator histone acetyltransferases (HATs) p300 and SRC-1 in angiotensin II (Ang II)-induced interleukin-6 (IL-6) gene expression in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Ang II increased IL-6 mRNA expression via NF-kappaB and CREB in an extracellular signal-regulated kinase (ERK)-dependent manner in rat VSMCs. It was also significantly enhanced by the histone deacetylase inhibitor, Trichostatin A. Chromatin immunoprecipitation (ChIP) assays showed that Ang II increased Histone H3 Lysine (K9/14) acetylation on the IL-6 promoter. Ang II-induced IL-6 promoter transactivation was significantly enhanced by p300 and SRC-1, with maximal activation in cells cotransfected with NF-kappaB (p65) and SRC-1. Nucleofection of VSMCs with either an ERK phosphorylation site mutant of SRC-1 or p300/CBP HAT deficient mutants significantly blocked Ang II-induced IL-6 expression. ChIP assays revealed that Ang II enhanced coordinate occupancy of p65, CREB, p300, and SRC-1 at the IL-6 promoter. An ERK pathway inhibitor blocked Ang-induced IL-6 promoter SRC-1 occupancy and histone acetylation. CONCLUSIONS: Ang II-induced IL-6 expression requires NF-kappaB and CREB as well as ERK-dependent histone acetylation mediated by p300 and SRC-1. These results provide new insights into nuclear chromatin mechanisms by which Ang II regulates inflammatory gene expression.
Asunto(s)
Angiotensina II/metabolismo , Proteína de Unión a CREB/metabolismo , Histona Acetiltransferasas/metabolismo , Interleucina-6/metabolismo , Músculo Liso Vascular/enzimología , Factores de Transcripción/metabolismo , Acetilación , Angiotensina II/farmacología , Animales , Células Cultivadas , Proteína p300 Asociada a E1A/metabolismo , Regulación de la Expresión Génica , Interleucina-6/genética , Masculino , Modelos Animales , FN-kappa B/metabolismo , Coactivador 1 de Receptor Nuclear , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
Bacillus thuringiensis (Bt) crystal proteins are pore-forming toxins used as insecticides around the world. Previously, the extent to which these proteins might also target the invertebrate phylum Nematoda has been mostly ignored. We have expressed seven different crystal toxin proteins from two largely unstudied Bt crystal protein subfamilies. By assaying their toxicity on diverse free-living nematode species, we demonstrate that four of these crystal proteins are active against multiple nematode species and that each nematode species tested is susceptible to at least one toxin. We also demonstrate that a rat intestinal nematode is susceptible to some of the nematicidal crystal proteins, indicating these may hold promise in controlling vertebrate-parasitic nematodes. Toxicity in nematodes correlates with damage to the intestine, consistent with the mechanism of crystal toxin action in insects. Structure-function analyses indicate that one novel nematicidal crystal protein can be engineered to a small 43-kDa active core. These data demonstrate that at least two Bt crystal protein subfamilies contain nematicidal toxins.