RESUMEN

2-Methoxyestradiol (2ME), an endogenous metabolite of 17ß-estradiol, has been previously reported to possess antiangiogenic and antitumor properties. Herein, we demonstrate that the effects of this antiangiogenic steroid can be readily assayed in live zebrafish, introducing a convenient and robust new model system as a screening tool for both single cell and collective cell migration assays. Using the in vitro mammalian endothelial cell line EA.hy926, we first show that cell migration and angiogenesis, as estimated by wound assay and tube formation respectively, are antagonized by 2ME. In zebrafish (Danio rerio) larvae, dose-dependent exposure to 2ME diminishes (1) larval angiogenesis, (2) leukocyte recruitment to damaged lateral line neuromasts and (3) retards the lateral line primordium in its migration along the body. Our results indicate that 2ME has an effect on collective cell migration in vivo as well as previously reported anti-tumorigenic activity and suggests that the molecular mechanisms governing cell migration in a variety of contexts are conserved between fish and mammals. Moreover, we exemplify the versatility of the zebrafish larvae for testing diverse physiological processes and screening for antiangiogenic and antimigratory drugs in vivo.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Estradiol/análogos & derivados , 2-Metoxiestradiol , Animales , Animales Modificados Genéticamente , Línea Celular , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Larva/genética , Larva/metabolismo , Larva/fisiología , Microscopía Fluorescente , Neovascularización Fisiológica/efectos de los fármacos , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología

RESUMEN

OBJECTIVE: To determine the functional role of Smad7 in granulosa cells. DESIGN: Granulosa cell culture and molecular biological techniques were used to investigate regulation and function of Smad7. SETTING: Research laboratory. ANIMAL(S): C57bl/j hybrid mouse. INTERVENTION(S): Primary mouse granulosa cells were isolated and grown in culture for all messenger RNA expression experiments. Smad7 promoter constructs were evaluated with a luciferase reporter system in SIGC cells to determine sites activating Smad7 expression. MAIN OUTCOME MEASURE(S): Overexpression (Smad7 complementary DNA) and downregulation (Smad7 small interfering RNA) of Smad7 in primary mouse granulosa cells were used to evaluate the functional role of Smad7 in granulosa cells. RESULT(S): Smad7 expression was upregulated by treatment with transforming growth factor-ß (TGF-ß) but not activin or activation of the cyclic adenosine monophosphate pathway. The promoter of Smad7 was activated by TGF-ß. Truncation of the promoter or mutation of the Smad response element at -141 eliminated TGF-ß activation of the promoter. Smad3 was not specifically required for TGF-ß-stimulated expression of Smad7, though activation of the TGFBR1 receptor was. When Smad7 was overexpressed in granulosa cells, apoptosis was markedly increased. When Smad7 expression was reduced with small interfering RNA, then the TGF-ß-induced apoptosis was blocked. CONCLUSION(S): Smad7 mediates apoptosis induced by TGF-ß in mouse granulosa cells, suggesting that dysregulation of Smad7 could impair folliculogenesis.

Asunto(s)

Apoptosis/fisiología , Células de la Granulosa , Oogénesis/fisiología , Proteína smad7/genética , Factor de Crecimiento Transformador beta/farmacología , Animales , Apoptosis/efectos de los fármacos , Femenino , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/fisiología , Luciferasas/genética , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética

RESUMEN

Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

Asunto(s)

Neoplasias de la Mama/patología , Carcinoma/patología , Movimiento Celular/efectos de los fármacos , Adhesiones Focales/efectos de los fármacos , Progesterona/farmacología , Receptor PAR-1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Evaluación Preclínica de Medicamentos , Femenino , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes erbB-1/fisiología , Humanos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 1/fisiología , Posmenopausia/genética , Posmenopausia/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Receptor PAR-1/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética

RESUMEN

Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.

Asunto(s)

Coagulación Sanguínea , Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/metabolismo , Progesterona/metabolismo , Tromboplastina/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Factor VIIa/metabolismo , Factor X/metabolismo , Femenino , Humanos , Microdominios de Membrana/metabolismo , Invasividad Neoplásica , Transporte de Proteínas , Receptores de Progesterona/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

2-Methoxyestradiol (2ME) is an endogenous metabolite of 17ß-estradiol with antiangiogenic and antitumor properties, although its mechanisms of action remain unclear. Progestins in hormone replacement therapy increase the risk of breast cancer. Progesterone also enhances the procoagulant activity and invasive potential of progesterone receptor (PR)-positive breast cancer cell lines, an effect largely mediated by induction of tissue factor (TF), the cellular activator of the coagulation cascade. Here we show that 2ME abrogates the induction TF expression in progesterone-treated breast cancer cells via a mechanism that does not involve the estrogen receptor. Instead, we demonstrate that by selectively antagonizing ERK1/2 signaling in breast cancer cells, 2ME limits the transactivation potential of ligand-bound PR and inhibits the expression of endogenous progesterone targets, such as TF and signal transducer and activator of transcription 5. We further demonstrate that 2ME can alter the phosphorylation status of PR. Thus, 2ME prevents progesterone-dependent increase in breast cancer cell invasiveness and procoagulant activity by uncoupling PR from the ERK1/2 signal transduction pathway.

Asunto(s)

Neoplasias de la Mama/metabolismo , Estradiol/análogos & derivados , Progesterona/metabolismo , Tromboplastina/biosíntesis , Moduladores de Tubulina/farmacología , 2-Metoxiestradiol , Western Blotting , Línea Celular Tumoral , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional/efectos de los fármacos , Transfección

RESUMEN

BACKGROUND: Gene expression profiling of normal receptive endometrium has been characterized, but intrinsic defects in endometrial gene expression associated with implantation failure have not been reported. METHODS: Women who had previously participated as recipients in oocyte donation cycles and repeatedly exhibited implantation failure (Group A, study group) or had at least one successful cycle (Group B, control group) and spontaneously fertile women (Group C, normal fertility group) were recruited. All were treated with exogenous estradiol and progesterone to induce an endometrial cycle, and an endometrial biopsy was taken on the seventh day of progesterone administration. RNA from each sample was analysed by cDNA microarrays to identify differentially expressed genes between groups. RESULTS: 63 transcripts were differentially expressed (>or=2-fold) between Groups A and B, of which 16 were subjected to real time RT-PCR. Eleven of these were significantly decreased in Group A with regard to Groups B and C. Among the dysregulated genes were MMP-7, CXCR4, PAEP and C4BPA. CONCLUSIONS: Repeated implantation failure in some oocyte recipients is associated with an intrinsic defect in the expression of multiple genes in their endometrium. Significantly decreased levels of several transcripts in endometria without manifest abnormalities is demonstrated for the first time and shown to be associated with implantation failure.

Asunto(s)

Implantación del Embrión , Endometrio/metabolismo , Perfilación de la Expresión Génica , Embarazo , ARN Mensajero/metabolismo , Adulto , Proteína de Unión al Complemento C4b , Femenino , Glicodelina , Glicoproteínas/genética , Antígenos de Histocompatibilidad/genética , Humanos , Metaloproteinasa 7 de la Matriz/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Gestacionales/genética , Receptores CXCR4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

OBJECTIVE: To compare mRNA and protein levels of proenkephalin A (PEA) and gamma-aminobutyric acid A receptor pi subunit (piGABA-R) in human secretory endometrium before and during receptivity and to determine the cell phenotypes where they are expressed. DESIGN: Prospective and observational, comparing prereceptive vs. receptive stages of secretory endometrium within the same nonconceptional menstrual cycle. SETTING: University and non-governmental organization (NGO)-based academic and clinical-research facilities. PATIENT(S): Seven healthy, multiparous, surgically sterilized women with spontaneous regular menstrual cycles. INTERVENTION(S): Endometrial biopsies were obtained on LH+3 and LH+7 within the same cycle. MAIN OUTCOME MEASURE(S): Levels of PEA and piGABA-R mRNA were determined by real-time PCR, and protein presence, by immunofluorescence. RESULT(S): The mRNA level of PEA fell, whereas that of piGABA-R increased, during endometrial receptivity. Positive immunostaining of PEA was found in the luminal and glandular epithelium, whereas that of piGABA-R was in luminal epithelium and stromal cells. CONCLUSION(S): The discrete cell-phenotype localization and timing of the changes in the level of PEA and of piGABA-R mRNA and protein suggest an important role for these molecules in switching the human endometrium from a refractory to a receptive state.

Asunto(s)

Endometrio/metabolismo , Encefalinas/metabolismo , Fase Luteínica/metabolismo , Precursores de Proteínas/metabolismo , Receptores de GABA-A/metabolismo , Adulto , Femenino , Expresión Génica/fisiología , Humanos , Subunidades de Proteína/metabolismo , Distribución Tisular