RESUMEN
The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta's smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.
Asunto(s)
Bradiquinina , Receptor de Bradiquinina B1 , Animales , Aorta , Bradiquinina/farmacología , Endotelio Vascular , Técnicas In Vitro , Lipopolisacáridos/farmacología , Potenciales de la Membrana , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Receptor de Bradiquinina B1/genética , Tapsigargina/farmacología , Receptor Toll-Like 4RESUMEN
Our aim was to evaluate whether endothelial overexpressing of the bradykinin B1 receptor could be associated with altered left ventricular and myocardial performance. Echocardiography and hemodynamic were employed to assess left ventricular morphology and function in Sprague Dawley transgenic rats overexpressing the endothelial bradykinin B1 receptor (Tie2B1 rats). The myocardial inotropism was evaluated on papillary muscles contracting in vitro. In Tie2B1 animals, an enlarged left ventricular cavity and lower fractional shortening coupled with a lower rate of pressure change values indicated depressed left ventricular performance. Papillary muscle mechanics revealed that both Tie2B1 and wild-type rat groups had the same contractile capacities under basal conditions; however, in transgenic animals, there was accentuated inotropism due to post-pause potentiation. Following treatment with the Arg(9)-BK agonist, Tie2B1 papillary muscles displayed a reduction in myocardial inotropism. Endothelial B1 receptor overexpression has expanded the LV cavity and worsened its function. There was an exacerbated response of papillary muscle in vitro to a prolonged resting pause, and the use of a B1 receptor agonist impairs myocardial inotropism.
Asunto(s)
Células Endoteliales/metabolismo , Contracción Miocárdica , Músculos Papilares/metabolismo , Receptor de Bradiquinina B1/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Función Ventricular Izquierda , Animales , Predisposición Genética a la Enfermedad , Masculino , Músculos Papilares/fisiopatología , Fenotipo , Ratas Sprague-Dawley , Ratas Transgénicas , Receptor de Bradiquinina B1/genética , Regulación hacia Arriba , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/fisiopatología , Remodelación VentricularRESUMEN
Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1-/- mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK's effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.
Asunto(s)
Compuestos de Bifenilo/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Lignanos/administración & dosificación , Proteínas Serina-Treonina Quinasas/genética , Factor de Transcripción STAT3/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica , Femenino , Humanos , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas Serina-Treonina Quinasas/biosíntesis , Factor de Transcripción STAT3/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Loss of HOXA5 expression occurs frequently in breast cancer and correlates with higher pathological grade and poorer disease outcome. However, how HOX proteins drive differentiation in mammalian cells is poorly understood. In this paper, we investigated cellular and molecular consequences of loss of HOXA5 in breast cancer, and the role played by retinoic acid in HOXA5 function. Analysis of global gene expression data from HOXA5-depleted MCF10A breast epithelial cells, followed by validation, pointed to a role for HOXA5 in maintaining several molecular traits typical of the epithelial lineage such as cell-cell adhesion, tight junctions and markers of differentiation. Depleting HOXA5 in immortalized MCF10A or transformed MCF10A-Kras cells reduced their CD24+/CD44lo population, enhanced self-renewal capacity and reduced expression of E-cadherin (CDH1) and CD24. In the case of MCF10A-Kras, HOXA5 loss increased branching and protrusive morphology in Matrigel, all features suggestive of epithelial to basal transition. Further, orthotopically implanted xenografts of MCF10A-Kras-scr grew as well-differentiated pseudo-luminal carcinomas, while MCF10A-Kras-shHOXA5 cells formed aggressive, poorly differentiated carcinomas. Conversely, ectopic expression of HOXA5 in aggressive SUM149 or SUM159 breast cancer cells reversed the cellular and molecular alterations observed in the HOXA5-depleted cells. Retinoic acid is a known upstream regulator of HOXA5 expression. HOXA5 depletion in MCF10A cells engineered to express doxycycline-induced shHOXA5 slowed transition of cells from a less differentiated CD24-/CD44+ to the more differentiated CD24+/CD44+ state. This transition was promoted by retinal treatment, which upregulated endogenous HOXA5 expression and caused re-expression of occludin and claudin-7 (CLDN7). Expression of CDH1 and CD24 was transcriptionally upregulated by direct binding of HOXA5 to their promoter sequences as demonstrated by luciferase and ChIP analyses. Thus, loss of HOXA5 in mammary cells leads to loss of epithelial traits, an increase in stemness and cell plasticity, and the acquisition of more aggressive phenotypes.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Antígeno CD24/genética , Cadherinas/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Animales , Antígenos CD , Cadherinas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Autorrenovación de las Células/genética , Análisis por Conglomerados , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Perfilación de la Expresión Génica , Xenoinjertos , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Clasificación del Tumor , Regiones Promotoras Genéticas , Unión Proteica , Células Madre/metabolismoRESUMEN
1. The effect of endogenous glucocorticoid hormones on the expression of rat B(1) receptors was examined by means of molecular and pharmacological functional approaches. 2. Rats were adrenalectomized (ADX), and 7 days after this procedure the intradermal injection of B(1) receptor agonist des-Arg(9)-BK produced a significant increase in the paw volume, while only a weak effect was observed in sham-operated animals. A similar increase in the contractile responses mediated by B(1) agonist des-Arg(9)-BK was also observed in the rat portal vein in vitro. 3. Chemical ADX performed with mitotane (a drug that reduces corticosteroid synthesis) produced essentially the same up-regulation of B(1) receptors as that observed in ADX rats. 4. The modulation of B(1) receptor expression was evaluated by ribonuclease protection assay, employing mRNA obtained from the lungs and paw of ADX rats. 5. Additionally, both paw oedema and contraction of portal vein mediated by B(1) agonist des-Arg(9)-BK in ADX rats, were markedly inhibited by treatment with dexamethasone, or COX-2 inhibitor meloxican, or with the NF-kappaB inhibitor PDTC. Interestingly, the same degree of inhibition was achieved when the animals were treated with a combination of submaximal doses of dexamethasone and PDTC. 6. The involvement of NF-kappaB pathway was further confirmed by mobility shift assay using nuclear extracts from lung, paw and heart of ADX rats. It was also confirmed that the treatment of ADX rats with dexamethasone, PDTC or dexamethasone plus PDTC completely inhibit NF-kappaB activation caused by absence of endogenous glucucorticoid. 7. Together, the results of the present study provide, for the first time, molecular and pharmacological evidence showing that B(1) kinin receptor expression can be regulated through endogenous glucocorticoids by a mechanism dependent on NF-kappaB pathway. Clinical significance of the present findings stem from evidence showing the importance of B(1) kinin receptors in the mediation of inflammatory and pain related responses.