RESUMEN
Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.
Asunto(s)
Proteínas Portadoras/metabolismo , Transformación Celular Neoplásica/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Neuroblastoma/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Inmunohistoquímica , Espectroscopía de Resonancia Magnética , Ratones , Ratones Desnudos , Modelos Moleculares , Mutación , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/genética , Metástasis de la Neoplasia , Neuroblastoma/genética , Neuroblastoma/patología , Péptidos/genética , Péptidos/metabolismo , Monoéster Fosfórico Hidrolasas , Unión Proteica , Estructura Terciaria de Proteína , Trasplante HeterólogoRESUMEN
Dipyridamole is a widely prescribed drug in ischemic disorders, and it is here investigated for potential clinical use as a new treatment for breast cancer. Xenograft mice bearing triple-negative breast cancer 4T1-Luc or MDA-MB-231T cells were generated. In these in vivo models, dipyridamole effects were investigated for primary tumor growth, metastasis formation, cell cycle, apoptosis, signaling pathways, immune cell infiltration, and serum inflammatory cytokines levels. Dipyridamole significantly reduced primary tumor growth and metastasis formation by intraperitoneal administration. Treatment with 15 mg/kg/day dipyridamole reduced mean primary tumor size by 67.5 % (p = 0.0433), while treatment with 30 mg/kg/day dipyridamole resulted in an almost a total reduction in primary tumors (p = 0.0182). Experimental metastasis assays show dipyridamole reduces metastasis formation by 47.5 % in the MDA-MB-231T xenograft model (p = 0.0122), and by 50.26 % in the 4T1-Luc xenograft model (p = 0.0292). In vivo dipyridamole decreased activated ß-catenin by 38.64 % (p < 0.0001), phospho-ERK1/2 by 25.05 % (p = 0.0129), phospho-p65 by 67.82 % (p < 0.0001) and doubled the expression of IkBα (p = 0.0019), thus revealing significant effects on Wnt, ERK1/2-MAPK and NF-kB pathways in both animal models. Moreover dipyridamole significantly decreased the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in primary tumors (p < 0.005), and the inflammatory cytokines levels in the sera of the treated mice. We suggest that when used at appropriate doses and with the correct mode of administration, dipyridamole is a promising agent for breast-cancer treatment, thus also implying its potential use in other cancers that show those highly activated pathways.
Asunto(s)
Neoplasias de la Mama/prevención & control , Dipiridamol/uso terapéutico , Neoplasias Pulmonares/prevención & control , Inhibidores de Fosfodiesterasa/uso terapéutico , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Medulloblastoma is one of the leading causes of morbidity and mortality in pediatric cancer. Wnt-active tumors, an independent molecular subgroup in medulloblastoma, are characterized by a distinct pattern of genomic aberrations. We assessed the anticancer activity of cantharidin and norcantharidin against medulloblastoma, as cell lines in vitro and in athymic nude mice in vivo. Cantharidin and norcantharidin treatment impaired the growth of DAOY and UW228 medulloblastoma cells and promoted the loss of ß-catenin activation and the ß-catenin nuclearization linked to N-cadherin impairment in vitro. Intra-peritoneal administration of norcantharidin inhibited the growth of intra-cerebellum tumors in orthotopic xenograft nude mice. Analysis of the xenograft tissues revealed enhanced neuronal differentiation and reduced ß-catenin expression. Our findings suggest that norcantharidin has potential therapeutic applications in the treatment of medulloblastoma as a result of its ability to cross the blood-brain barrier and its impairment of Wnt-ß-catenin signaling.
