RESUMEN
Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.
Asunto(s)
Humanos , Movimiento Celular/fisiología , Proteínas de Unión al GTP rho/fisiología , Factores de Virulencia/genética , Células Epiteliales/microbiología , Escherichia coli Enteropatógena/patogenicidad , Sistemas de Secreción Tipo III/fisiología , Western Blotting , Apoptosis , Factores de Virulencia/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Citometría de FlujoRESUMEN
Las Rho GTPasas son una familia de proteínas que actúan como interruptores moleculares en diversas vías de señalización coordinando la regulación de distintos procesos celulares. La desregulación de dichas proteínas se vincula con transformación maligna y progresión tumoral en distintos tipos de cáncer. Por estos motivos, en los últimos años las Rho GTPasas fueron postuladas como blancos moleculares interesantes. En este trabajo describimos las distintas estrategias estudiadas utilizando a las Rho GTPasas como blanco y su grado de avance, mostrando una estrategia novedosa para el tratamiento del cáncer.
Rho GTPases are molecular switches that control the different cellular processes. Deregulation of these proteins is associated to transformation and malignant progression in several cancer types. Given the evidence available of the role of Rho GTPases in cancer it is suggested that these proteins can serve as potential therapeutic targets. This review focuses on the strategies used to develop Rho GTPases modulators and their potential use in therapeutic settings.
Asunto(s)
Humanos , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Proteínas de Unión al GTP rho/fisiología , Neoplasias/enzimologíaRESUMEN
Las Rho GTPasas son una familia de proteínas clave en la transmisión de señales provenientes del exterior celular hacia efectores intracelulares tanto citoplasmáticos como nucleares. En los últimos año ha habido un desarrollo vertiginoso de múltiples herramientas genéticas y farmacológicas, lo que ha permitido establecer de manera mucho más precisa las funciones específicas de estas proteínas. El objetivo de la presente revisión es hacer foco en las múltiples funciones celulares reguladas por las Rho GTPasas, describiendo en detalle el mecanismo molecular involucrado. Se discute además la participación de estas proteínas en diversas enfermedades humanas haciendo énfasis en su vinculación con el cáncer. Por último, se hace una actualización detallada sobre las estrategias terapéuticas en experimentación que tienen a las Rho GTPasas como blancos moleculares.
Rho GTPases are a key protein family controlling the transduction of external signals to cytoplasmatic and nuclear effectors. In the last few years, the development of genetic and pharmacological tools has allowed a more precise definition of the specific roles of Rho GTPases. The aim of this review is to describe the cellular functions regulated by these proteins with focus on the molecular mechanism involved. We also address the role of Rho GTPases in the development of different human diseases such as cancer. Finally, we describe different experimental therapeutic strategies with Rho GTPases as molecular targets.