RESUMEN
Changes in cellular biomechanical properties affect cell migration and invasion. The natural compound Cucurbitacin B (CuB) has potent anticancer activity; however, the mechanism underlying its inhibitory effect on breast cancer metastasis needs further study. Here, we showed that low-dose CuB inhibited adhesion and altered the viscoelasticity of breast cancer cells, thereby, reducing cell deformability. In vitro and in vivo experiments proved that CuB effectively inhibited the migration and invasion of breast cancer cells. Further studies have found that CuB downregulated the expression of F-actin/vimentin/FAK/vinculin in breast cancer cells, altering the distribution and reorganization of cytoskeletal proteins in the cells. CuB inhibited signaling by the Rho family GTPases RAC1/CDC42/RhoA downstream of integrin. These findings indicate that CuB has been proven to mediate the reorganization and distribution of cytoskeletal proteins of breast cancer cells through RAC1/CDC42/RhoA signaling, which improves the mechanical properties of cell adhesion and deformation and consequently inhibits cell migration and invasion.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Triterpenos/farmacología , Animales , Fenómenos Biomecánicos , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
Signaling through the Rho family of small GTPases has been intensely investigated for its crucial roles in a wide variety of human diseases. Although RhoA and Rac1 signaling pathways are frequently exploited with the aid of effective small molecule modulators, studies of the Cdc42 subclass have lagged because of a lack of such means. We have applied high-throughput in silico screening and identified compounds that are able to fit into the surface groove of Cdc42, which is critical for guanine nucleotide exchange factor binding. Based on the interaction between Cdc42 and intersectin (ITSN), a specific Cdc42 guanine nucleotide exchange factor, we discovered compounds that rendered ITSN-like interactions in the binding pocket. By using in vitro binding and imaging as well as biochemical and cell-based assays, we demonstrated that ZCL278 has emerged as a selective Cdc42 small molecule modulator that directly binds to Cdc42 and inhibits its functions. In Swiss 3T3 fibroblast cultures, ZCL278 abolished microspike formation and disrupted GM130-docked Golgi structures, two of the most prominent Cdc42-mediated subcellular events. ZCL278 reduces the perinuclear accumulation of active Cdc42 in contrast to NSC23766, a selective Rac inhibitor. ZCL278 suppresses Cdc42-mediated neuronal branching and growth cone dynamics as well as actin-based motility and migration in a metastatic prostate cancer cell line (i.e., PC-3) without disrupting cell viability. Thus, ZCL278 is a small molecule that specifically targets Cdc42-ITSN interaction and inhibits Cdc42-mediated cellular processes, thus providing a powerful tool for research of Cdc42 subclass of Rho GTPases in human pathogenesis, such as those of cancer and neurological disorders.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/química , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/química , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/fisiología , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Células 3T3 Swiss , Interfaz Usuario-Computador , Cicatrización de Heridas/efectos de los fármacos , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/fisiologíaRESUMEN
Metastatic squamous cell carcinoma of the head and neck (SCCHN) has been shown to express chemokine receptor 7 (CCR7), which activates phosphoinositide-3 kinase (PI3K) to promote invasion and survival of SCCHN cells. We hypothesized that Cdc42 might be involved in the CCR7-PI3K pathway. Adhesion assays, migration assays, immunofluorescence staining, Western blotting and immunohistochemical analysis were used to find whether Cdc42 can be activated by CCL19 (the CCR7 ligand) and its role in SCCHN. Results showed that CCL19 induced polarized localization of Cdc42 and actin polymerization in the leading edge of migrating cells. The level of activated membrane-bound Cdc42 was elevated, as measured by the GTPase activity pull-down assay. The increased membrane localization and membrane-bound activity of Cdc42 were abolished by CCR7 and PI3K inhibition, indicating the involvement of Cdc42 in the CCR7-PI3K cascade. Knockdown of Cdc42 by small interfering RNA (siRNA) led to significant reduction in the activation of Rac, filamentous actin (F-actin) accumulation as well as in the migration and invasion induced by CCL19. Taken together, our data indicate the important role played by Cdc42 in CCL19-induced migration and invasion of SCCHN cells.
Asunto(s)
Quimiocina CCL19/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Receptores CCR7/agonistas , Receptores CCR7/fisiología , Proteína de Unión al GTP cdc42/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patología , Carcinoma de Células Escamosas , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Quimiocina CCL19/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Receptores CCR7/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología , Carcinoma de Células Escamosas de Cabeza y Cuello , Células Tumorales Cultivadas , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
UNLABELLED: The intracellular signaling pathways controlling chondrocyte physiology are largely unknown. Here we show that the small GTPases, Rac1 and Cdc42, accelerate the rate of chondrocyte differentiation and apoptosis, thereby antagonizing the activity of RhoA. These results identify Rac1 and Cdc42 pathways as novel regulators of cartilage development. INTRODUCTION: Proliferation, hypertrophic differentiation, and ultimate apoptosis of chondrocytes regulate endochondral bone growth and development, but the intracellular signaling pathways controlling chondrocyte biology are incompletely understood. In this study, we investigated the role of the small GTPases Rac1 and Cdc42 in chondrocytes. MATERIALS AND METHODS: Rac1 and Cdc42 expression during chondrogenic differentiation was assessed by RT-PCR and Western blotting. Effects of Rac1 and Cdc42 on parameters of chondrocyte biology were studied using transient transfections into primary mouse chondrocytes and stable transfections of the chondrogenic cell line ATDC5. Luciferase assays, RT-PCR, cell proliferation, alkaline phosphatases assays, staining procedures, TUNEL assays, and caspase activity assays were performed to study the chondrocyte response to overexpression of Rac1 and Cdc42 proteins. Activation of the p38 pathway was analyzed using Western blotting with phospho-specific antibodies, and mitogen-activated protein (MAP) kinase pathways were inhibited using pharmacological approaches. RESULTS AND CONCLUSIONS: Rac1 and Cdc42 activities are required for maximal activity of the collagen X promoter, a hypertrophic marker, in primary chondrocytes, suggesting essential roles of these GTPases in chondrocyte hypertrophy. Overexpression of Rac1 or Cdc42 in chondrogenic ATDC5 cells results in reductions in cell numbers and marked acceleration of hypertrophic differentiation, thus opposing the effects of the related GTPase RhoA. Rac1 and Cdc42 also induce accelerated chondrocyte apoptosis, as shown by TUNEL and caspase activity assays and changes in cell morphology and actin organization. Rac1 and Cdc42 overexpression results in activation of the p38 MAP kinase pathway in ATDC5 cells, and pharmacological inhibition of p38 signaling blocks the effects of Rac1 and Cdc42 overexpression on hypertrophy and apoptosis. Our results therefore suggest that Rac1 and Cdc42 signaling accelerates progression through the chondrocyte life cycle in a p38-dependent fashion and antagonizes RhoA signaling pathways in chondrocyte proliferation, hypertrophy, and apoptosis.
Asunto(s)
Condrocitos/citología , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rac1/fisiología , Proteína de Unión al GTP rhoA/fisiología , Actinas/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Apoptosis , Western Blotting , Diferenciación Celular , Línea Celular , Proliferación Celular , Colágeno Tipo X/genética , ADN Complementario/metabolismo , Activación Enzimática , Hipertrofia , Etiquetado Corte-Fin in Situ , Luciferasas/metabolismo , Ratones , Regiones Promotoras Genéticas , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.
Asunto(s)
Endotelio Vascular/fisiología , Fosfatidilcolinas/farmacología , Esfingosina/análogos & derivados , Proteína de Unión al GTP cdc42/fisiología , Proteínas de Unión al GTP rac/fisiología , Hidroxitolueno Butilado/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , ADN Complementario/genética , Dimiristoilfosfatidilcolina/farmacología , Impedancia Eléctrica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Isoprostanos/aislamiento & purificación , Isoprostanos/farmacología , Lisofosfolípidos/farmacología , Oxidación-Reducción , Fosfatidilcolinas/aislamiento & purificación , Arteria Pulmonar/citología , ARN Interferente Pequeño/farmacología , Espectrometría de Masa por Ionización de Electrospray , Esfingosina/farmacología , Relación Estructura-Actividad , Trombina/farmacología , Transfección , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.