RESUMEN
Arterial and venous endothelial cells exhibit distinct molecular characteristics at early developmental stages. These lineage-specific molecular programs are instructive to the development of distinct vascular architectures and physiological conditions of arteries and veins, but their roles in angiogenesis remain unexplored. Here, we show that the caudal vein plexus in zebrafish forms by endothelial cell sprouting, migration and anastomosis, providing a venous-specific angiogenesis model. Using this model, we have identified a novel compound, aplexone, which effectively suppresses venous, but not arterial, angiogenesis. Multiple lines of evidence indicate that aplexone differentially regulates arteriovenous angiogenesis by targeting the HMG-CoA reductase (HMGCR) pathway. Treatment with aplexone affects the transcription of enzymes in the HMGCR pathway and reduces cellular cholesterol levels. Injecting mevalonate, a metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenous-dependent requirement for protein prenylation in zebrafish and human endothelial cells.
Asunto(s)
Arterias/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Sulfonamidas/farmacología , Venas/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Animales , Animales Modificados Genéticamente , Arterias/fisiología , Células Cultivadas , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Embrión no Mamífero , Humanos , Terapia Molecular Dirigida , Neovascularización Fisiológica/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Especificidad por Sustrato/efectos de los fármacos , Venas/fisiología , Pez Cebra/embriología , Pez Cebra/metabolismo , Pez Cebra/fisiologíaRESUMEN
Small molecule inhibitors of protein geranylgeranyltransferase-I (GGTase-I) provide a promising type of anticancer drugs. Here, we first report the identification of a novel tetrahydropyridine scaffold compound, P61-E7, and define effects of this compound on pancreatic cancer cells. P61-E7 was identified from a library of allenoate-derived compounds made through phosphine-catalyzed annulation reactions. P61-E7 inhibits protein geranylgeranylation and blocks membrane association of geranylgeranylated proteins. P61-E7 is effective at inhibiting both cell proliferation and cell cycle progression, and it induces high p21(CIP1/WAF1) level in human cancer cells. P61-E7 also increases p27(Kip1) protein level and inhibits phosphorylation of p27(Kip1) on Thr187. We also report that P61-E7 treatment of Panc-1 cells causes cell rounding, disrupts actin cytoskeleton organization, abolishes focal adhesion assembly and inhibits anchorage independent growth. Because the cellular effects observed pointed to the involvement of RhoA, a geranylgeranylated small GTPase protein shown to influence a number of cellular processes including actin stress fiber organization, cell adhesion and cell proliferation, we have evaluated the significance of the inhibition of RhoA geranylgeranylation on the cellular effects of inhibitors of GGTase-I (GGTIs). Stable expression of farnesylated RhoA mutant (RhoA-F) results in partial resistance to the anti-proliferative effect of P61-E7 and prevents induction of p21(CIP1/WAF1) and p27(Kip1) by P61-E7 in Panc-1 cells. Moreover, stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation caused by P61-E7. Taken together, these findings suggest that P61-E7 is a promising GGTI compound and that RhoA is an important target of P61-E7 in Panc-1 pancreatic cancer cells.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Leucina/análogos & derivados , Fosfinas/química , Piridinas/química , Piridinas/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Actinas/metabolismo , Transferasas Alquil y Aril/metabolismo , Animales , Catálisis , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Inhibidores Enzimáticos/síntesis química , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat , Leucina/síntesis química , Leucina/química , Leucina/farmacología , Ratones , Células 3T3 NIH , Prenilación de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Piridinas/síntesis química , Pirroles/química , Sulfonamidas/síntesis química , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Protein geranylgeranyltransferase-I (GGTase-I) catalyzes protein geranylgeranylation, which is critical for the function of proteins such as Rho, Rac, and Ral. We previously identified several small-molecule inhibitors of GGTase-I from an allenoate-derived compound library and showed that these compounds exhibit specific inhibition of GGTase-I resulting in the inhibition of proliferation associated with the induction of G(1) cell cycle arrest of a variety of cancer cell lines. Because inhibition of GGTase-I is expected to suppress tumor growth, we investigated in vivo effects of one of these GGTase-I inhibitors (GGTI), P61A6, by using a human pancreatic cancer xenograft model in mice. The new compound GGTI P61A6 showed an excellent antitumor effect. I.p. administration of P61A6 significantly suppressed tumor growth of the PANC-1 xenograft. Even once per week administration of GGTI was enough to suppress tumor growth. Immunohistochemical examination indicated the inhibition of cell proliferation in the tumors by P61A6 treatment, but neither apoptosis nor antiangiogenesis was observed. Increased cytosolic localization of proteins such as Rap1 and RhoA in tumors was observed. In addition, the enzyme activity of GGTase-I in tumors was inhibited. Pharmacokinetic analysis showed that the plasma half-life of GGTI is â¼6 h, suggesting its prolonged effect. These data suggest that the novel GGTI compound P61A6 is an excellent chemotherapeutic drug candidate for human pancreatic cancer. They also provide evidence that protein GGTase-I may be a valid target for cancer therapy.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/metabolismo , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Pancreáticas/metabolismo , Fenilalanina/análogos & derivados , Sulfonamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , Ratones , Ratones SCID , Neoplasias Pancreáticas/patología , Fenilalanina/administración & dosificación , Fenilalanina/química , Fenilalanina/metabolismo , Fenilalanina/farmacología , Prenilación de Proteína/efectos de los fármacos , Sulfonamidas/administración & dosificación , Sulfonamidas/química , Sulfonamidas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein geranylgeranylation is critical for the function of a number of proteins such as RhoA, Rac, and Rab. Protein geranylgeranyltransferase I (GGTase-I) and Rab geranylgeranyltransferase (RabGGTase) catalyze these modifications. In this work, we first describe the identification and characterization of small molecule inhibitors of GGTase-I (GGTI) with two novel scaffolds from a library consisting of allenoate-derived compounds. These compounds exhibit specific inhibition of GGTase-I and act by competing with a substrate protein. Derivatization of a carboxylic acid emanating from the core ring of one of the GGTI compounds dramatically improves their cellular activity. The improved GGTI compounds inhibit proliferation of a variety of human cancer cell lines and cause G(1) cell cycle arrest and induction of p21(CIP1/WAF1). We also report the identification of novel small molecule inhibitors of RabGGTase. These compounds were identified first by screening our GGTI compounds for those that also exhibited RabGGTase inhibition. This led to the discovery of a common structural feature for RabGGTase inhibitors: the presence of a characteristic six-atom aliphatic tail attached to the penta-substituted pyrrolidine core. Further screening led to the identification of compounds with preferential inhibition of RabGGTase. These compounds inhibit RabGGTase activity by competing with the substrate protein. These novel compounds may provide valuable reagents to study protein geranylgeranylation.