RESUMEN
Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/ß-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/ß-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in ß-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/ß-catenin signaling addicted tumors. E7449 is currently in early clinical development.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Isoquinolinas/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Quinazolinonas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Compuestos Azo , Western Blotting , Carboplatino/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoquinolinas/química , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , Neoplasias/genética , Neoplasias/patología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Quinazolinonas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tanquirasas/antagonistas & inhibidores , Tanquirasas/genética , Tanquirasas/metabolismo , Temozolomida , Carga Tumoral/efectos de los fármacos , Vía de Señalización Wnt/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor necrosis factor α (TNF-α) influences endothelial cell viability by altering the regulatory molecules involved in induction or suppression of apoptosis. However, the underlying mechanisms are still not completely understood. In this study, we demonstrated that A20 (also known as TNFAIP3, tumor necrosis factor α-induced protein 3, and an anti-apoptotic protein) regulates the inhibitor of apoptosis protein-2 (cIAP-2) expression upon TNF-α induction in endothelial cells. Inhibition of A20 expression by its siRNA resulted in attenuating expression of TNF-α-induced cIAP-2, yet not cIAP-1 or XIAP. A20-induced cIAP-2 expression can be blocked by the inhibition of phosphatidyl inositol-3 kinase (PI3-K), but not nuclear factor (NF)-κB, while concomitantly increasing the number of endothelial apoptotic cells and caspase 3 activation. Moreover, TNF-α-mediated induction of apoptosis was enhanced by A20 inhibition, which could be rescued by cIAP-2. Taken together, these results identify A20 as a cytoprotective factor involved in cIAP-2 inhibitory pathway of TNF-α-induced apoptosis. This is consistent with the idea that endothelial cell viability is dependent on interactions between inducers and suppressors of apoptosis, susceptible to modulation by TNF-α.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Apoptosis/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Western Blotting , Caspasa 3/metabolismo , Bovinos , Células Cultivadas , Cromonas/farmacología , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Morfolinas/farmacología , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina-Proteína LigasasRESUMEN
OBJECTIVE: Related transcriptional enhancer factor 1 (RTEF-1) is a key transcriptional regulator in endothelial function. In this study, we investigated a possible role for RTEF-1 in the regulation of microvascular relaxation and the underlying mechanism involved. Activation of fibroblast growth factor receptor 1 (FGFR1) by FGFs increases vasodilation, although transcriptional control of the molecular mechanisms underlying FGFR1 is still unclear. MATERIALS AND METHODS: We demonstrated that RTEF-1 stimulated FGFR1 expression at the transcriptional level, specifically an area including Sp1 elements, as evidenced by promoter assays. Additionally, RTEF-1 increased FGFR1 mRNA and protein expression in vitro and in VE-cadherin-promoted RTEF-1 (VE-Cad/RTEF-1) transgenic mice, whereas RTEF-1 siRNA blocked the upregulation of FGFR1 expression. Furthermore, increased endothelial-dependent microvessel relaxation was observed in the coronary arteries of VE-Cad/RTEF-1 mice, and increased proliferation was observed in RTEF-1-overexpressing cells, both of which correlated to increased FGF/FGFR1 signaling and endothelial nitric oxide synthase (eNOS) upregulation. Our results indicate that RTEF-1 acts as a transcriptional stimulator of FGFR1 and is involved in FGF pathways by increasing microvessel dilatation via eNOS. CONCLUSIONS: These findings suggest that RTEF-1 plays an important role in FGFR1- stimulated vasodilatation. Understanding the effect of RTEF-1 in microvessel relaxation may provide beneficial knowledge in improving treatments in regards to ischemic vascular disorders.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Endotelio Vascular/fisiología , Microvasos/fisiología , Proteínas Musculares/fisiología , Factores de Transcripción/fisiología , Vasodilatación , Animales , Bovinos , Proliferación Celular , Células Cultivadas , Humanos , Óxido Nítrico Sintasa de Tipo III/fisiología , Regiones Promotoras Genéticas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/análisis , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Factores de Transcripción de Dominio TEARESUMEN
We report here that des-methyl, des-amino pateamine A (DMDA-PatA), a structurally simplified analogue of the marine natural product pateamine A, has potent antiproliferative activity against a wide variety of human cancer cell lines while showing relatively low cytotoxicity against nonproliferating, quiescent human fibroblasts. DMDA-PatA retains almost full in vitro potency in P-glycoprotein-overexpressing MES-SA/Dx5-Rx1 human uterine sarcoma cells that are significantly resistant to paclitaxel, suggesting that DMDA-PatA is not a substrate for P-glycoprotein-mediated drug efflux. Treatment of proliferating cells with DMDA-PatA leads to rapid shutdown of DNA synthesis in the S phase of the cell cycle. Cell-free studies show that DMDA-PatA directly inhibits DNA polymerases α and γ in vitro albeit at concentrations considerably higher than those that inhibit cell proliferation. DMDA-PatA shows potent anticancer activity in several human cancer xenograft models in nude mice, including significant regressions observed in the LOX and MDA-MB-435 melanoma models. DMDA-PatA thus represents a promising natural product-based anticancer agent that warrants further investigation.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Epoxi/farmacología , Macrólidos/farmacología , Tiazoles/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Compuestos Epoxi/química , Compuestos Epoxi/uso terapéutico , Femenino , Humanos , Macrólidos/química , Macrólidos/uso terapéutico , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Tiazoles/química , Tiazoles/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fibroblast growth factor receptor-1 (FGFR-1) has been implicated in the process of cardiogenesis, although the underlying molecular mechanisms are poorly understood. In this study, we report the regulation of FGFR-1 expression by related transcriptional enhancer factor-1 (RTEF-1) in vitro (endothelial cells) and in vivo (RTEF-1 transgenic mice). FGFR-1 promoter activity, FGFR-1 mRNA and protein level were measured in bovine aortic endothelial cells (BAEC) in response to RTEF-1 and in endothelial cells isolated from livers in RTEF-1 transgenic mice. RTEF-1 stimulated FGFR-1 promoter activity in a dose-dependent manner. RTEF-1 enhanced FGFR-1 mRNA (4-fold) and protein expression (3.5-fold) whereas RTEF-1 siRNA decreased FGFR-1 protein expression (4-fold). FGFR-1 mRNA and protein expression were also increased in endothelial cells isolated from livers of RTEF-1 transgenic mice. Furthermore, RTEF-1 enhanced tubule formation whereas this was decreased by RTEF-1 knockdown. Moreover, increased relaxation of microvessels was found in RTEF-1 transgenic mice compared to wild-type mice. Our results indicate that RTEF-1 acts as a transcriptional stimulator of FGFR-1 in endothelial cells through its activation of the FGFR-1 promoter. RTEF-1 thus plays an important role in the regulation of FGFR-1 expression. These findings help further understand FGFR activity in angiogenesis and may lead to new therapeutic targets in ischemic vascular disorders.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Proteínas Musculares/metabolismo , Neovascularización Fisiológica/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Factores de Transcripción/metabolismo , Animales , Bovinos , Células Cultivadas , Proteínas de Unión al ADN/genética , Humanos , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genéticaRESUMEN
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor known to be up-regulated in ischemic heart and hypoxic endothelial cells. However, the transcriptional regulation of VEGF in hypoxia-induced angiogenesis is not fully understood. Transcriptional enhancer factor-1 (TEF-1) is a transcriptional factor family that can regulate many genes expressed in cardiac and skeletal muscle cells by binding to myocyte-specific chloramphenicol acetyltransferase heptamer elements in the promoters of these genes. In this study, we demonstrated that related TEF-1 (RTEF-1), a member of the TEF-1 family, is up-regulated in hypoxic endothelial cells. Overexpression of RTEF-1 increases VEGF promoter activity and VEGF expression. Sequential deletion and site-directed mutation analyses of the VEGF promoter demonstrated that a GC-rich region containing four Sp1 response elements, located between -114 and -50, was essential for RTEF-1 function. This region is beyond the hypoxia-inducible factor-1alpha binding site and does not consist of M-CAT-related elements. By electrophoretic mobility shift assay, RTEF-1 was found to interact with the first Sp1 residue (-97 to -87) of the four consecutive Sp1 elements. Binding activity of RTEF-1 to VEGF promoter is also confirmed by chromatin immunoprecipitation. In addition, induction of VEGF promoter activity by RTEF-1 results in an increase of angiogenic processes including endothelial cells proliferation and vascular structure formation. These results indicate that RTEF-1 acts as a transcriptional stimulator of VEGF by regulating VEGF promoter activity through binding to Sp1 site. In addition, RTEF-1-induced VEGF promoter activity was enhanced in a hypoxic condition, indicating that RTEF-1 may play an important role in the regulation of VEGF under hypoxia.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Bovinos , Hipoxia de la Célula , Células Cultivadas , Neovascularización Fisiológica , Regiones Promotoras Genéticas , Factor de Transcripción Sp1 , Factores de Transcripción de Dominio TEARESUMEN
There is increasing evidence that cyclooxygenase (COX)-2 possess both angiogenic and cardioprotective properties. We examined the effects of hypoxic cardiac myocytes (H9c2 cells) on COX-2 expression in human umbilical vein endothelial cells (HUVECs) to determine the pathway involved in COX-2 regulation. The medium from hypoxic (<1% O2) cardiac myocytes (HMCM) or normoxic cardiac myocytes (21% O2) was added to HUVEC cultures. HMCM induced a transient increase of COX-2 mRNA expression at 1 and 3 h without affecting the COX-1 mRNA level. A similar effect also observed in HMCM from cultured primary cardiac myocytes (rat neonatal cardiac myocytes). The increased COX-2 mRNA was associated with a time-dependent increase in COX-2 protein expression. COX-2 was significantly induced by VEGF (4.86 +/- 1.03-fold) and IL-1beta (3.93 +/- 0.89-fold) and slightly increased by TNF-alpha but not by FGF2, IGF-1, or PDGFs. Analysis of proteins secreted in HMCM showed increased levels of VEGF but not IL-1 beta or TNF-alpha. The HMCM-induced COX-2 expression was inhibited by the addition of an anti-VEGF neutralizing antibody. VEGF induced endothelial cell COX-2 expression by both increasing COX-2 transcription and prolonging the COX-2 mRNA half-life. Furthermore, staurosporine, a nonselective PKC inhibitor, prevented the induction of VEGF by hypoxia. Both a selective PKC-alpha and -beta inhibitor and an inducible nitric oxide synthase (NOS) inhibitor decreased the induction of COX-2 by HMCM and VEGF. Finally, HMCM-induced upregulation of COX-2 was accompanied by upregulation of PGI2 and PGE2. These results suggest that VEGF is one of the principal factors produced by hypoxic myocytes that is responsible for the induction of endothelial cell COX-2 expression. This process likely involves both PKC and NOS pathways. Our findings have important implications regarding the cardiac protection of COX-2 in ischemic heart disease.
Asunto(s)
Células Endoteliales/enzimología , Hipoxia/metabolismo , Isoenzimas/metabolismo , Miocitos Cardíacos/enzimología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular , Medios de Cultivo Condicionados/farmacología , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Células Endoteliales/citología , Epoprostenol/biosíntesis , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/genética , Proteínas de la Membrana , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/citología , Prostaglandina-Endoperóxido Sintasas/genética , Ratas , Transducción de Señal/fisiología , Venas Umbilicales/citologíaRESUMEN
Gut-enriched Krüppel-like factor (GKLF/KLF4), a member of the Krüppel-like factor family of transcription factors, has recently been shown to be associated with growth arrest in the colonic mucosa. Our laboratory has previously demonstrated that GKLF is one of the down-stream targets of interferon-gamma (IFN-gamma) in colon cancer cells, but the signaling pathway regulating GKLF expression is not known. In this report, we showed that IFN-gamma increased GKLF and interferon regulatory factor-1 mRNA levels in a parallel fashion and these effects were abolished in STAT1 knockout mouse fibroblast cell lines, indicating that STAT1 mediated IFN-gamma-stimulated GKLF gene expression. IFN-gamma treatment induced phosphorylation of STAT1 and IFN-gamma-induced GKLF mRNA expression was attenuated by tyrosine protein kinase inhibitors, suggesting that phosphorylation of STAT1 played an essential role in this process. To further evaluate the effect of STAT1 on GKLF gene expression, a 2622-bp mouse GKLF promoter was isolated from a liver genomic library. In a transient transfection system, IFN-gamma treatment increased GKLF promoter activity by 3.5-fold. Sequential deletion and mutation analysis of the GKLF promoter has identified the sequence between -1675 and -1580, a region containing a GAS element, to be essential for IFN-gamma function. By electrophoretic mobility gel shift assay, nuclear extracts from IFN-gamma-stimulated HT-29 cells were found to bind to the GAS motif on the GKLF promoter and this protein-DNA complex was supershifted by the STAT1 antiserum. These results indicate that IFN-gamma-induced GKLF expression required phosphorylated STAT1 and that these effects were mediated, in part, through interaction of STAT1 with the GAS element on the GKLF promoter.
Asunto(s)
Neoplasias del Colon/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Interferón gamma/farmacología , Transactivadores/fisiología , Factores de Transcripción/genética , Células Tumorales Cultivadas/efectos de los fármacos , Secuencia de Bases , Northern Blotting , Western Blotting , Neoplasias del Colon/patología , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Expresión Génica , Humanos , Factor 1 Regulador del Interferón , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Factor de Transcripción STAT1 , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas/metabolismo , Tirosina/metabolismo , Regulación hacia ArribaRESUMEN
Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor that expresses in the gastrointestinal tract and mediates growth arrest of colonic epithelium. The molecular mechanisms governing its growth inhibitory effect have not been fully elucidated. In the present study, we showed that induction of GKLF mRNA and protein expression by interferon-gamma treatment was associated with reduction of ornithine decarboxylase (ODC) gene expression and enzyme activity in colon cancer HT-29 cells. Overexpression of GKLF in HT-29 cells significantly reduced ODC mRNA and protein levels as well as enzyme activity and resulted in growth arrest, indicating that ODC might be a downstream target of GKLF. This conclusion was further supported by data showing that GKLF mRNA and protein concentrations were the highest at the G(1)/S boundary of the cell cycle, where ODC mRNA and protein levels were the lowest and that overexpression of GKLF resulted in cell arrested at the G(1) phase. Reporter gene transfection studies and electrophoretic mobility gel shift assays demonstrated that GKLF repressed ODC promoter activity and that these effects appeared to be mediated through interaction with a GC box in the proximal portion of the promoter. Transfection studies using reporter constructs and chromatin immunoprecipitation assays also demonstrated that GKLF inhibited transactivation of the ODC gene by interfering with the binding of Sp1 to the ODC promoter. These results indicate that GKLF may function as a G(1)/S checkpoint regulator and exert its growth arrest effect through down-regulation of ODC gene expression. Furthermore, GKLF is a transcriptional repressor of the ODC gene, and these effects are mediated by interaction with the GC-rich region on the promoter.