RESUMEN
Polyphosphate (polyP) is abundant in bone but its roles in signaling and control of gene expression remain unclear. Here, we investigate the effect of extracellular polyP on proliferation, migration, apoptosis, gene and protein expression in human osteoblast-like SaOS-2 cells. Extracellular polyP promoted SaOS-2 cell proliferation, increased rates of migration, inhibited apoptosis and stimulated the rapid phosphorylation of extracellular-signal-regulated kinase (ERK) directly through basic fibroblast growth factor receptor (bFGFR). cDNA microarray revealed that polyP induced significant upregulation of interleukin 11 (IL-11) at both RNA and protein levels.
Asunto(s)
Interleucina-11/genética , Interleucina-11/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Polifosfatos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/citología , Fosforilación , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Inorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzyme's catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC(50) of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC(50) of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/enzimología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Clonación Molecular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , G-Cuádruplex , Cinética , Modelos Moleculares , Mutación , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND & AIMS: Nilotinib is a novel tyrosine kinase inhibitor of Bcr-Abl and other kinases. In this study, we have examined its activity as an anti-fibrotic agent. METHODS: The in vitro effect of Nilotinib on rat and human HSCs was assessed using proliferation assays and Western blotting. The in vivo antifibrotic efficacy of Nilotinib was assessed in mice with liver fibrosis induced by CCl(4) and bile duct ligation (BDL). RESULTS: Nilotinib inhibited proliferation, migration, and actin filament formation, as well as the expression of α-SMA and collagen in activated HSCs. Nilotinib induced apoptosis of HSCs, which was correlated with reduced bcl-2 expression, increased p53 expression, cleavage of PARP, as well as increased expression of PPARγ and TRAIL-R. Nilotinib also induced cell cycle arrest, accompanied by increased expression of p27 and downregulation of cyclin D1. Interestingly, Nilotinib not only inhibited activation of PDGFR, but also TGFRII through Src. Nilotinib significantly inhibited PDGF and TGFß-simulated phosphorylation of ERK and Akt. Furthermore, PDGF- and TGFß-activated phosphorylated form(s) of Abl in human HSCs were inhibited by Nilotinib. In vivo, Nilotinib reduced collagen deposition and α-SMA expression in CCl(4) and BDL-induced fibrosis. These beneficial effects were associated with suppressed expression of procollagen-(I), TIMP-1, CD31, CD34, VEGF, and VEGFR. Nilotinib could induce HSC undergoing apoptosis in vivo, which was correlated with downregulation of bcl-2. We also observed reduced expression of phosphorylated ERK, Akt, and Abl in the Nilotinib-treated CCl(4) and BDL livers. In addition to its antifibrotic activity, the drug was hepatoprotective and reduced the elevations of ALT and AST after CCl(4) and BDL. CONCLUSIONS: These studies uncover a novel role of Bcr-Abl activity in treatment of liver fibrosis through multiple mechanisms and indicate that Nilotinib represents a potentially effective antifibrotic agent.
Asunto(s)
Genes abl/efectos de los fármacos , Cirrosis Hepática/patología , Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/efectos de los fármacos , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Conductos Biliares , Tetracloruro de Carbono , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ligadura , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , PPAR gamma/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
Stimulation of hepatic stellate cells (HSCs) by platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) is an essential pathway of proliferation and fibrogenesis, respectively, in liver fibrosis. We provide evidence that PTK787/ZK222584 (PTK/ZK), a potent tyrosine kinase inhibitor that blocks vascular endothelial growth factor receptor (VEGFR), significantly inhibits PDGF receptor expression, as well as PDGF-simulated HSC proliferation, migration and phosphorylation of ERK1/2, Akt and p70S6 kinase. Interestingly, PTK/ZK also antagonizes the TGF-beta1-induced expression of VEGF and VEGFR1. Furthermore, PTK/ZK downregulates TGF-beta receptor expression, which is associated with reduced Akt, ERK and p38MAPK phosphorylation. Furthermore, PDGF-induced TGF-beta1 expression is inhibited by PTK/ZK. These findings provide evidence that PTK/ZK targets multiple essential pathways of stellate cell activation that provoke proliferation and fibrogenesis. Our study underscores the potential use of PTK/ZK as an antifibrotic drug in chronic liver disease.
Asunto(s)
Células Estrelladas Hepáticas/efectos de los fármacos , Ftalazinas/farmacología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Ftalazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/uso terapéutico , ARN Mensajero/metabolismo , Ratas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasas raf/metabolismoRESUMEN
Liver fibrosis due to hepatic stellate cell (HSC) activation represents a common response to chronic liver injury. PTK787/ZK222584 (PTK/ZK) is a pan-VEGFR tyrosine kinase inhibitor. The aim of this study was to examine the effect of PTK/ZK in liver fibrosis. In primary HSCs, PTK/ZK inhibited the expression of alpha-smooth muscle actin (alpha-SMA), collagen, tissue inhibitor of metalloproteinase-1 (TIMP-1), as well as cell proliferation, migration and actin filament formation. PTK/ZK-induced apoptosis of HSCs, which was correlated with increased caspase-3 activation and suppressed Bcl-2 expression. PTK/ZK also induced cell cycle arrest, accompanied by increasing the expression of p27(Kip1) and downregulation of cyclin D1 and cyclin E. PTK/ZK significantly inhibited vascular endothelial growth factor (VEGF) expression, as well as VEGF-simulated cell proliferation and phosphorylation of Akt in activated HSCs. In a murine fibrotic liver, PTK/ZK attenuated collagen deposition and alpha-SMA expression in carbon tetrachloride-induced fibrosis in both a 'prevention' and 'treatment' dosing scheme. These beneficial effects were associated with reduced phosphorylation of Akt and suppressed mRNA expression of procollagen-(I), TIMP-1, matrix metalloproteinase-9 and CD31. These findings provide novel insights into the potential value of blocking VEGF signaling by a small molecule tyrosine kinase inhibitor in treating hepatic fibrosis.
Asunto(s)
Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática/prevención & control , Ftalazinas/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Piridinas/toxicidad , Factor A de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Citoesqueleto/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , ARN Mensajero/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND/AIM: p27(Kip1) (p27) is a member of the Cip/Kip family of cyclin/cyclin-dependent kinase inhibitors (CKIs), and its CKI-independent function regarding cell motility modulation has been discovered. However, it is controversial whether p27 promotes or inhibits cell migration. This study investigates the migration regulatory role of p27 in metastatic hepatocellular carcinoma (HCC) cells. METHODS: RNA interference, RhoA-GTP pull-down assay, Western blots, immunostaining, transwell and wound-healing assays were used. RESULTS: High levels of p27 and phosphorylated p27 (Ser10) were detected in metastatic HCC cells, MHCC97L and MHCC97H, when compared with nonmetastatic HCC cells, PLC and Hep3B. We hypothesized that p27 is responsible for metastasis-related migration in HCC cells and tested the hypothesis by using the p27 RNA interference approach. Increased RhoA activity was observed when p27 was knocked down in MHCC97L cells, which further led to stress fiber formation and decreased cell migration and wound healing. Moreover, high p27 and low stathmin expression of metastatic HCC cells indicated that migration inhibition by p27-stathmin interaction might not be the major regulatory pathway in metastatic HCC cells. CONCLUSION: p27 promotes cell migration in metastatic HCC cells through the regulation of RhoA activity.