RESUMO
Inhibition of the mitogen-activated protein kinase/extracellular signal-regulated (MAPK/ERK) pathway is an attractive therapeutic approach for human cancer therapy. In the course of evaluating structurally distinct small molecule inhibitors that target mitogen-activated protein kinase kinase (MEK) and ERK kinases in this pathway, we observed an unusual, dose-related increase in the incidence of green serum in preclinical safety studies in rats. Having ruled out changes in bilirubin metabolism, we demonstrated a 2- to 3-fold increase in serum ceruloplasmin levels, likely accounting for the observed green color. This was not associated with an increase in α-2-macroglobulin, the major acute phase protein in rats, indicating that ceruloplasmin levels increased independently of an inflammatory response. Elevated serum ceruloplasmin was also not correlated with changes in total hepatic copper, adverse clinical signs, or pathology findings indicative of copper toxicity, therefore discounting copper overload as the etiology. Both ERK and MEK inhibitors led to increased ceruloplasmin secretion in rat primary hepatocyte cultures in vitro, and this increase was associated with activation of the Forkhead box, class O1 (FOXO1) transcription factor. In conclusion, increased serum ceruloplasmin induced by MEK and ERK inhibition is due to increased synthesis by hepatocytes from FOXO1 activation and results in the nonadverse development of green serum in rats.
Assuntos
Ceruloplasmina/análise , Cobre/sangue , Inibidores Enzimáticos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Soro/química , Bibliotecas de Moléculas Pequenas/toxicidade , Animais , Circulação Sanguínea , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Feminino , Fígado/química , Fígado/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Prostaglandins (PGs), bioactive lipid molecules produced by cyclooxygenase enzymes (COX-1 and COX-2), have diverse biological activities, including growth-promoting actions on gastrointestinal mucosa. They are also implicated in the growth of colonic polyps and cancers. However, the precise mechanisms of these trophic actions of PGs remain unclear. As activation of the epidermal growth factor receptor (EGFR) triggers mitogenic signaling in gastrointestinal mucosa, and its expression is also upregulated in colonic cancers and most neoplasms, we investigated whether PGs transactivate EGFR. Here we provide evidence that prostaglandin E2 (PGE2) rapidly phosphorylates EGFR and triggers the extracellular signal-regulated kinase 2 (ERK2)--mitogenic signaling pathway in normal gastric epithelial (RGM1) and colon cancer (Caco-2, LoVo and HT-29) cell lines. Inactivation of EGFR kinase with selective inhibitors significantly reduces PGE2-induced ERK2 activation, c-fos mRNA expression and cell proliferation. Inhibition of matrix metalloproteinases (MMPs), transforming growth factor-alpha (TGF-alpha) or c-Src blocked PGE2-mediated EGFR transactivation and downstream signaling indicating that PGE2-induced EGFR transactivation involves signaling transduced via TGF-alpha, an EGFR ligand, likely released by c-Src-activated MMP(s). Our findings that PGE2 transactivates EGFR reveal a previously unknown mechanism by which PGE2 mediates trophic actions resulting in gastric and intestinal hypertrophy as well as growth of colonic polyps and cancers.
Assuntos
Neoplasias do Colo/metabolismo , Dinoprostona/metabolismo , Receptores ErbB/metabolismo , Mucosa Gástrica/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Ativação Transcricional/fisiologia , Animais , Linhagem Celular , Dinoprostona/farmacologia , Inibidores Enzimáticos/farmacologia , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Genes src/fisiologia , Humanos , Hipertrofia/patologia , Metaloproteinases da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Quinazolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Tirfostinas/farmacologiaRESUMO
Aging is associated with impaired angiogenesis (new blood vessels formation from the endothelial cells of pre-existing vessels) in a variety of tissues. The precise mechanisms of aging-related impairment of angiogenesis are not known. PTEN is a dual-specificity phosphatase that antagonizes in some cells the PI3K/Akt signaling pathway, important for cell survival, function and angiogenesis. PTEN's role in aging-related impairment of angiogenesis is not known. In this study, we investigated whether expression of PTEN in endothelial cells may play a mechanistic role in aging-related impairment of angiogenesis. We demonstrated that human microvascular endothelial cells (HMVEC) derived from aging individuals (Aged-HMVEC) have: (1) significantly increased PTEN mRNA and protein levels and (2) impaired in vitro angiogenesis vs. neonatal derived HMVEC (Neo-HMVEC), and that (3) downregulation of PTEN using specific siRNA restores angiogenesis in Aged-HMVEC to normal. This is the first demonstration of increased PTEN expression in human microvascular endothelial cells derived from aging tissues and that elevated PTEN is a major factor responsible for aging-related impairment of in vitro angiogenesis.
Assuntos
Envelhecimento/genética , Endotélio Vascular/metabolismo , Inativação Gênica , Neovascularização Fisiológica/genética , PTEN Fosfo-Hidrolase/genética , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Capilares/metabolismo , Capilares/patologia , Células Cultivadas , Regulação para Baixo , Endotélio Vascular/patologia , Humanos , Masculino , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/biossíntese , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND & AIMS: Aging gastric mucosa has impaired mucosal defense and increased susceptibility to injury. Our aims were to determine the mechanisms responsible for above abnormalities. METHODS: We used Fisher F-344 rats, 3 and 24 months of age. We measured gastric mucosal blood flow; visualized mucosal hypoxia; examined expression of early growth response-1 transcription factor and phosphatase and tensin homologue deleted on chromosome 10 (PTEN); assessed apoptosis; and determined expression of caspase-3, caspase-9, and survivin. We also examined susceptibility of gastric mucosa of young and aging rats to ethanol injury and whether down-regulation of PTEN affects susceptibility of aging gastric mucosa to injury. To determine human relevance, we examined expression of PTEN and survivin in human gastric specimens of young and aging individuals. RESULTS: Gastric mucosa of aging (vs young) rats has a 60% reduction in mucosal blood flow; prominent hypoxia; and increased early growth response-1 transcription factor and PTEN messenger RNAs, and proteins. It also has increased expression of proapoptotic proteins caspase-3 and capase-9, reduced survivin, and a 6-fold increased apoptosis vs mucosa of young rats. Ethanol-induced gastric mucosal injury in aging (vs young) rats was significantly increased. The down-regulation of PTEN in gastric mucosa of aging rats completely reversed its increased susceptibility to ethanol injury. In aging human gastric mucosa, PTEN expression was significantly increased, whereas survivin was significantly reduced. CONCLUSIONS: (1) Gastric mucosa of aging rats has significantly reduced blood flow, tissue hypoxia, activation of Egr-1, PTEN; increased caspases; and reduced survivin. (2) These changes increase susceptibility of aging gastric mucosa to injury.
Assuntos
Envelhecimento/fisiologia , Mucosa Gástrica/fisiopatologia , Hipóxia/fisiopatologia , PTEN Fosfo-Hidrolase/metabolismo , Gastropatias/fisiopatologia , Animais , Apoptose , Caspases/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Mucosa Gástrica/irrigação sanguínea , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Ratos Endogâmicos F344 , Survivina , Regulação para CimaRESUMO
Although Interleukin-22 (IL-22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL-22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL-22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL-22 recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies. The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross-species translatability of the biological response in activating the IL-22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on-target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.
Assuntos
Hepatócitos/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Interleucinas/toxicidade , Proteínas Recombinantes de Fusão/toxicidade , Animais , Ensaios Clínicos Fase I como Assunto , Avaliação Pré-Clínica de Medicamentos , Feminino , Hepatócitos/metabolismo , Humanos , Interleucinas/administração & dosagem , Macaca fascicularis , Masculino , Camundongos , Cultura Primária de Células , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Fator de Transcrição STAT3/metabolismo , Suínos , Porco Miniatura , Interleucina 22RESUMO
Colorectal cancer is often lethal when invasion and/or metastasis occur. Tumor progression to the metastatic phenotype is mainly dependent on tumor cell invasiveness. Secondary bile acids, particularly deoxycholic acid (DCA), are implicated in promoting colon cancer growth and progression. Whether DCA modulates beta-catenin and promotes colon cancer cell growth and invasiveness remains unknown. Because beta-catenin and its target genes urokinase-type plasminogen activator receptor (uPAR) and cyclin D1 are overexpressed in colon cancers, and are linked to cancer growth, invasion, and metastasis, we investigated whether DCA activates beta-catenin signaling and promotes colon cancer cell growth and invasiveness. Our results show that low concentrations of DCA (5 and 50 microM) significantly increase tyrosine phosphorylation of beta-catenin, induce urokinase-type plasminogen activator, uPAR, and cyclin D1 expression and enhance colon cancer cell proliferation and invasiveness. These events are associated with a substantial loss of E-cadherin binding to beta-catenin. Inhibition of beta-catenin with small interfering RNA significantly reduced DCA-induced uPAR and cyclin D1 expression. Blocking uPAR with a neutralizing antibody significantly suppressed DCA-induced colon cancer cell proliferation and invasiveness. These findings provide evidence for a novel mechanism underlying the oncogenic effects of secondary bile acids.
Assuntos
Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ácido Desoxicólico/farmacologia , Transdução de Sinais , Transativadores/metabolismo , Bioensaio , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Neoplasias do Colo/patologia , Ciclina D1/genética , Ciclina D1/metabolismo , Ácido Desoxicólico/metabolismo , Regulação da Expressão Gênica , Humanos , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Tirosina/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , beta CateninaRESUMO
PRO304186, a humanized monoclonal antibody targeting soluble interleukin-17 A and F, was developed for autoimmune and inflammatory disease indications. When administered to cynomolgus monkeys PRO304186 induced unexpected adverse effects characterized by clinical signs of hematemesis, hematochezia, and moribundity. Pathology findings included hemorrhage throughout the gastrointestinal tract without any evidence of vascular wall damage or inflammatory cellular infiltration. Mechanistic investigation of these effects revealed mild elevations of serum MCP-1 and IL-12/23 but without a classical proinflammatory profile in PRO304186-treated animals. In vitro studies demonstrated off-target effects on vascular endothelial cells including activation of nitric oxide synthase leading to production of nitric oxide (NO) accompanied by increased mitochondrial membrane depolarization, glutathione depletion, and increased paracellular permeability. Additionally, endothelial cell-PRO304186-conditioned medium reduced myosin light chain phosphorylation in vascular smooth muscle cells. Furthermore, an ex vivo study utilizing segments from cynomolgus aorta and femoral artery confirmed PRO304186-induced endothelium-dependent smooth muscle relaxation and vasodilation mediated via NO. Finally, a single dose of PRO304186 in cynomolgus monkeys induced a rapid and pronounced increase in NO in the portal circulation that preceded a milder elevation of NO in the systemic circulation and corresponded temporally with systemic hypotension; findings consistent with NO-mediated vasodilation leading to hypotension. These changes were associated with non-inflammatory, localized hemorrhage in the gastrointestinal tract consistent with hemodynamic vascular injury associated with intense local vasodilation. Together, these data demonstrate that PRO304186-associated toxicity in monkeys was due to an off-target effect on endothelium that involved regional NO release resulting in severe systemic vasodilation, hypotension, and hemorrhage.
Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Artérias/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Hemorragia Gastrointestinal/induzido quimicamente , Hipotensão/induzido quimicamente , Óxido Nítrico/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Artérias/metabolismo , Artérias/fisiopatologia , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Hemorragia Gastrointestinal/metabolismo , Hemorragia Gastrointestinal/fisiopatologia , Hematemese/induzido quimicamente , Hematemese/metabolismo , Hematemese/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipotensão/metabolismo , Hipotensão/fisiopatologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/imunologia , Interleucina-17/metabolismo , Macaca fascicularis , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Fatores de TempoRESUMO
Colorectal cancer is the second most frequent cancer in the Western world, often lethal when invasion and/or metastasis occur. In addition to hepatocyte growth factor (HGF), colon cancer invasion may be driven by prostaglandins, especially the E2 series (PGE2), generated by the cyclooxygenase-2 (Cox-2) enzyme. While concentration of PGE2 as well as expression of Cox-2, HGF receptor (c-Met-R), epidermal growth factor receptor (EGFR), and beta-catenin are all dramatically increased in colon cancers and implicated in their growth and invasion, the precise role of PGE2 in the latter process remains unclear. Here we provide evidence that PGE2 transactivates c-Met-R (contingent upon functional EGFR), increases tyrosine phosphorylation and nuclear accumulation of beta-catenin, and induces urokinase-type plasminogen activator receptor (uPAR) mRNA expression. This is accompanied by increased beta-catenin association with c-Met-R and enhanced colon cancer cell invasiveness. Inactivation of EGFR and c-Met-R significantly reduced PGE2-induced cancer cell invasiveness. Clinical relevance of these findings is confirmed by our immunohistochemical studies demonstrating that cancer cells in the invasive front overexpress Cox-2, c-Met-R, and beta-catenin. Our findings explain a functional relationship between prostaglandins, EGFR, and c-Met-R in colon cancer growth and invasion.
Assuntos
Neoplasias do Colo/fisiopatologia , Dinoprostona/farmacologia , Receptores ErbB/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor Cross-Talk , Transdução de Sinais , Células CACO-2 , Movimento Celular , Núcleo Celular/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2 , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/metabolismo , Cinética , Proteínas de Membrana , Invasividade Neoplásica , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/biossíntese , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transativadores/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , beta CateninaRESUMO
Repair of superficial gastric mucosal injury is accomplished by the process of restitution-migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.
Assuntos
Actinas/metabolismo , Indometacina/farmacologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Estômago/efeitos dos fármacos , Fibras de Estresse/metabolismo , Animais , Apoptose , Caspase 3 , Caspases/metabolismo , Morte Celular , Linhagem Celular , Movimento Celular , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Mucosa Gástrica/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Mitomicina/farmacologia , Necrose , Fosforilação , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , Tensinas , Fatores de Tempo , Tirosina/metabolismoRESUMO
Fibroblast growth factor 19 (FGF19) represses cholesterol 7α-hydroxylase (Cyp7α1) and inhibits bile acid synthesis in vitro and in vivo. Previous studies have shown that anti-FGF19 antibody treatment reduces growth of colon tumor xenografts and prevents hepatocellular carcinomas in FGF19 transgenic mice and thus may be a useful cancer target. In a repeat dose safety study in cynomolgus monkeys, anti-FGF19 treatment (3-100 mg/kg) demonstrated dose-related liver toxicity accompanied by severe diarrhea and low food consumption. The mechanism of anti-FGF19 toxicity was investigated using in vitro and in vivo approaches. Our results show that anti-FGF19 antibody had no direct cytotoxic effect on monkey hepatocytes. Anti-FGF19 increased Cyp7α1, as expected, but also increased bile acid efflux transporter gene (bile salt export pump, multidrug resistant protein 2 [MRP2], and MRP3) expression and reduced sodium taurocholate cotransporting polypeptide and organic anion transporter 2 expression in liver tissues from treated monkeys and in primary hepatocytes. In addition, anti-FGF19 treatment increased solute transporter gene (ileal bile acid-binding protein, organic solute transporter α [OST-α], and OST-ß) expression in ileal tissues from treated monkeys but not in Caco-2 cells. However, deoxycholic acid (a secondary bile acid) increased expression of FGF19 and these solute transporter genes in Caco-2 cells. Gas chromatography-mass spectrometry analysis of monkey feces showed an increase in total bile acids and cholic acid derivatives. These findings suggest that high doses of anti-FGF19 increase Cyp7α1 expression and bile acid synthesis and alter the expression of bile transporters in the liver resulting in enhanced bile acid efflux and reduced uptake. Increased bile acids alter expression of solute transporters in the ileum causing diarrhea and the enhanced enterohepatic recirculation of bile acids leading to liver toxicity.
Assuntos
Anticorpos/imunologia , Ácidos e Sais Biliares/biossíntese , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Íleo/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Fatores de Crescimento de Fibroblastos/imunologia , Perfilação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/metabolismo , Absorção Intestinal , Macaca fascicularisRESUMO
MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target in oncology research. MEK small molecule inhibitors cause soft tissue mineralization in rats secondary to serum inorganic phosphorus (iP) elevation, but the molecular mechanism for this toxicity remains undetermined. We performed investigative studies with structurally distinct MEK inhibitors GEN-A and PD325901 (PD-901) in Sprague-Dawley rats. Our data support a mechanism that involves FGF-23 signal blockade in the rat kidney, causing transcriptional upregulation of 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (Cyp27b1), the rate-limiting enzyme in vitamin D activation, and downregulation of 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1), the enzyme that initiates the degradation of the active form of vitamin D. These transcriptional changes increase serum vitamin D levels, which in turn drive the increase in serum iP, leading to soft tissue mineralization in the rat.
Assuntos
Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Rim/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fósforo/sangue , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Cálcio/sangue , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Homeostase/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Masculino , Estrutura Molecular , Hormônio Paratireóideo/sangue , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Espectrometria de Massas em Tandem , Vitamina D/sangueRESUMO
Fibroblast growth factors (FGF) play important roles in development, angiogenesis, and cancer. FGF19 uniquely binds to FGF receptor 4 (FGFR4). Our previous study has shown that FGF19 transgenic tumors have an activated Wnt-pathway phenotype. Wnt signaling is implicated in initiating or promoting FGF signaling in various cell types and organs. In this study, we examined whether FGF19 or inhibition of FGF19 affects the beta-catenin signaling pathway using human colon cancer cell lines (HCT116, Colo201). Our results show that FGF19 increases tyrosine phosphorylation of beta-catenin and causes loss of beta-catenin-E-cadherin binding. FGF19 increases p-GSK3beta and active beta-catenin levels and anti-FGF19 antibody (1A6) treatment abrogates this effect of FGF19. Anti-FGF19 antibody treatment increases S33/S37/T41 phosphorylation and ubiquitination of beta-catenin. Ion-trap mass spectrometric analysis confirmed that 1A6 increases phosphorylation of beta-catenin in the NH(2) terminus. Using HCT116-paired beta-catenin knockout cells, we show that FGF19 induces TCF/LEF reporter activity in parental (WT/Delta45) and in WT/--but not in mutant (-/Delta45) cells, and that inhibition of endogenous FGF19 reduces this reporter activity, indicating that wild-type beta-catenin is accessible for modulation. FGFR4 knockdown using inducible short hairpin RNA significantly reduces the colony-forming ability in vitro and tumor growth in vivo. Although cleaved caspase-3 immunoreactivity remains unchanged, the number of ki67-positive nuclei is reduced in FGFR4 knockdown tumor xenograft tissues. Consistent with the reduced beta-catenin activation, Taqman analyses show that FGF19/FGFR4 inhibition reduced beta-catenin target gene (cyclin D1, CD44, c-jun, Cox-2, UPAR) expression. These findings highlight that FGF19/FGFR4 cross-talk with beta-catenin and that pathway intervention reduces tumor growth.
Assuntos
Anticorpos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , beta Catenina/metabolismo , Animais , Domínio Catalítico , Feminino , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/imunologia , Fatores de Crescimento de Fibroblastos/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Fosforilação , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Treonina/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rebamipide is an antiulcer drug used in Japan, Korea, China, Philippines, and other Asian countries for treatment of gastritis and peptic ulcer. Its effect on gastric cancer cell growth and its regulatory mechanisms remain unknown. We examined whether rebamipide affects human gastric cancer cell proliferation and activation of Smad signaling pathway. Gastric cancer (AGS) cells were treated with either (a) medium (control), (b) medium-containing rebamipide (0.5-2 mg/mL), or (c) PD98059+rebamipide. We determined cell proliferation, expression of p21, phosphorylation of ERK2, JNK p38, and Smad2/3, formation of Smad2/3-Smad4 complex, and nuclear translocation of Smad2/3. Rebamipide treatment inhibited AGS cell proliferation and increased p21, Smad2/3 phosphorylation, and Smad2/3-Smad4 complex formation. Rebamipide induced phosphorylation of ERK2 but not JNK or p38. Inactivation of ERK2 by PD98059 partly attenuated rebamipide-induced p21 expression. These data demonstrate that rebamipide activates Smad signaling pathway and suppresses human gastric cancer cell growth. Inactivation of ERK2 partly inhibited rebamipide-induced p21 expression, indicating a crosstalk between ERK and Smad signaling pathways.
Assuntos
Alanina/análogos & derivados , Antiulcerosos/farmacologia , Quinolonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Neoplasias Gástricas/metabolismo , Alanina/farmacologia , Proliferação de Células , Quinases Ciclina-Dependentes/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Fosforilação , Células Tumorais CultivadasRESUMO
BACKGROUND: Infective endocarditis is one of the most serious complications of bacteremia in patients undergoing chronic hemodialysis and is more frequent than previously recognized. The aim of our study was to describe the clinical characteristics, outcome, and factors predicting mortality of infective endocarditis in hemodialysis patients. METHODS: In this retrospective review, all patients on chronic hemodialysis admitted to a 600-bed urban teaching hospital with infective endocarditis over a 15-year period (1990 to 2004), were identified using discharge codes. Modified Duke criteria were retrospectively applied, and patients fulfilling the criteria for definite endocarditis were included in the study. RESULTS: Sixty-nine patients on hemodialysis with definite endocarditis were identified. The predominant type of vascular access was double-lumen catheter (66.7%). The mean duration of dialysis was 37 +/- 32 months. The predominant organism was Staphylococcus aureus (57.9%), of which 57.5% were methicillin susceptible. The most frequently infected valve was mitral (49.3%), followed by aortic (21.7%) and tricuspid (10.1%) valves. The cardiac and neurologic complication rates were 40.6% and 37.7%, respectively. Fifteen patients underwent valvular heart surgery. The overall in-hospital mortality was 49.3% (34 of 69). More patients who had surgery survived than patients who did not (12 of 15 versus 23 of 54; p = 0.018, odds ratio = 5.39, 95% confidence interval: 1.3 to 17.6). On logistic regression, valve surgery was the only independent factor predicting survival (p = 0.023). CONCLUSIONS: The prognosis of infective endocarditis in hemodialysis patients is poor, with surgery serving as an independent predictor of survival.
Assuntos
Endocardite Bacteriana/epidemiologia , Endocardite Bacteriana/terapia , Diálise Renal , Adulto , Idoso , Antibacterianos/uso terapêutico , Endocardite Bacteriana/complicações , Feminino , Implante de Prótese de Valva Cardíaca , Hospitais de Ensino , Humanos , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Mortalidade , Estudos Retrospectivos , Fatores de Risco , Análise de Sobrevida , Resultado do Tratamento , População UrbanaRESUMO
Although leptin is known to induce proliferative response in gastric cancer cells, the mechanism(s) underlying this action remains poorly understood. Here, we provide evidence that leptin-induced gastric cancer cell proliferation involves activation of STAT and ERK2 signaling pathways. Leptin-induced STAT3 phosphorylation is independent of ERK2 activation. Leptin increases SHP2 phosphorylation and enhances binding of Grb2 to SHP2. Inhibition of SHP2 expression with siRNA but not SHP2 phosphatase activity abolished leptin-induced ERK2 activation. While JAK inhibition with AG490 significantly reduced leptin-induced ERK2, STAT3 phosphorylation, and cell proliferation, SHP2 inhibition only partially reduced cancer cell proliferation. Immunostaining of gastric cancer tissues displayed local overexpression of leptin and its receptor indicating that leptin might be produced and act locally in a paracrine or autocrine manner. These findings indicate that leptin promotes cancer growth by activating multiple signaling pathways and therefore blocking its action at the receptor level could be a rational therapeutic strategy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Leptina/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Neoplasias Gástricas/patologia , Transativadores/metabolismo , Proliferação de Células , Ativação Enzimática , Humanos , Fosforilação , Fator de Transcrição STAT3 , Transdução de Sinais , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/metabolismoRESUMO
PURPOSE: Gallbladder cancer is a malignancy with poor prognosis, predominantly resulting from invasion and metastasis. Our previous studies have demonstrated that prostaglandin E(2) (PGE(2)), generated by cyclooxygenase 2 (Cox-2), transactivates epidermal growth factor receptor (EGFR), c-Met and beta-catenin; thus, enhancing colon cancer cell growth and invasiveness in vitro. To determine whether these findings are applicable to clinical conditions, we examined the expression and cellular localization/co-localization of Cox-2, c-Met, beta-catenin, EGFR and c-erbB2 in gallbladder cancer. MATERIALS AND METHODS: Thirty-five specimens of invasive gallbladder cancer, 8 in situ carcinoma and 7 adenoma specimens were immunostained with specific antibodies against Cox-2, c-Met, beta-catenin, EGFR and c-erbB2. The cellular distribution, localization and colocalization were examined, and the signal intensities quantified in: a) the central area of gallbladder cancer and b) cancer cells forming the invasive front. RESULTS: Cox-2, c-Met, beta-catenin, c-erbB2 and EGFR were over-expressed in 80, 74, 71, 62 and 11% of invasive gallbladder cancers, respectively. beta-catenin was expressed in 80% of non-malignant specimens, exclusively in the cell membrane, while the cancer specimens showed cytoplasmic and/or nuclear staining. Significantly higher Cox-2, c-Met and beta-catenin expressions were present in cancer cells of the invasive front than in the tumor central areas (p<0.001), and these expressions were significantly (p=0.01) associated with the invasion depth. Co-expressions of Cox-2, c-Met, beta-catenin and c-erbB2 were present in 42% of the specimens in cancer cells forming the invasive front. CONCLUSION: The overexpressions, and often co-localizations, of Cox-2, c-Met and beta-catenin in cancer cells forming the invasive front indicate their local interactions and important roles in invasion.
RESUMO
We have recently shown that Neuropeptide S (NPS) can promote arousal and induce anxiolytic-like effects after central administration in rodents. Another study reported a number of natural polymorphisms in the human NPS receptor gene. Some of these polymorphisms were associated with increased risk of asthma and possibly other forms of atopic diseases, but the physiological consequences of the mutations remain unclear. One of the polymorphisms produces an Asn-Ile exchange in the first extracellular loop of the receptor protein, and a C-terminal splice variant of the NPS receptor was found overexpressed in human asthmatic airway tissue. We sought to study the pharmacology of the human receptor variants in comparison with the murine receptor protein. Here, we report that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing binding affinity in NPSR Ile107. In contrast, the C-terminal splice variant of the human NPS receptor shows a pharmacological profile similar to NPSR Asn107. The mouse NPS receptor, which also carries an Ile residue at position 107, displays an intermediate pharmacological profile. Structure-activity relationship studies show that the amino terminus of NPS is critical for receptor activation. The altered pharmacology of the Ile107 isoform of the human NPS receptor implies a mechanism of enhanced NPS signaling that might have physiological significance for brain function as well as peripheral tissues that express NPS receptors.
Assuntos
Variação Genética , Neuropeptídeos/farmacologia , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Asparagina/química , Células CACO-2 , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/análise , AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Éxons , Genes Reporter , Células HT29 , Humanos , Isoleucina/química , Luciferases/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Polimorfismo Genético , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ensaio Radioligante , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Relação Estrutura-AtividadeRESUMO
BACKGROUND & AIMS: Cellular and molecular mechanisms of esophageal ulcer healing remain unexplored. We studied the sequential cellular events and the expression of keratinocyte growth factor (KGF) and its receptor (KGF-R) during the healing of experimental esophageal ulcers. METHODS: Esophageal ulcers were produced in rats by local application of acetic acid. Studies included (1) ulcer size, (2) quantitative histology, and (3) KGF and KGF-R messenger RNA and protein expression by reverse-transcription polymerase chain reaction, Western blotting, and immunostaining. In separate groups, ulcer size and esophageal epithelial proliferation were evaluated after a single injection of recombinant human KGF (1 mg/kg) around the ulcer. RESULTS: Ulcers were fully developed 3 days after induction, and 58% of ulcers were re-epithelialized by 9 days. At 3 days, in esophageal tissue bordering the ulcers, KGF messenger RNA and protein were increased by 191% and 151%, respectively, and KGF-R messenger RNA and protein were increased by 357% and 237%, respectively. KGF was expressed in stromal cells, whereas KGF-R was expressed in epithelial cells. At 6 days, epithelial proliferation at the ulcer margin was increased by 216%, and treatment with KGF further enhanced cell proliferation and accelerated ulcer healing. CONCLUSIONS: KGF is a likely mediator of esophageal epithelial proliferation and ulcer healing.
Assuntos
Doenças do Esôfago/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Úlcera/metabolismo , Cicatrização/fisiologia , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Doenças do Esôfago/tratamento farmacológico , Doenças do Esôfago/patologia , Fator 7 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Imunofluorescência , Expressão Gênica/fisiologia , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/análise , Receptores de Fatores de Crescimento de Fibroblastos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Úlcera/tratamento farmacológico , Úlcera/patologia , Cicatrização/efeitos dos fármacosRESUMO
Nonsteroidal anti-inflammatory drugs, both nonselective and cyclooxygenase-2 (COX-2) selective, delay gastric ulcer healing. Whether they affect esophageal ulcer healing remains unexplored. We studied the effects of the COX-2 selective inhibitor, celecoxib, on esophageal ulcer healing as well as on the cellular and molecular events involved in the healing process. Esophageal ulcers were induced in rats by focal application of acetic acid. Rats with esophageal ulcers were treated intragastrically with either celecoxib (10 mg/kg, once daily) or vehicle for 2 or 4 days. Esophageal ulceration triggered increases in: esophageal epithelial cell proliferation; expression of COX-2 (but not COX-1); hepatocyte growth factor (HGF) and its receptor, c-Met; and activation of extracellular signal-regulated kinase 2 (ERK2). Treatment with celecoxib significantly delayed esophageal ulcer healing and suppressed ulceration-triggered increases in esophageal epithelial cell proliferation, c-Met mRNA and protein expression, and ERK2 activity. In an ex vivo organ-culture system, exogenous HGF significantly increased ERK2 phosphorylation levels in esophageal mucosa. A structural analog of celecoxib, SC-236, completely prevented this effect. These findings indicate that celecoxib delays esophageal ulcer healing by reducing ulceration-induced esophageal epithelial cell proliferation. These actions are associated with, and likely mediated by, down-regulation of the HGF/c-Met-ERK2 signaling pathway.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Doenças do Esôfago/metabolismo , Esôfago/metabolismo , Esôfago/patologia , Isoenzimas/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Sulfonamidas/farmacologia , Úlcera/metabolismo , Animais , Celecoxib , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Doenças do Esôfago/patologia , Esôfago/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Prostaglandina-Endoperóxido Sintases , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirazóis , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Úlcera/patologia , Cicatrização/efeitos dos fármacosRESUMO
Activation of endothelial nitric oxide synthase (eNOS) in portal hypertensive (PHT) gastric mucosa leads to hyperdynamic circulation and increased susceptibility to injury. However, the signaling mechanisms for eNOS activation in PHT gastric mucosa and the role of TNF-alpha in this signaling remain unknown. In PHT gastric mucosa we studied (1) eNOS phosphorylation (at serine 1177) required for its activation; (2) association of the phosphatidylinositol 3-kinase (PI 3-kinase), and its downstream effector Akt, with eNOS; and, (3) whether TNF-alpha neutralization affects eNOS phosphorylation and PI 3-kinase-Akt activation. To determine human relevance, we used human microvascular endothelial cells to examine directly whether TNF-alpha stimulates eNOS phosphorylation via PI 3-kinase. PHT gastric mucosa has significantly increased (1) eNOS phosphorylation at serine 1177 by 90% (P <.01); (2) membrane translocation (P <.05) and phosphorylation (P <.05) of p85 (regulatory subunit of PI 3-kinase) by 61% and 85%, respectively; (3) phosphorylation (P <.01) and activity (P <.01) of Akt by 40% and 52%, respectively; and (4) binding of Akt to eNOS by as much as 410% (P <.001). Neutralizing anti-TNF-alpha antibody significantly reduced p85 phosphorylation, phosphorylation and activity of Akt, and eNOS phosphorylation in PHT gastric mucosa to normal levels. Furthermore, TNF-alpha stimulated eNOS phosphorylation in human microvascular endothelial cells. In conclusion, these findings show that in PHT gastric mucosa, TNF-alpha stimulates eNOS phosphorylation at serine 1177 (required for its activation) via the PI 3-kinase-Akt signal transduction pathway.