RESUMEN
OBJECTIVE: To investigate the roles of exogenous basic fibroblast growth factor (bFGF) on the repair of full-thickness articular cartilage defects in rabbits. DESIGN: In the present study, a double-layered collagen membrane sandwiched with bFGF-loaded-nanoparticles between a dense layer and a loose layer was implanted into full-thickness articular cartilage defects in rabbits. By grafting the membrane in a different direction, the dense layer or the loose layer facing the surface of the subchondral bone, the effects of the released bFGF on the defects and the profiles of nine growth factors (GFs) in synovial fluid (SF) were investigated using histological methods and antibody arrays, respectively. RESULTS: In the group with the loose layer facing the surface of the subchondral bone, fast release of bFGF was observed, and early high levels of endogenous transforming growth factor-ß2 (TGF-ß2), vascular endothelial growth factor (VEGF), bFGF, bone morphogenetic protein 2 (BMP-2), BMP-3, and BMP-4 in SF were detected by antibody arrays, especially on day 3. Chondrocyte-like cells were also observed in this group at an early stage. As a result, this group showed better levels of repair, as compared to the other groups in which low GF levels were detected at an early stage, and chondrocyte-like cells appeared much later. CONCLUSIONS: Our study suggests that exogenous bFGF promotes articular cartilage repair by up-regulating the levels of multiple GFs, but administration at an early stage is required.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Sustancias de Crecimiento/biosíntesis , Animales , Cartílago Articular/lesiones , Cartílago Articular/metabolismo , Cartílago Articular/ultraestructura , Colágeno/efectos de los fármacos , Colágeno/ultraestructura , Evaluación Preclínica de Medicamentos/métodos , Microscopía Electrónica de Rastreo , Nanopartículas , Conejos , Líquido Sinovial/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
OBJECTIVE: To screen a library of small chemicals for compounds that activate the DPC4 signal transduction pathway in a human pancreatic cancer cell line. SUMMARY BACKGROUND DATA: Various tumor-suppressor genes are mutated in all human cancers. Specifically, DPC4 (deleted in pancreatic carcinoma, locus 4 or MADH4/SMAD4) is a tumor-suppressor gene mutated in approximately 50% of human pancreatic adenocarcinomas. DPC4 plays an important role in the well-studied transforming growth factor-beta (TGFbeta) signaling pathway. It would be useful to identify therapies that augment or restore the downstream functions of this critical signal transduction pathway, in hopes that such therapy would have a rational role in anticancer therapy. METHODS: Using a commercially available plasmid vector with a luciferase reporter gene already incorporated, a DPC4-specific reporter construct was genetically engineered. This was done by inserting six copies of the palindromic Smad binding element (6SBE), which is a DNA binding element specific for DPC4, in front of the minimal promoter in the plasmid. This construct was then stably integrated into the genome of a human pancreatic cancer cell line (PANC-1) that has wild-type DPC4. Several stably transfected clones were tested for basal luciferase expression and inducibility with TGFbeta, which is known to activate the DPC4 signal transduction pathway. A single transfected clone was chosen for the drug screen based on basal luciferase (reporter) expression and TGFbeta inducibility. A systematic screen of the chemical library was then performed, using luciferase activity to detect DPC4 activity and induction of the signaling pathway. RESULTS: A high-throughput system based on this stably integrated reporter system was used to screen a library of 16,320 random compounds to identify agents that conferred robust augmentation of the DPC4 signal transduction pathway. Of the 16,320 compounds screened, 11 were associated with a 2- to 5-fold induction of luciferase activity, and one with a 12-fold activation. The latter compound was shown to be a novel histone deacetylase inhibitor and was further characterized. CONCLUSIONS: These results confirm the feasibility of a specific high-throughput reporter system to screen a large compound library in human cells efficiently. The screening identified several compounds capable of augmenting DPC4-specific luciferase reporter activity, and a specific mechanism for one compound was identified. The discovery of such agents will aid our understanding of complex tumor-suppressive signaling pathways and may identify other potential therapeutic targets within this critical signaling pathway. In addition, random drug screening provides an unbiased method for identifying drugs or lead compounds for potential therapeutic use.
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Adenocarcinoma/genética , Proteínas de Unión al ADN/genética , Genes Reporteros , Genes Supresores de Tumor/genética , Neoplasias Pancreáticas/genética , Transducción de Señal , Transactivadores/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Evaluación Preclínica de Medicamentos , Biblioteca de Genes , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteína Smad4 , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Lipopolysaccharide-binding protein (LBP) and CD14 play key intermediary roles in the activation of cells by endotoxin. As endotoxin has been postulated to participate in promoting pathological liver injury in alcoholic liver disease, we investigated the role of LBP and CD14 in alcoholic liver injury. Rats were fed intragastrically ethanol or dextrose and either medium-chain triglycerides, corn oil, or fish oil for 4 weeks. Kupffer cells, endothelial cells, and hepatocytes were isolated. LBP and CD14 mRNA levels were measured in liver and individual cell types. The highest levels of LBP and CD14 mRNA levels in the liver were found in the fish oil/ethanol group, which was also the group with the greatest degree of pathological injury and inflammation. CD14 mRNA levels were also significantly elevated in groups fed unsaturated fatty acids with dextrose. CD14 expression was localized to the Kupffer cells and LBP expression to the hepatocytes. Expression of CD14 mRNA was also found in nonmyeloid cells in the two experimental groups (fish oil/ethanol and corn oil/ethanol) that had liver necrosis and inflammation. Our results suggest that enhanced LBP and CD14 expression correlates with the presence of pathological liver injury in alcoholic liver injury. Furthermore, unsaturated fatty acids may prime cells to respond to endotoxin by enhancing CD14 expression.
Asunto(s)
Proteínas de Fase Aguda , Proteínas Portadoras/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Cirrosis Hepática Alcohólica/metabolismo , Cirrosis Hepática Experimental/metabolismo , Glicoproteínas de Membrana , Animales , Northern Blotting , Western Blotting , Proteínas Portadoras/genética , Grasas de la Dieta/administración & dosificación , Etanol/toxicidad , Macrófagos del Hígado/metabolismo , Receptores de Lipopolisacáridos/genética , Hígado/metabolismo , Cirrosis Hepática Alcohólica/etiología , Cirrosis Hepática Alcohólica/patología , Cirrosis Hepática Experimental/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Folate deficiency has been associated with dysplasia in human cancer models. Patients with ulcerative colitis commonly have decreased folate levels, which are partially due to sulfasalazine, a competitive inhibitor of folate absorption. To study the effect of folate supplementation on the risk of dysplasia or cancer (neoplasia) in ulcerative colitis, records from 99 patients with pancolitis for greater than 7 yr and enrolled in a surveillance program were reviewed. Thirty-five patients with neoplasia were compared with 64 patients in whom dysplasia was never found to determine the effect of folate supplementation on the rate of development of neoplasia using case-control methodology. At the time of the index colonoscopy, patients with neoplasia were older (43 +/- 11 vs. 39 +/- 12 yr) and had disease of longer duration (20 +/- 8 vs. 15 +/- 7 yr, p less than 0.05). Folate supplementation was associated with a 62% lower incidence of neoplasia compared with individuals not receiving supplementation (odds ratio, 0.38; 95% confidence interval, 0.12-1.20). There was no appreciable change in this effect when models were fit to adjust for sulfasalazine dose, duration of disease, age at symptom onset, prednisone dose, sulfa allergy, sex, race, or family history of colon cancer. The statistical power of the association between folate supplementation and neoplasia was 72%. Correction of risk factors before the development of neoplasia may prevent this serious complication. Pending a larger case-control study, folate supplementation during sulfasalazine administration is recommended to possibly prevent the complication of dysplasia or cancer in ulcerative colitis.