RESUMEN
The keloid fibroblast (KF) is known to have higher proliferative capacity than normal dermal fibroblast (NF). Metallothionein (MT), a metal-binding protein, has been reported to promote cell proliferation. In this study, we evaluated the expression of MT isoforms at the mRNA level in fetal bovine serum (FBS)-stimulated proliferating KF. Although the morphological appearance of NF and KF was similar when viewed under light, confocal and transmission electron microscopy, there was surprisingly a generally lower expression of MT isoforms in KF when compared with NF and also reduced MT staining in dermal fibroblasts of keloids as opposed to normal skin. Primary cultures of KF grown in 5% FBS or 10% FBS compared to without FBS demonstrated significantly higher proliferative activity and more abundant deposition of collagen. Contrary to expectation, MT-1A, -1F, -1G, -1X and -2A isoforms were significantly down-regulated in proliferating KF. Moreover, stimulating KF with TGF ß1, which is known to promote collagen synthesis and keloid formation, increased expression of Collagen 1A and 3A genes accompanied by reduction in MT-2A gene expression. Furthermore, down-regulation of the MT-2A gene in proliferating KF by siRNA-mediated silencing enhanced cell proliferation with concomitant up-regulation of the NF-κB gene and 10 of 13 other NF-κB pathway-related genes analysed but no alteration of the Collagen 1 and Collagen 3 gene expression. It would appear that down-regulation of MT isoforms in proliferating KF, in particular MT-2A, enhances keloidogenesis with the possible involvement of the NF-κB signalling pathway.
Asunto(s)
Proliferación Celular , Colágeno/metabolismo , Fibroblastos/metabolismo , Queloide/patología , Metalotioneína/metabolismo , Isoformas de Proteínas/metabolismo , Células Cultivadas , Colágeno/genética , Medio de Cultivo Libre de Suero/farmacología , Regulación hacia Abajo/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Técnicas In Vitro , Queratinocitos/metabolismo , Queratinocitos/patología , Metalotioneína/genética , FN-kappa B/genética , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genética , Suero/fisiología , Transducción de Señal/genética , Piel/metabolismo , Piel/patología , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/genéticaRESUMEN
Gold nanoparticles (AuNPs) are one of the most versatile and widely researched materials for novel biomedical applications. However, the current knowledge in their toxicological profile is still incomplete and many on-going investigations aim to understand the potential adverse effects in human body. Here, we employed two dimensional gel electrophoresis to perform a comparative proteomic analysis of AuNP treated MRC-5 lung fibroblast cells. In our findings, we identified 16 proteins that were differentially expressed in MRC-5 lung fibroblasts following exposure to AuNPs. Their expression levels were also verified by western blotting and real time RT-PCR analysis. Of interest was the difference in the oxidative stress related proteins (NADH ubiquinone oxidoreductase (NDUFS1), protein disulfide isomerase associate 3 (PDIA3), heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and thioredoxin-like protein 1 (TXNL1)) as well as proteins associated with cell cycle regulation, cytoskeleton and DNA repair (heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and Secernin-1 (SCN1)). This finding is consistent with the genotoxicity observed in the AuNP treated lung fibroblasts. These results suggest that AuNP treatment can induce oxidative stress-mediated genomic instability.
Asunto(s)
Fibroblastos/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Oro/farmacología , Pulmón/citología , Nanopartículas del Metal/efectos adversos , Línea Celular , Rotura Cromosómica/efectos de los fármacos , Ensayo Cometa , Regulación hacia Abajo/efectos de los fármacos , Electroforesis en Gel Bidimensional , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Hibridación Fluorescente in Situ , Proteínas/genética , Proteínas/metabolismo , Proteómica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Regulación hacia Arriba/efectos de los fármacosRESUMEN
AIM: To determine the expression pattern and prognostic value of heparan sulfate in gastric cancer. METHOD: The 10E4 antiheparan sulfate monoclonal antibody was used to examine the expression pattern of heparan sulfate in tissue microarrays consisting of 162 cases of gastric carcinoma by immunohistochemistry. The immunoreactivities of both epithelial and stromal components of the specimens were examined and analysed statistically for significant associations with clinicopathological parameters, including histological grade of the tumour, extent of cancer infiltration and presence of lymph-node metastases, lymphovascular invasion, perineural invasion, perforation of gastric wall and stromal reaction. The potential use of heparan sulfate as a predictive factor for patient survival was also evaluated. RESULTS: Reduced expression of heparan sulfate in the epithelial component was associated with higher histological grades of gastric cancer as well as the presence of more extensive tumour infiltration. Furthermore, this decrease in heparan sulfate expression was found to be predictive of reduced patient survival after tumour recurrence. CONCLUSION: The data suggest that heparan sulfate may play an important role in regulating the biology of gastric cancer, and that it may be a useful prognostic marker of this tumour.