RESUMO
BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) pathophysiology involves neurovascular proteolysis and inflammation. How these 2 phenomena are related remains unclear. We hypothesize that matrix metalloproteinases (MMPs) mediate the depletion of anti-inflammatory plasma-type gelsolin (pGSN). METHODS: We enrolled 42 consecutive SAH subjects and sampled cerebrospinal fluid (CSF) and blood on post-SAH Days 2 to 3, 4 to 5, 6 to 7, and 10 to 14. Control subjects were 20 consecutive non-SAH hydrocephalus patients with lumbar drains. Enzyme-linked immunosorbent assay, Western blotting, and zymography were used to quantify pGSN and MMP-9. RESULTS: In CSF, pGSN was lower in SAH compared with control subjects on post-SAH Days 2 to 3 (P=0.0007), 4 to 5 (P=0.041), and 10 to 14 (P=0.007). In blood, pGSN decreased over time (P=0.001) and was lower in SAH compared with control subjects on post-SAH Days 4 to 5 (P=0.037), 6 to 7 (P=0.006), and 10 to 14 (P=0.006). Western blots demonstrated that SAH CSF had novel bands at 52 and 46 kDa, representing cleaved pGSN fragments. Gelatin zymography showed that CSF MMP-9 was elevated in SAH compared with control subjects. Higher CSF MMP-9 correlated with lower CSF pGSN on post-SAH Day 7 (r=-0.38; P=0.05). CONCLUSIONS: SAH is associated with decreased CSF and blood pGSN and elevated CSF MMP-9. Novel cleaved pGSN fragments are present in CSF of SAH subjects, consistent with pGSN cleavage by MMPs. Because pGSN is known to inhibit inflammatory mediators, these findings suggest that MMPs may reduce pGSN and exacerbate inflammation after SAH. Further studies are warranted to investigate the mechanisms underlying MMP-pGSN signaling in SAH.
Assuntos
Gelsolina/metabolismo , Hemorragia Subaracnóidea/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Feminino , Gelsolina/sangue , Gelsolina/líquido cefalorraquidiano , Humanos , Hidrocefalia/sangue , Hidrocefalia/líquido cefalorraquidiano , Masculino , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/líquido cefalorraquidiano , Pessoa de Meia-Idade , Hemorragia Subaracnóidea/sangue , Hemorragia Subaracnóidea/líquido cefalorraquidianoRESUMO
Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase. In this work, we demonstrated that these anti-heparanase aptamers are capable of inhibiting tissue invasion of tumour cells associated with oral cancer and verified that such inhibition is due to inhibition of the enzyme and not due to other potentially cytotoxic effects of the aptamers. Furthermore, we have identified a short 30 bases aptamer as a potential candidate for further studies, as this showed a higher ability to inhibit tissue invasion than its longer counterpart, as well as a reduced potential for complex formation with other non-specific serum proteins. Finally, the aptamer was found to be stable and therefore suitable for use in human models, as it showed no degradation in the presence of human serum, making it a potential candidate for both diagnostic and therapeutic use.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Glucuronidase/antagonistas & inibidores , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/tratamento farmacológico , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Regulação Neoplásica da Expressão Gênica , Glucuronidase/metabolismo , Humanos , Neoplasias Bucais/enzimologia , Neoplasias Bucais/patologia , Invasividade NeoplásicaRESUMO
BACKGROUND: The use of mesoporous silica for cancer targeting is increasing rapidly. The association between rigid model of nanoparticles such as mesoporous silica and biological compounds with affinity for oncological diseases is a very promising drug targeting system nowadays. METHODS: In this study, we used the mesoporous silica (SBA-15) associated with aptamer (functionalized for the tumor marker MUC-1). RESULTS: The results obtained in the characterization were quite interesting and demonstrated that the silica produced were very uniform and with a size range of 50-100 nm. Thus, the results of cytotoxicity demonstrated that there is no cytotoxicity related to the nanoparticle. CONCLUSION: We conclude that although further studies are required, the nanoparticle mesoporous silica model loaded with aptamer is very functional and its use can be widespread for other application especially in nuclear medicine.
Assuntos
Aptâmeros de Peptídeos/administração & dosagem , Aptâmeros de Peptídeos/química , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/tratamento farmacológico , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Mucina-1/metabolismo , Neoplasias/metabolismo , Tamanho da Partícula , Porosidade , Técnica de Seleção de Aptâmeros/métodos , Dióxido de Silício/administração & dosagem , Dióxido de Silício/química , Distribuição TecidualRESUMO
Heparanase is an enzyme involved in extracellular matrix remodelling and heparan sulphate proteoglycan catabolism. It is secreted by metastatic tumour cells, allowing them to penetrate the endothelial cell layer and basement membrane to invade target organs. The release of growth factors at the site of cleaved heparan sulphate chains further enhance the potential of the tumour by encouraging the process of angiogenesis. This leads to increased survival and further proliferation of the tumour. Aptamers are single or double stranded oligonucleotides that recognise specific small molecules, peptides, proteins, or even cells or tissues and have shown great potential over the years as diagnostic and therapeutic agents in anticancer treatment. For the first time, single stranded DNA aptamers were successfully generated against the active heterodimer form of heparanase using a modified SELEX protocol, and eluted based on increasing affinity for the target. Sandwich ELISA assays showed recognition of heparanase by the aptamers at a site distinct from that of a polyclonal HPSE1 antibody. The binding affinities of aptamer to immobilised enzyme were high (7 × 10(7) to 8 × 10(7) M(-1)) as measured by fluorescence spectroscopy. Immunohistochemistry and immunofluorescence studies demonstrated that the aptamers were able to recognise heparanase with staining comparable or in some cases superior to that of the HPSE1 antibody control. Finally, matrigel assay demonstrated that aptamers were able to inhibit heparanase. This study provides clear proof of principle concept that nucleic acid aptamers can be generated against heparanase. These reagents may serve as useful tools to explore the functional role of the enzyme and in the future development of diagnostic assays or therapeutic reagents.