RESUMO
Strong chelating ligands as oxodiacetate (oda) are model systems to study the process of metal trapping by living organisms. Vanadium compounds display interesting biological and pharmacological actions. In vertebrates, vanadium is stored mainly in bones. In the present study we report the effects of the complex of oda with vanadyl(IV) cation, VO(oda), on two osteoblast cell lines, one normal (MC3T3E1) and the other tumoral (UMR106). VO(oda) exerted cytotoxic actions in osteoblasts as it was determined through a dose-dependent decrease in cell proliferation, and morphological and actin alterations. The putative mechanisms underlying VO(oda) deleterious effects were also investigated. The complex increased the level of ROS which correlated with a decreased in GSH/GSSG ratio. Besides, VO(oda) induced a dissipation of the mitochondria membrane potential (MMP) and promoted an increase in ERK cascade phosphorylation, which is involved in the regulation of cellular death and survival. All the effects were more pronounced in MC3T3-E1 than in UMR106 cells. ERK activation was inhibited by PD98059, Wortmanin and the ROS scavenger NAC (N-acetyl cysteine). These results suggest that VO(oda) stimulated ERKs phosphorilation by induction of free radicals involving kinases upstream of ERK pathway. The inhibitory effect of the complex on cell proliferation was partially reversed in both cell lines by NAC. Moreover, PD98059 and Wortmanin also partially reversed the inhibition of cell proliferation in the tumoral osteoblasts. The use of specific inhibitors and ROS scavengers suggested the involvement of oxidative stress, MMP alterations and ERK pathway in the apoptotic actions of this complex.
Assuntos
Citotoxinas/química , Citotoxinas/farmacologia , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Osteoblastos/efeitos dos fármacos , Oxigênio/química , Vanádio/química , Actinas/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Espaço Intracelular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Vermelho Neutro , Osteoblastos/citologia , Osteoblastos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The tissue accumulation of protein-bound advanced glycation endproducts (AGE) may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS) and nitric oxide synthase (NOS) expression on these AGE-collagen mediated effects. RESULTS: AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP) activity. In preosteoblastic MC3T3E1 cells (24-hour culture), proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts) AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture) AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. CONCLUSIONS: These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.
Assuntos
Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Osteoblastos/citologia , Estresse Oxidativo , Animais , Calcificação Fisiológica , Adesão Celular/efeitos dos fármacos , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Glicosilação , Camundongos , Óxido Nítrico Sintase/metabolismo , Osteoblastos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
The cytotoxicity and free radical production induced by vanadium compounds were investigated in an osteoblast (MC3T3E1) and an osteosarcoma (UMR106) cell lines in culture. Vanadate induced cell toxicity, reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) increased in a concentration-dependent manner (0.1-10 mM) after 4 h. The concentration-response curve of vanadate-induced cytotoxicity and oxidative stress in MC3T3E1 cells was shifted to the left of the UMR106 curve, suggesting a greater sensitivity of the non-transformed cells in comparison to the osteosarcoma UMR106 cells. Supplementing with vitamin E acetate (80 microM) significantly inhibited ROS and TBARS formation but did not improve the vanadate-dependent decrease in cell number. Other vanadium compounds (vanadyl, pervanadate, and VO/Aspi, a complex of vanadyl(IV) with aspirin) showed different degrees of cell toxicity and induced oxidative stress. Altogether these results suggest that oxidative stress is involved in vanadium induced osteoblastic cytotoxicity, although the mechanism is unknown.
Assuntos
Neoplasias Ósseas/patologia , Osteoblastos/efeitos dos fármacos , Osteossarcoma/patologia , Estresse Oxidativo/efeitos dos fármacos , Vanádio/toxicidade , Antioxidantes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes , Violeta Genciana , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Corantes de Rosanilina , Substâncias Reativas com Ácido Tiobarbitúrico , Células Tumorais Cultivadas , Vitamina E/farmacologiaRESUMO
Hyperglycaemia in poorly controlled diabetic patients induces non-enzymatic glycosylation (glycation) of proteins, altering their structure and physiological bioactivity. Alkaline phosphatase (ALP) is a membrane-bound exoenzyme which faces the extracellular compartment. We have investigated the glycation of intestinal alkaline phosphatase in vitro and the consequences of such molecular modifications on certain structural and functional characteristics. The effect of glycation on alkaline phosphatase specific activity was determined after incubation of the enzyme with different sugars for various periods of time. The formation of early reversible glycation products was determined by the measurement of fructosamine levels, while the appearance of advanced glycation end products was estimated by spectrofluorometric analysis. A decrease in the specific activity of ALP was associated both with an increase in fructosamine levels and with the appearance of AGE-characteristic fluorescence. Changes in these parameters were found to depend on the incubation time, and on the concentration and glycating capability of the sugar employed. Co-incubation with aminoguanidine slowed down the appearance of protein-linked fluorescence, and additionally curbed the decrease in enzymatic specific activity. A significant correlation between the levels of ALP-fructosamine and ALP-advanced glycation end product was observed. Patterns of protein bands fractionated by SDS-PAGE were essentially identical for the nonglycated controls and the glycated samples. The electrophoretic mobility of the band of alkaline phosphatase on cellulose acetate gels increased as a function of the incubation time and the glycosylating power of the carbohydrate used. The present study provides evidence for the in vitro glycation of alkaline phosphatase, and for the consecutive alteration of its activity and structure.
Assuntos
Fosfatase Alcalina/metabolismo , Animais , Metabolismo dos Carboidratos , Bovinos , Frutosamina/análise , Glicosilação , Intestinos/enzimologia , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Two different lines of osteoblast-like cells were used to investigate the effect of advanced glycation end-products of bovine serum albumin on cell proliferation and differentiation. These parameters were found to be both dose- and time-dependent. Cell proliferation remained unchanged after a 24 h incubation period, it increased after intermediate periods of incubation with advanced glycation end-products, but was found to be depressed after several days incubation. Cellular alkaline phosphatase activity followed a similar pattern: an initial increase induced by advanced glycation end-products was generally followed, after relatively long incubation periods, by a slight but significant decrease in this parameter. 45Ca2+ uptake was only significantly inhibited by advanced glycation end-products after 24 h incubation. These results suggest that advanced glycation end-products directly regulate osteoblast proliferation and differentiation in a dose and time dependent manner.