RESUMEN
BACKGROUND: Cell surface plasmin is involved in tumor growth and metastatic dissemination by regulating cancer cells adhesion, migration and invasion. Plasmin-induced cell detachment is accompanied by an increased rate of reactive oxygen species (ROS) generation and cell death. However, cancer cells acquire the ability to develop adaptive mechanisms to resist ROS-mediated apoptosis. AIM: To establish the role of adaptor protein Ruk/CIN85 in the control of viability and redox balance in breast adenocarcinoma cells exposed to plasmin(ogen). MATERIALS AND METHODS: Mouse 4T1 cells with the stable overexpression of adaptor protein Ruk/CIN85 (RukUp subline) and corresponding control (Mock subline) were treated with Glu-plasminogen (1-100 nM). Plasminogen to plasmin conversion was monitored spectrophotometrically by cleavage of the specific chromogenic substrate S2251. Specific uPA inhibitor BC11 was used to verify the uPA-mediated mechanism of plasminogen pericellular activation by 4T1 cells. Cell survival rate was assessed by MTT-test and cell proliferation was estimated by colony formation assay. Enzymatic activities of catalase, glutathione peroxidase, superoxide dismutase, as well as hydrogen peroxide (H2O2) levels were measured by spectrophotomertric and fluorometric assays. The intracellular ROS generation was monitored by flow cytometry using H2DCF-DA fluorescent probe. RESULTS: Plasminogen was shown to be converted into an active proteinase plasmin on the surface of carcinoma cells in uPA-dependent manner. Plasmin(ogen) suppressed proliferation and affected survival of both studied 4T1 sublines. However, RukUp cells displayed higher resistance to plasmin(ogen)-induced cytotoxicity than Mock cells. Plasmin(ogen) promoted significant elevation in ROS generation rate in cells with the basal level of Ruk/CIN85 expression. In contrast, RukUp cells appear to be more effective in counteracting prooxidant changes due to the activation of some enzymes of the glutathione system, in particular glutathione peroxidase, and a concomitant decrease of H2O2 accumulation. CONCLUSION: Adaptor protein Ruk/CIN85 is involved in the regulation of redox homeostasis in cancer cells to maintain levels of ROS, thus promoting redox adaptation in cancer cells exposed to plasmin(ogen). Thus, Ruk/CIN85 may represent one of the relevant targets in order to diminish the resistance of cancer cells to ROS-mediated apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama , Fibrinolisina , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Estrona/análogos & derivados , Femenino , Fibrinolisina/metabolismo , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Ratones , Oxidación-Reducción , Plasminógeno/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In this study we have tested an idea on the important role of amine oxidases (semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase) as an additional source of oxidative/carbonyl stress under glycerol-induced rhabdomyolysis, since the enhanced formation of reactive oxygen species and reactive carbonyl species in a variety of tissues is linked to various diseases. In our experiments we used the sensitive fluorescent method devised for estimation of amine oxidases activity in the rat kidney and thymus as targeted organs under rhabdomyolysis. We have found in vivo the multiple rises in activity of semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase (2-4.5 times) in the corresponding cell fractions, whole cells or their lysates at the 3-6th day after glycerol injection. Aberrant antioxidant activities depended on rhabdomyolysis stage and had organ specificity. Additional treatment of animals with metal chelator 'Unithiol' adjusted only the activity of antioxidant enzymes but not amine oxidases in both organs. Furthermore the in vitro experiment showed that Fenton reaction (hydrogen peroxide in the presence of iron) products alone had no effect on semicarbazide-sensitive amine oxidase activity in rat liver cell fraction whereas supplementation with methylglyoxal resulted in its significant 2.5-fold enhancement. Combined action of the both agents had additive effect on semicarbazide-sensitive amine oxidase activity. We can assume that biogenic amine and polyamine catabolism by amine oxidases is upregulated by oxidative and carbonyl stress factors directly under rhabdomyolysis progression, and the increase in catabolic products concentration contributes to tissue damage in glycerol-induced acute renal failure and apoptosis stimulation in thymus.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Monoaminooxidasa/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rabdomiólisis/enzimología , Animales , Quelantes/farmacología , Glicerol , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/patología , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/patología , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Especificidad de Órganos , Oxidación-Reducción , Carbonilación Proteica , Piruvaldehído/antagonistas & inhibidores , Piruvaldehído/farmacología , Ratas , Ratas Wistar , Rabdomiólisis/inducido químicamente , Rabdomiólisis/tratamiento farmacológico , Rabdomiólisis/patología , Semicarbacidas/antagonistas & inhibidores , Semicarbacidas/farmacología , Timo/efectos de los fármacos , Timo/enzimología , Timo/patología , Unitiol/farmacología , Poliamino OxidasaRESUMEN
This work is devoted to the study of the features of oxidative metabolism of hepatocytes in diabetic mice and those under the vitamin D3 action. We found out that a 2.5-fold decrease of 25OHD3 content in the serum was caused by chronic hyperglycemia in diabetes. Intensification of the reactive oxygen species (ROS) and nitrogen monoxide (NO) production, protein oxidative modifications (detected by the contents of carbonyl groups and 3-nitrotyrosine), accumulation of diene conjugates and TBA-reactive products of lipid peroxidation, and the decreased level of free SH-groups of low molecular weight compounds in the liver were accompanied by development of vitamin D3 deficient state. It was shown that there was a decrease in the key antioxidant enzymes activity (catalase, SOD), while the activity of prooxidant enzymes NAD(P)H: quinone oxidoreductase, xanthine oxidase and NAD(P)H oxidase was increased. The identified oxidative metabolism lesions caused the elevation of the hepatocytes necrotic death that was tested for the ability of their nuclei to accumulate propidium iodide. Prolonged vitamin D3 administration (during 2 months) at a dose of 20 IU to diabetic mice helps to reduce the ROS formation and biomacromolecules oxidative damage, normalizes the antioxidant system state in the liver and increases survival of hepatocytes. The results suggest that vitamin D3 is a key player in the regulation of the oxidative metabolism in diabetes.
Asunto(s)
Colecalciferol/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hígado/metabolismo , Estrés Oxidativo/fisiología , Animales , Antioxidantes/metabolismo , Supervivencia Celular/efectos de los fármacos , Colecalciferol/farmacología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Ratones Endogámicos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Subchronic administration of semicarbazide in the experiment with the rats was used to reduce the formaldehyde level in the organism in order to reveal the interaction between formaldehyde metabolism and biochemical parameters, which define the oxidant-antioxidant system condition and NO metabolism. It has been found that under semicarbazide impact the generation of free radicals, ROS, nitrite and nitrate were enhanced while aldehydes level was reduced that resulted from not only semicarbazide effect like the aldehydes acceptor, but the formaldehyde synthesis slowdown and acceleration of its transformation into format as well. We suppose that formaldehyde plays certain role in the development of connective tissue pathology.
Asunto(s)
Equilibrio Ácido-Base/efectos de los fármacos , Formaldehído/metabolismo , Óxido Nítrico/metabolismo , Semicarbacidas/toxicidad , Animales , Enfermedades del Tejido Conjuntivo/metabolismo , Formiatos/sangre , Formiatos/orina , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/metabolismo , Miocardio/enzimología , Miocardio/metabolismo , Nitritos/sangre , Nitritos/orina , Ratas , Ratas WistarRESUMEN
The intracellular redox state is of importance for cell growth, differentiation, and apoptosis through reactive oxygen species (ROS) functioning as metabolic fine-tuner. Optimal levels of polyamines are necessary for growth, differentiation, and apoptotic cell death while they also protect cell from ROS accumulation. We have carried out studies to find out the interrelation between these two distant metabolic pathways. For that purpose, the glucocorticoid-triggered programmed cell death of rat thymocytes has been used. Our data confirm that SOD activity (which testifies both to the level of ROS generation and antioxidative defense state) changes in response to programmed cell death conditions and to alteration of intracellular polyamines level. Thymocytes death induced by dexamethasone is partially mediated by polyamines content. Our data prove that one of the molecular mechanisms of thymocytes population resistance after dexamethasone treatment is an enhanced level of antioxidant defense. It is evident that in dexamethasone-treated rat thymocytes polyamines modulate signal transduction processes to apoptosis development via changes in cellular redox status.
Asunto(s)
Apoptosis/efectos de los fármacos , Dexametasona/efectos adversos , Glucocorticoides/efectos adversos , Poliaminas/farmacología , Superóxido Dismutasa/metabolismo , Timo/efectos de los fármacos , Animales , Catalasa/metabolismo , Células Cultivadas , Femenino , Oxidación-Reducción , Ratas , Especies Reactivas de Oxígeno/metabolismo , Timo/citología , Timo/enzimologíaRESUMEN
The polypeptide sequence of the unmodified catalase from Penicillium vitale containing 696 amino acid residues was deduced. The sequences of 76 tryptic peptides of the unmodified catalase, 63 tryptic peptides of the catalase with modified Lys residues, 48 peptides resulting from catalase cleavage by the Staphylococcus aureus V8 protease, and 9 fragments obtained by BrCN-treatment were considered, and a comparison with the sequences of other catalases was made.
Asunto(s)
Catalasa/química , Penicillium/enzimología , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno/química , Endopeptidasas/química , Lisina/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Homología de Secuencia de Aminoácido , Staphylococcus aureus/enzimologíaRESUMEN
The steady-state kinetics of catalytic action of Penicillium vitale catalase has been studied. The enzyme reaction conforms to the Michaelis-Menten equation, which is shown by considering the initial velocity of the enzyme-catalytic reaction with increased concentrations of hydrogen-peroxide and sodium perborate. The parameter Km value is rather large (231-259) mM. On the other hand the Pen, vitale catalase is one of the more active enzymes and shows kcat values equal to 0.8-3.0 x 10(-6) s-1 and kcat/Km ratio of the order of 0.4-1.2 x 10(7) M-1 s-1. The enzyme reaction conforms to the Arrhenius equation when the temperature varied from 0 to 60 degrees C. It shows a slight temperature dependence and extremely low Ea value: 1.48 kcal/mol.
Asunto(s)
Catalasa/metabolismo , Penicillium/enzimología , Catalasa/análisis , Catalasa/aislamiento & purificación , Catálisis , Concentración de Iones de Hidrógeno , Cinética , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
Preparations of Penicillium vitale catalase immobilized by aminoethyl cellulose (AE-cellulose) are obtained using two methods: by the enzyme covalent cross-linking with the carrier by glutaric aldehyde and by the covalent binding of catalase to the carrier aminogroups through the carbohydrate enzyme component. A dependence is established for the degree of catalase binding and catalase activity of the immobilized enzyme on the enzyme carrier in immobilization ratio. The optimal enzyme-carrier ratio in both cases is 10 mg of the enzyme per 1 g of the carrier. With such a ratio and binding by means of glutaric aldehyde 9.5 +/- 0.23 mg of protein is added to 1 g of the carrier with the 68.7 +/- 3.45% of the activity preserved, and in binding through the carbohydrate component of catalase 9.2 +/- 0.29 mg/g is added with preservation of 84.7 +/- 4.42% of the activity. Studies in properties of soluble and AE-cellulose immobilized catalase of P. vitale and determination of thermodynamic parameters of inactivation showed that the immobilized enzyme is more stable to the effect of temperature and extreme pH values than the soluble one.
Asunto(s)
Catalasa/metabolismo , Enzimas Inmovilizadas/metabolismo , Penicillium/enzimología , Celulosa/análogos & derivados , Cinética , MétodosRESUMEN
The process of hydrogen peroxide continuous decomposition by the preparation of the fungus Penicillium vitale catalase immobilized by aminoorganosilica which were activated by glutaric aldehyde, cyanuric chloride or 2,4-toluylene diisocyanate. Catalase with an oxidized carbohydrate component was used as well. Such a modified enzyme was directly bound with the surface of aminocontaining silica and alumina. It is shown that in the process of H2O2 decomposition the preparations of immobilized catalase are inactivated. The decrease in its activity is described by a model which suggests that rates of hydrogen peroxide decomposition and enzyme inactivation are described by the first order equations. A method for calculation and prediction of mean time of continuous operation of columns with bound catalase and other immobilized enzymes is suggested in terms of the given model.
Asunto(s)
Catalasa/metabolismo , Enzimas Inmovilizadas/metabolismo , Peróxido de Hidrógeno/metabolismo , Penicillium/enzimología , Estabilidad de Medicamentos , CinéticaRESUMEN
An efficient method is developed for P. vitale catalase immobilization through the oxidized carbohydrate enzyme component, using silochrome. The method provides the enzyme binding without losing its catalytic capacity in the immobilized preparation. When the enzyme is immobilized by high-dispersed silica containing isocyanate, aldehyde groups or active atoms of chlorine, 8, 15, and 20 mg of the enzyme is bounded per 1 g of the carrier, respectively, its catalytic capacity being completely retained. A dependence is established for the degree of catalase bonding and catalytic capacity of the immobilized enzyme of the enzyme carrier ratio in immobilization. The catalytic activity of the immobilized catalase preparations reaches 2 000 Becker units/l g. The preparations are stable in storage. Some of their properties are studied.