RESUMEN
BACKGROUND AND AIMS: Control of cell proliferation is important for cancer prevention since cell proliferation has an essential role in carcinogenesis. In rodent carcinogenesis models, antioxidant agents suppress carcinogen-induced cellular hyper proliferation in the target organs. Strict control of cell division is an essential process to ensure that DNA synthesis and mitotic division are accurately and coordinately executed. We studied the interplay between cell cycle and heme oxygenase-1 (HO-1) and the effect of the acetylsalicylic acid (ASA) in hepatic carcinogenesis. METHODS: Male CF1 mice pre-treated with dietary p-dimethylaminoazobenzene (DAB; 0.5%, w/w) were fed with ASA (0.16%, w/w). We investigated the hepatic expression of cyclin D1, cyclin E, Cdk2, Cdk4, p21, p27, p53; the level of bcl-2, an antiapoptotic protein and of heme oxygenase-1 (HO-1), a marker of oxidative stress, by Western blot analysis. RESULTS: The treatment with ASA produced an important attenuation in the induction of cyclin E and cyclin D1 provoked by DAB. p21 and p27 levels were increased when animals received both drugs. The administration of ASA to DAB treated animals induced Cdk2 (29%). HO-1 induction (65%) provoked by DAB was diminished by ASA administration reaching lower induction levels (23%). CONCLUSION: The deregulation of cyclin/CDK expression and the up-regulation of p21 and p27 with the administration of ASA, post-treatment of the carcinogen administration, would block the pass through out to the G0/G1 check point to permit the cells to repair their DNA and HO-1 protected the liver from reactive oxygen species produced from DAB.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Ácido Salicílico/farmacología , Animales , Antiinflamatorios/farmacología , Western Blotting , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Hemo-Oxigenasa 1 , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Proteínas de la Membrana , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , p-Dimetilaminoazobenceno/administración & dosificación , p-Dimetilaminoazobenceno/farmacologíaRESUMEN
Rhodanese (thiosulphate:cyanide sulphurtransferase) shows distinctive mitochondrial and cytoplasmic activities in several models of tumorigenesis. To investigate the basis for these differences, the enzyme was purified from mitochondrial and cytosolic liver fractions of mice treated with the carcinogen p-dimethyl-aminoazobenzene (DAB) and some inhibition kinetic studies were carried out. When both substrates were assayed at inhibitory levels, non-competitive inhibition was observed for the second substrate at variable concentrations, the reversible connection between both substrates was attained by the instability of the second enzyme form. It is suggested that the enzyme might be changing from an unstable ES form to a more stable sulphur substituted intermediate as a consequence of DAB treatment. Sulphite was a competitive inhibitor vs thiosulphate for rhodanese isolated from normal liver and a hyperbolic activator for the enzyme isolated from liver of DAB-treated animals.
Asunto(s)
Carcinógenos/farmacología , Inhibidores Enzimáticos/farmacocinética , Hígado/efectos de los fármacos , Hígado/enzimología , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Tiosulfato Azufretransferasa/antagonistas & inhibidores , Tiosulfato Azufretransferasa/metabolismo , p-Dimetilaminoazobenceno/farmacología , Animales , Cianuros/farmacología , Citosol/efectos de los fármacos , Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Cinética , Masculino , Ratones , Ratones Endogámicos , Tiosulfatos/farmacologíaRESUMEN
Rhodanese (thiosulfate:cyanide sulfurtransferase, E.C. 2.8.1.1), an enzyme involved in heme regulation, showed distinctive mitochondrial and cytoplasmic activities in several models of tumorigenesis. To investigate the basis for these differences, the enzyme was partly purified and characterized from the mitochondrial and cytosolic liver fraction of mice treated with the carcinogen p-dimethyl-aminoazobenzene (DAB). A linear relationship between incubation time and specific activity was observed up to about 30 min for cytosolic enzyme and 15 min for mitochondrial enzyme irrespective of whether or not the enzyme was derived from treated or untreated animals. The optimum incubation temperature was 3 degrees C for the enzyme of both fractions in control animals and 30 degrees C for treated animals in both cases. In control and DAB treated animals the cytoplasmic rhodanese exhibited a maximum at a lower pH than for the mitochondrial enzyme. The enzyme showed typical Michaelis-Menten behavior with cyanide inhibition at concentrations higher than 25 mM for controls and 10 mM for treated animals for both fractions and thiosulfate inhibition at concentrations higher than 100 mM in all cases studied. Km values of 190 and 65.66 mM were obtained for thiosulfate and 6.37 and 9.79 mM for cyanide for both mitochondrial and cytosolic fractions of control animals; while Km values of 31.75 and 4.58 mM were obtained for thiosulfate and 0.61 and 1.11 mM for cyanide in both fractions of treated animals. We demonstrated differences in the kinetics for rhodanese derived from mitochondrial and cytoplasmic fractions of livers taken from tumor bearing mice. These differences might provide an explanation for the abnormalities of heme synthesis previously reported during hepatocarcinogenesis.