RESUMO
An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI approximately 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.
Assuntos
Antineoplásicos/farmacologia , Bothrops , Carcinoma de Ehrlich/tratamento farmacológico , Venenos de Crotalídeos/enzimologia , L-Aminoácido Oxidase/farmacologia , Aminoácidos/metabolismo , Animais , Carcinoma de Ehrlich/enzimologia , Carcinoma de Ehrlich/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Venenos de Crotalídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Peróxido de Hidrogênio/metabolismo , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/isolamento & purificação , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Oxirredução , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade da EspécieRESUMO
The cytotoxic activity of amino (3a-e), aza-1-antraquinone (4a-e) lapachol derivatives against Ehrlich carcinoma and human K562 leukemia cells was investigated. Cell viability was determined using MTT assay, after 48 (Ehrlich) or 96 h (K562) of culture, and vincristine (for K562 leukemia) and quercetin (for Ehrlich carcinoma) were used as positive controls. The results showed dose-dependent growth-inhibiting activities and that the amino derivatives were active against the assayed cells, whereas the 4a-e derivatives were not. The allylamine derivative 3a was the most active against Ehrlich carcinoma, with IC50 = 16.94 ± 1.25 muM, and against K562 leukemia, with IC50 = 14.11 ± 1.39 muM. The analogous lawsone derivative, 5a, was also active against Ehrlich carcinoma (IC50 = 23.89 ± 2.3 muM), although the 5d and 5e derivatives showed lower activity. The interaction between 3a-d and calf thymus DNA was investigated by fluorimetric titration and the results showed a hyperchromic effect indicating binding to DNA as presented of ethidium bromide, used as positive control. The inhibitory action on DNA-topoisomerase II-a was also evaluated by a relaxation assay of supercoiled DNA plasmid, and the etoposide (200 muM) was used as positive control. Significant inhibitory activities were observed for 3a-d at 200 muM and a partial inhibitory action was observed for lapachol and methoxylapachol.
Assuntos
Animais , Humanos , Camundongos , Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Ehrlich/enzimologia , DNA Topoisomerases Tipo II/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Naftoquinonas/farmacologia , Antineoplásicos Fitogênicos/química , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , /efeitos dos fármacos , Naftoquinonas/química , Quercetina/farmacologia , Vincristina/farmacologiaRESUMO
The cytotoxic activity of amino (3a-e), aza-1-antraquinone (4a-e) lapachol derivatives against Ehrlich carcinoma and human K562 leukemia cells was investigated. Cell viability was determined using MTT assay, after 48 (Ehrlich) or 96 h (K562) of culture, and vincristine (for K562 leukemia) and quercetin (for Ehrlich carcinoma) were used as positive controls. The results showed dose-dependent growth-inhibiting activities and that the amino derivatives were active against the assayed cells, whereas the 4a-e derivatives were not. The allylamine derivative 3a was the most active against Ehrlich carcinoma, with IC50 = 16.94 +/- 1.25 microM, and against K562 leukemia, with IC50 = 14.11 +/- 1.39 microM. The analogous lawsone derivative, 5a, was also active against Ehrlich carcinoma (IC50 = 23.89 +/- 2.3 microM), although the 5d and 5e derivatives showed lower activity. The interaction between 3a-d and calf thymus DNA was investigated by fluorimetric titration and the results showed a hyperchromic effect indicating binding to DNA as presented of ethidium bromide, used as positive control. The inhibitory action on DNA-topoisomerase II-a was also evaluated by a relaxation assay of supercoiled DNA plasmid, and the etoposide (200 microM) was used as positive control. Significant inhibitory activities were observed for 3a-d at 200 microM and a partial inhibitory action was observed for lapachol and methoxylapachol.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Ehrlich/enzimologia , Inibidores Enzimáticos/farmacologia , Naftoquinonas/farmacologia , Inibidores da Topoisomerase II , Animais , Antineoplásicos Fitogênicos/química , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Células K562/efeitos dos fármacos , Camundongos , Naftoquinonas/química , Quercetina/farmacologia , Vincristina/farmacologiaRESUMO
Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.
Assuntos
Bothrops/metabolismo , Fosfolipases A/metabolismo , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Animais , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Carcinoma de Ehrlich/induzido quimicamente , Carcinoma de Ehrlich/enzimologia , Carcinoma de Ehrlich/patologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Ensaio de Imunoadsorção Enzimática , Hemoglobinas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Agregação Plaquetária/efeitos dos fármacos , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/farmacologiaRESUMO
Treatment of tumor-bearing mice with LD12.5 values of iodoacetate; IAA (1.84 mg/100 g b.w.) and/or dimethylsulphoxide; DMSO (350 mg/100 g b.w.) significantly increased the cumulative mean survival time and percentage of survivors and reduced the mean tumor weight, compared to tumor-bearing controls, however, a more pronounced effect is recorded in the combined treatment. Also, an increase in the life span (ILS%) and tumor growth inhibition ratio (T/C%) are reported and amounted to 145.78 and 43.80%, 195.54 and 61.30% and 220.77 and 78.40% in IAA, DMSO and combined-treated groups, respectively. Results obtained from biochemical studies reveal that a single IAA treatment of tumor-bearing mice significantly increased the levels of plasma lactate dehydrogenase (LDH) activity, while it also significantly decreased the levels of plasma glucose and liver total protein, RNA and DNA, compared to normal controls. On the other hand, a single DMSO treatment significantly elevated the activities of blood antioxidant enzymes, i.e. glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PDH) and decreased the liver RNA and DNA levels. Combined treatment increased significantly the levels of plasma LDH and erythrocytes G6PDH activities, as well as liver glycogen, and in contrast it decreased the levels of liver total protein, RNA and DNA, compared to normal controls.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Ehrlich/tratamento farmacológico , Dimetil Sulfóxido/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Iodoacetatos/uso terapêutico , Animais , Carcinoma de Ehrlich/sangue , Carcinoma de Ehrlich/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , CamundongosRESUMO
Topoisomerase inhibitors are agents with anticancer activity. 7"-O-Methyl-agathisflavone (I) and amentoflavone (II) are biflavonoids and were isolated from the Brazilian plants Ouratea hexasperma and O. semiserrata, respectively. These biflavonoids and the acetyl derivative of II (IIa) are inhibitors of human DNA topoisomerases I at 200 microM, as demonstrated by the relaxation assay of supercoiled DNA, and only agathisflavone (I) at 200 microM also inhibited DNA topoisomerases II-alpha, as observed by decatenation and relaxation assays. The biflavonoids showed concentration-dependent growth inhibitory activities on Ehrlich carcinoma cells in 45-h culture, assayed by a tetrazolium method, with IC50 = 24 +/- 1.4 microM for I, 26 +/- 1.1 microM for II and 10 +/- 0.7 microM for IIa. These biflavonoids were assayed against human K562 leukemia cells in 45-h culture, but only I showed 42% growth inhibitory activity at 90 microM. Our results suggest that biflavonoids are targets for DNA topoisomerases and their cytotoxicity is dependent on tumor cell type.