RESUMO
The effect of ten phosphorohydrazone derivatives of chromone on the fibrin formation process was evaluated in the presence of basic fibroblast growth factor (bFGF), which plays an important role in tumor angiogenesis. The relationship between the chemical properties of the derivatives and the resulting fibrin structure was also examined. The structure of fibrin was analyzed by UV spectrophotometry and the degree of bFGF binding to fibrin was estimated by Western blotting. The measurements were taken at 3 different pH values: 6.64, 7.60 and 8.40. Three of the analyzed phosphorohydrazone derivatives (compounds 2, 7 and 10) demonstrated the most significant influence on reduction of polymerization at the studied pH values.
Assuntos
Cromonas/química , Fibrina/química , Fator 2 de Crescimento de Fibroblastos/química , Hidrazonas/química , DimerizaçãoRESUMO
Obtained benzimidazole derivatives, our next synthesized heterocyclic compounds, belong to a new group of chemical bondings with potential anticancer properties (Blaszczak-Swiatkiewicz & Mikiciuk-Olasik, 2006, J Liguid Chrom Rel Tech 29: 2367-2385; Blaszczak-Swiatkiewicz & Mikiciuk-Olasik, 2008, Wiad Chem 62: 11-12, in Polish; Blaszczak-Swiatkiewicz & Mikiciuk-Olasik, 2011, J Liguid Chrom Rel Tech 34: 1901-1912). We used HPLC analysis to determine stability of these compounds in 0.2% DMSO (dimethyl sulfoxide). Optimisation of the chromatographic system and validation of the established analytical method were performed. Reversed phases (RP-18) and a 1:1 mixture of acetate buffer (pH 4.5) and acetonitrile as a mobile phase were used for all the analysed compounds at a flow rate 1.0 mL/min. The eluted compounds were monitored using a UV detector, the wavelength was specific for compounds 6 and 9 and compounds 7 and 10. The retention time was specific for all four compounds. The used method was found to have linearity in the concentration range of (0.1 mg/mL-0.1 µg/mL) with a correlation coefficient not less than r(2)=0.9995. Statistical validation of the method proved it to be a simple, highly precise and accurate way to determine the stability of benzimidazole derivatives in 0.2% DMSO. The recoveries of all four compounds examined were in the range 99.24-100.00%. The developed HPLC analysis revealed that the compounds studied remain homogeneous in 0.2% DMSO for up to 96 h and that the analysed N-oxide benzimidazole derivatives do not disintegrate into their analogues - benzimidazole derivatives. Compounds 8, 6 and 9 exhibit the best cytotoxic properties under normoxic conditions when tested against cells of human malignant melanoma WM 115.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/isolamento & purificação , Benzimidazóis/síntese química , Benzimidazóis/isolamento & purificação , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Cristalização , Cristalografia por Raios X , Dimetil Sulfóxido/química , Estabilidade de Medicamentos , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Limite de Detecção , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Solventes/químicaRESUMO
Neoplastic cells produce procoagulants responsible for hypercoagulation states frequently observed in cancer patients. It is accepted that two major procoagulants from malignant tissue are tissue factor (TF) and a direct activator of coagulation factor X called cancer procoagulant (CP). Direct factor X-activating activity of cultured human malignant melanoma WM 115 cells has been analyzed in the cell extracts, whole cells and in the medium after the cell culture. The factor X-activating activity was detected in the malignant cell lysates but not in the cultured medium or intact malignant cells. The lysates contained no TF as determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) using anti-TF monoclonal antibody. The enzymatic characteristics of the activity was typical for CP. The results suggest that cancer procoagulant is an intracellular protein.
Assuntos
Cisteína Endopeptidases/metabolismo , Fator X/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Tromboplastina/metabolismo , Animais , Linhagem Celular TumoralRESUMO
Additional carboxylation of glutamic acid by vitamin K-dependent gamma-carboxylase is a common posttranslational modification of many proteins, including some of blood clotting factors. Vitamin K-antagonists, such as warfarin, are often included in the therapy of malignant disease, decreasing the blood coagulation potential. Cancer procoagulant, a direct blood coagulation factor X activator from malignant tissue, is considered as a vitamin K-dependent protein, so it could serve as one of possible targets for the therapy with warfarin. However, there is still no experimental data demonstrating directly the presence of gamma-carboxyglutamic acid (Gla) in a cancer procoagulant molecule. The presence of Gla in cancer procoagulant isolated from human amnion-chorion membranes and from human malignant melanoma WM 115 cell line was analyzed directly, using specific anti-Gla monoclonal antibodies. There was no detectable amount of Gla in cancer procoagulant isolated from fetal or malignant tissue. Cancer procoagulant from human tissues does not contain Gla-rich domain. The finding indicates that cancer procoagulant is rather a poor target for warfarin therapy of malignant disease.
