Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom |
| |
Authors: | Gustavo B Naumann Liliana F Silva Luciana Silva Gilson Faria Michael Richardson Karla Evangelista Markus Kohlhoff Celia MF Gontijo Alexei Navdaev Flavia F de Rezende Johannes A Eble Eladio F Sanchez |
| |
Institution: | 1. Research and Development Center, Ezequiel Dias Foundation, 30510-0103, Belo Horizonte, Brazil;2. Research Center Rene Rachou, Belo Horizonte, Dept. Vascular Matrix Biology Excellence Cluster CardioPulmonary System, Frankfurt University Hospital, Frankfurt am Main, Germany;3. Center for Molecular Medicine, Dept. Vascular Matrix Biology Excellence Cluster CardioPulmonary System, Frankfurt University Hospital, Frankfurt am Main, Germany |
| |
Abstract: | BackgroundMultifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms.MethodsThe l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.ResultsBl-LAAO is a 60 kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H2O2. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC50 of 0.07 μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H2O2 in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.ConclusionBl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H2O2 which kill the cells.General significanceThese results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism. |
| |
Keywords: | MTT [3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetra-zolium] IC50 inhibitory concentration that causes 50% inhibition OPD o-phenylenediamine DMC dimethylcasein Bl-LAAO l-amino acid oxidase from Bothrops leucurus venom i p intraperitoneal ACN acetonitrile TFA trifluoroacetic acid 2-DE two-dimensional gel electrophoresis |
本文献已被 ScienceDirect 等数据库收录! |
|