ß-micrustoxin (Mlx-9), a PLA2 from Micrurus lemniscatus snake venom: biochemical characterization and anti-proliferative effect mediated by p53

Background: Endogenous phospholipases A2 (PLA2 ) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM médium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.(AU)

Alternative methods and animal experimentation: an innovative approach / Métodos substitutivos e a experimentação animal: um enfoque inovador

The scientific community believes that tests on animal models increase the chances of promising drug identification to warrant use in human clinical trials. However, science has shown that there is little evidence available to support this view, by advancing knowledge of pathologies, interspecies and species-specific differences, and technological advancement with alternative methods. The researchers, institutions and pharmacology industry increase their investments on the evidence presented by alternative methods, especially in vitro and in silico methods, and in the integration of databases based on the ethical principles of the 3Rs and those involved in the speed of a slow and costly process that is the production of a new drug and the failure of its reproducibility in clinical trials. Therefore, the importance of establishing new methods, their validation, the knowledge of the models, experimental designs, evaluation of the predictive studies, systematic reviews, meta-analyzes and the best understanding of the molecules before starting any research project should be employed. The main objective of the present study was to present the most recent data regarding validated alternative methods and the problems related to animal experimentation, especially in concern about the animal model and its predictive value and reproducibility for the human species. The research presented data indicating that the evidence that may predict the animal as a model for the research is insufficient. The alternative methods are a new paradigm and an innovative tool for the questions of the species-specific discrepancies found between human species and animals, especially regards to the toxicology and efficacy of a new target molecule. [...]

A comunidade científica acredita que testes em modelos animais aumentam as chances de identificação de drogas promissoras para justificar a utilização em ensaios clínicos humanos. Contudo, a ciência tem mostrado que há poucas evidências disponíveis que sustentem este ponto de vista, através do avanço do conhecimento das patologias, das diferenças interespécies e espécie-específicas e do avanço tecnológico com os métodos substitutivos. Partindo dos princípios éticos dos 3Rs e daqueles envolvidos na celeridade de um processo lento e oneroso que é a produção de um fármaco e na falência de sua reprodutibilidade, cada vez mais os pesquisadores, as instituições e a indústria apostam nas evidências apresentadas pelos métodos substitutivos, em especial nos métodos in vitro e in silico e na integração dos bancos de dados. Portanto, a importância de se estabelecer novos métodos, validá-los, conhecer os modelos, desenhos experimentais, avaliar os estudos preditivos, revisões sistemáticas, meta--análises e o melhor entendimento das moléculas antes de se iniciar qualquer projeto de pesquisa devem ser empregados. O estudo apresentado teve por objetivo principal apresentar os dados mais recentes no que tange aos métodos substitutivos validados e os complicadores relacionados à experimentação animal, especialmente, em relação ao modelo animal e seu valor preditivo e reprodutibilidade para a espécie humana. A pesquisa apresentou dados indicativos de que as evidências que possam predizer o animal como modelo para a pesquisa são insuficientes. Os métodos substitutivos apresentam-se hoje como um novo paradigma e uma ferramenta inovadora para as questões das discrepâncias espécie-específicas encontradas entre o homem e o animal, principalmente no que concerne à toxicologia e eficácia de uma nova molécula-alvo.[...]

Alternative methods and animal experimentation: an innovative approach / Métodos substitutivos e a experimentação animal: um enfoque inovador

The scientific community believes that tests on animal models increase the chances of promising drug identification to warrant use in human clinical trials. However, science has shown that there is little evidence available to support this view, by advancing knowledge of pathologies, interspecies and species-specific differences, and technological advancement with alternative methods. The researchers, institutions and pharmacology industry increase their investments on the evidence presented by alternative methods, especially in vitro and in silico methods, and in the integration of databases based on the ethical principles of the 3Rs and those involved in the speed of a slow and costly process that is the production of a new drug and the failure of its reproducibility in clinical trials. Therefore, the importance of establishing new methods, their validation, the knowledge of the models, experimental designs, evaluation of the predictive studies, systematic reviews, meta-analyzes and the best understanding of the molecules before starting any research project should be employed. The main objective of the present study was to present the most recent data regarding validated alternative methods and the problems related to animal experimentation, especially in concern about the animal model and its predictive value and reproducibility for the human species. The research presented data indicating that the evidence that may predict the animal as a model for the research is insufficient. The alternative methods are a new paradigm and an innovative tool for the questions of the species-specific discrepancies found between human species and animals, especially regards to the toxicology and efficacy of a new target molecule. [...](AU)

A comunidade científica acredita que testes em modelos animais aumentam as chances de identificação de drogas promissoras para justificar a utilização em ensaios clínicos humanos. Contudo, a ciência tem mostrado que há poucas evidências disponíveis que sustentem este ponto de vista, através do avanço do conhecimento das patologias, das diferenças interespécies e espécie-específicas e do avanço tecnológico com os métodos substitutivos. Partindo dos princípios éticos dos 3Rs e daqueles envolvidos na celeridade de um processo lento e oneroso que é a produção de um fármaco e na falência de sua reprodutibilidade, cada vez mais os pesquisadores, as instituições e a indústria apostam nas evidências apresentadas pelos métodos substitutivos, em especial nos métodos in vitro e in silico e na integração dos bancos de dados. Portanto, a importância de se estabelecer novos métodos, validá-los, conhecer os modelos, desenhos experimentais, avaliar os estudos preditivos, revisões sistemáticas, meta--análises e o melhor entendimento das moléculas antes de se iniciar qualquer projeto de pesquisa devem ser empregados. O estudo apresentado teve por objetivo principal apresentar os dados mais recentes no que tange aos métodos substitutivos validados e os complicadores relacionados à experimentação animal, especialmente, em relação ao modelo animal e seu valor preditivo e reprodutibilidade para a espécie humana. A pesquisa apresentou dados indicativos de que as evidências que possam predizer o animal como modelo para a pesquisa são insuficientes. Os métodos substitutivos apresentam-se hoje como um novo paradigma e uma ferramenta inovadora para as questões das discrepâncias espécie-específicas encontradas entre o homem e o animal, principalmente no que concerne à toxicologia e eficácia de uma nova molécula-alvo.[...](AU)