Estudo da relaÃÃo entre a atividade anti-tumoral in vitro do Ãcido Ãsnico e a ativaÃÃo da via metabÃlica p53

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Usnic acid is a metabolite of lichen that exerts a wide variety of biological activities including cytotoxicity against cells derived from human malignant tumours. Although cytotoxicity of usnic acid has been extensively reviewed, its mechanism of action is still unclear. There are no data in the literature about the involvement of the tumour suppressor p53 with the effects of usnic acid. Wild type p53 responds to different forms of cellular stress by acting as a transcription factor, regulating the expression of genes that will lead to inhibition of cellular proliferation by inducing cell cycle arrest or apoptosis. p53 actions include the down regulation of genes that encode proteins associated to the polymerization and stabilization of microtubules. These functions are lost when mutations occur in the p53 pathway, which happens in more than 50% of human cancers. The objective of this work was to investigate the relationship between the antineoplastic activity of usnic acid and p53 pathway. To establish the sensitivity of cancer cell lines to usnic acid, MTT (3-[4,5-dimetiltiazol-2-il]-2,5- difeniltetrazÃlio) assays were performed using a variety of drug concentrations (1 to 60 μM) for 72h against the following cell lines: MCF7 (oestrogen positive, wild type for p53) and MDA-MB-231 (oestrogen negative, non-functional p53) and the lung cancer cell line H1299 (p53 null). To investigate the involvement of p53 in the cytotoxic activity of usnic acid, the protein levels of p53 and p21 (a cyclin dependent kinase inhibitor whose expression is under the control of p53) in MCF7 cells treated with 29 μM usnic acid for 24h were determined with western blot assays using the antibodies DO-12 (specific for p53) and WAF1 (specific for p21). To establish whether the anticancer activity of usnic acid results in DNA damage, the phosphorylation of p53 SER15 (a DNA damage marker) was investigated following MCF7 cells treatment with 29 μM usnic acid for 24h. For this study the FPS15 antibody (polyclonal, specific for phosphor-SER15) was used. To determine if the transcriptional activity of p53 correlated with the increase in its protein level the Ã-Gal assay was performed. T22 mouse fibroblast cells bearing the reporter plasmid RGFos-LacZ (containing a 36 bp p53 binding site) were treated with different concentrations of usnic acid. To investigate the effects of usnic acid on the polymerization and stabilization of microtubules, MCF7 cells were exposed to 29 μM usnic acid for 24h, fixed with methanol and treated with monoclonal anti- Ã-tubulin antibody. Usnic acid showed cytotoxic activity against the several human cancer cells tested, with increase in the p53 and p21 protein levels. However, this was not correlated with increase in p53 transcriptional activity or with p53 SER15 phosphorylation. Furthermore, no modifications in the microtubule formation were detected. The property of usnic acid as a non-genotoxic anticancer agent that works in a p53-independent manner makes it a potential candidate for novel cancer therapy

ASSUNTO(S)

genotoxicidade proteÃna p53 Ãcido Ãsnico p53 activity usnic acid microtubulos linhagens celulares tumorais: mcf7, mda-mb-231 e h1299 microtubules atividade gene p53 genotoxicity ciencias biologicas mcf7, mda-mb-231, h1299 cells

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