DM43, A SNAKE VENOM METALLOPROTEINASE INHIBITOR, INDUCES CELLULAR DEATH ON FIBROBLAST CELL LINE (3T3)

Jurgilas, P.B.(1), DE Meis, J.(2), Mendes-da-Cruz, D.(2), Farias-de-Oliveira, D.(2), Savino, W.(2), Perales, J.(1)

(1)Departamento de Fisiologia e Farmacodinâmica, Laboratório de Toxinologia, IOC, FIOCRUZ, Rio de Janeiro, Brasil. (2)Departamento de Imunologia, Laboratório de Pesquisas Sobre o Timo, IOC, FIOCRUZ, Rio de Janeiro, Brasil.

Natural resistance of marsupials against the toxic effects of venoms is due to the presence of soluble neutralizing proteins in their sera. We have isolated from opossum serum (Didelphis marsupialis) an acidic glycoprotein with 43 kDa (DM43), with an effective inhibition action to metalloproteinases snake venoms, by non-covalent complex formation (Neves-Ferreira et al., 2000). Recently, it was showed that, the tissue inhibitors of metalloproteinases (TIMPs), are multifunctional factors which, besides their inhibitory effect, have others activities including regulation of proliferation, pro-MMP-2 activation, inhibition of angiogenesis, cell transformation, and apoptosis (Brew et al., 2000). The aim of this work was to study the ability of DM43 to produce some cellular effect, as TIMPs do. Celularity, viability and proliferation were carried out on 3T3 fibroblasts, incubating three doses of DM43 (10, 250 e 1000ng) for 20 hours at 37°C. Apoptosis and necrosis were detected using flow cytometry by light scatter and analyzed on a FACScan. Cells were also reviewed microscopically for morphological evidence. Our results demonstrated a celularity decrease, in all doses used of DM43. This decrease could be promoted by the increase of cellular death and/or change of cellular cycle. Cell cycle analysis demonstrated similar results in control and in DM43 treated cells. The measured  of the cellular death by 7-Actinomicin D (7AAD) labeling showed a significative increase of cell death in all doses analyzed, but preferentially with 10ng of DM43. Interestingly, DM43 in this lowest dose seems to be responsible for the lower celularity and the higher cell death. Our data suggests that the snake-venom metalloproteinase inhibitor (DM43) promotes cell death in fibroblast (3T3). Studies are under way to understand the mechanisms involved in this phenomenon.

Supported by: CAPES, CNPQ, FIOCRUZ.

CORRESPONDENCE TO:

Patricia Barbosa Jurgilas, Avenida Francisco Neves, 50 BL 3, Apto. 109, Ilha do Governador, Rio de Janeiro, RJ, Brasil, Email: jurgilas@ioc.fiocruz.br