DM43 an inhibitor of involved metalloproteases of matrix on Osteoarthritis and Neoplasms.
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). In this study, we have been investigating the effect of DM43 against MMPs involved in some pathologic processes, like osteoarthritis and cancer. In order to test if DM43 is a potential inhibitor of MMPs, we submitted supernatants of stromal cells (3T3), adenocarcinoma cell line (prostate and breast) and synovial fluid from osteoarthritis to an affinity column coupled with DM43. Bound samples were analyzed on SDS-PAGE and electrotransferred to a PVDF membrane, which was incubated with anti-MMP2, anti-MMP3 or anti-MMP9 monoclonal antibodies. Positive staining was obtained. Enzymes were inhibited by ortophenantroline and DM43, using fibronectin and casein as substrates. Number of cells, cycle and cellular death were carried out on fibroblasts (3T3) and adenocarcinoma (MDA and MCF7) with three doses of DM43 (10, 250 e 1000ng/mL). Our results demonstrated a decrease on 3T3 and MCF7 numbers, using all these DM43 doses. Cell cycle analysis demonstrated no differences in control and DM43 treated cells. The decrease of celularity was promoted by the increase in cellular death by an already unknown pathway. The cellular death on 3T3 and MCF7 was increased 2 and 7x respectively, compared with the control. MDA cell line was not affected by DM43 treatment. Imunocytochemistry was carried out on 3T3 cells, which were incubated with antibodies raised against ECM (laminin, fibronectin and collagen IV) followed by FITC-conjugated goat anti-rabbit for staining procedure. DM43 in all doses used was able to increase the fibronectin deposition leading to adhesion increase. The inhibition of MMPs activities by DM43 can result on induction of cellular death on stromal and tumoral cells, beyond the deposition of MEC. These results suggest, the use of DM43, as a new candidate for anti-cancer therapy, just like, for patients with osteoarthritis.
Advisor:Gilberto Barbosa Domont; Jonas Enrique Aguilar Perales
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Date of Publication:10/01/2004