Chimeric Manganese-Super Oxide Dismutase-2 (Mn-SOD-2) Secreted from a Human Liposarcoma with Specific and Selective Cytotoxic Activity on Tumor Cells Expressing Estrogen Receptors.

A human liposarcoma derived cell line (LSA), growing in a chemical defined medium, releases in this conditioned medium (LSA-CM), proteins for an outocrine growth. From 40 liters of LSA-CM we have isolated, purified and sequenced a mutated form of Manganese Super Oxide Dismutase-2 displaying a specific and selective cytotoxic activity on tumor cells expressing Estrogen Receptors (RE). The recombinant Mn-SOD-2, expressed in E. coli, presents the same characteristics of pure extractive molecule. The Mn-SOD-2 can be secreted, as from LSA cells, and can be internalized by tumor cells expressing ER. TEM observations by using immunocytochemistry, confirm an intense secretion of this molecule that occurs in Golgi apparatus and in the large amount of secretion vesicles surrounding the plasmatic membrane. Mass Spectrometric analysis of rec-Mn-SOD-2 shows a M.W. of 26666 0.9, about 1800 Da higher than the wild type protein, suggesting the occurrence of an additional 15–20 aminoacids peptide. Particularly interesting was the discovery that the rec-Mn-SOD-2 contains a moiety of the Estrogen Receptors (16349 fmol/mg protein), and confirmed by Immunoblotting, by using two different antibodies raised against the and estrogen receptors. The in vitro Pharmacology analysis of rec-Mn-SOD-2 confirm the tumor suppressive activity on many tumor cell lines expressing ER and low levels of Catalase enzyme. Moreover, when rec-Mn-SOD-2 (1.5 M) is combined with low amount of Cisplatin (25 M), a synergic effect on the cytotoxic activity was observed, producing the necrosis of tumor cells in a few hrs. We retain that per se the rec-Mn-SOD-2 is toxic for tumor cells, but when it is combined with the Cisplatin, this drug turning off completely the gene for Catalase, amplifies the necrotic effect of rec-Mn-SOD-2. On these basis a mechanism for the cytotoxicicty effect of rec-Mn-SOD-2 might be suggested: rec-Mn-SOD-2 can be internalised into tumor cells due to the presence of the RE moiety and transforms all the free radicals into hydrogen peroxide. This reaction is dose-dependent, and only a normal levels of Catalase might be effective to neutralize the cytotoxicity of hydrogen peroxide. Since many tumors express low levels of Catalase (20 to 40 fold lower than the normal cells), the necrosis produced by rec-Mn-SOD-2 might be ascribed to the excess of hydrogen peroxide. This hypothesis is also confirmed by the observation that the addition of low amount (25 M) of Cisplatin, which is able to turn off completely the gene for Catalase, drammatically increases the citotoxic effect of rec-SOD-2. L.M. and TEM immunocytochemistry on tumor cells, after rec-Mn-SOD-2 adding to culture medium, displays internalisation of rec-Mn-SOD-2 in cytoplasm, mitochondria and nucleus and a clear necrotic feature of these cells. These results were confirmed by in vivo experiments. Rec-Mn-SOD-2 was daily injected (2 g/s.c.) into balb-C-fR-iii affected by mammary adenocarcinoma, inducing the necrosis of the tumor mass, as monitored by NMR and histological examination. Moreover, the treated animals did not develope lung metastasis. At time Mn-SOD-2 is under R&D from FIDIA-Pharmaceutical Co. to explore possible terapeutic of this antitumoral agent.