Potentiation of Doxorubicin by DETA-NO in a Murine Mammary Adenocarcinoma LM3 Cell Line.

SCHÖPFER F; JASNIS MA; BAL DE KIER JOFFÉ; PODEROSO JJ

San Francisco, EEUU

Congreso; First International Conference, Biology, Chemistry and Therapeutic Applications of Nitric Oxide.; 2000

NO Society

Doxorubicin (DOX) is an anthracycline antitumor agent extensively used to treta acute leukemias, lymphomas and a wide variety of solid tumors including breast cancer. DOX exerts its cytotoxic actions by two different mechanisms including binding to DNA and superoxide production. Superoxide anion reacts with nitrc oxide in a diffusion-limited reaction forming the powerful oxidant peroxynitrite. The aim of this work was to induce intracellular peroxynitrite formation by combining DOX with DETA-NO to enhance tumor cell killing. The superoxide anion formation by chemically reduced DOX, using the adenochrome method, showed a linear dependence on DOX concentration. Nitrotyrosine formation, measured by western blot on BSA previously exposed to 0-4 uM reduced DOX and a pulse of pure 1.7 mM NO showed DOX-dependent nitration. Combined treatments were studied in the LM3 cell line. Cells were grown up to 75% confluence and then incubated during 24 or 48 hs in culture medium containing 2% FCS in the presence of 0-4 uM DOX, 0-2 mM EDTA, 0-2 mM DETA-NO or both. Cell survival was analyzed using the MTS method. DOX treatment resulted in a concentration dependent increase in cell killing. Treatment of cells with increasing DETA-NO concentration increased DOX toxicity, resulting a synergic effect. Similar results were obtained by counting apoptotic/necrotic cells stained with acridine orange and ethidium bromide in a fluorescence microscopy. Percentages of cell death in controls, and after 1 mM DETA-NO, 1 uM DOX or its combination were 1.8±0.9, 15.9±0.8, 15.6±2.5 and 48.7±1.1% respectively. The study supports that NO reacts with doxorubicin-derived superoxide anion. Synergism of treatments is due to the formation of peroxynitrite and encourages to test lower less toxic doxorubicin concentrations, associated to nitric oxide.