Nitric oxide increases doxorubicin toxicity in the murine mammary adenocarcinoma LM3 cell line.

SCHÖPFER F; SKVERER S; JASNIS M; BOVERIS AD; BOVERIS A; BAL DE KIER JOFFÉ E; PODEROSO JJ

Mar del Plata

Congreso; II Congress South American Group for Free Radical Research; 2001

South American Group for Free Radical Research

Doxorubicin (DOX) is an anthracycline antitumor agent used to treat 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 nitric 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 killing. The superoxide anion formation by chemically reduced DOX showed a linear dependence on DOX concentration. DOX autoxidation showed also the formation of the DMPO:OOH adduct. The addition of Mn(III)TBAP resulted in the formation of the DMPO:OH adduct, whereas NO totally abolished the formation of both signals. Nitrotyrosine formation, measured by western blot on BSA previously exposed to 0-4 mM 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 mM DOX, 0,2 mM DETA-NO or both. Cells survival was analyzed using the MTS method. DOX treatment resulted in a concentration dependent increase in cell killing. Treatments 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 1mM DETA-NO 1 mM DOX or its combination were 1.8±0.9, 15.9±0.8, 15.6±2.5 and 48.7±1.1 % respectively. The cotreatment also resulted in the cellular protein nitration and the addition of Mn(III)TBAP restored the control nitration levels. 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.