Doxorubicin-mediated free radical generation in intact human tumor cells enhances nitroxide electron paramagnetic...

The decay of nitroxide spin label electron paramagnetic resonance (EPR) absorption intensity was used to investigate the doxorubicin-mediated intracellular generation of free radicals. The effects of 50-500 micrograms/ml doxorubicin on human tumor cells (MCF-7, breast cancer cells, and HL-60, promyelocytic leukemia, cells) were studied by measuring 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) absorption intensity decay (TAID) at a TEMPO concentration of 10 microM. Doxorubicin accelerated the TAID in both cell lines with a detection limit of 50 micrograms/ml for MCF-7 cells and 500 micrograms/ml doxorubicin for HL-60 cells. Preincubation of cells with the iron chelating agent, deferoxamine (5 mM), partially prevented the effects of doxorubicin on the TAID. Catalase and copper, zinc-superoxide dismutase (Cu,Zn-SOD) had no influence on the effects of doxorubicin on the TAID in intact cells. However, Cu,Zn-SOD completely abolished the effects of doxorubicin on the TAID in a MCF-7 cell-free system. Our findings suggest that doxorubicin mediates the intracellular generation of O2.- and that iron is involved in this process.