Doxorubicin enhances complement susceptibility of human melanoma cells by extracellular oxygen radical formation

In two recent publications we showed that rapid inactivation of cell-bound C3b is a protective mechanism of human melanoma cells against killing by the R24 monoclonal antibody and human complement (Panneerselvam, M., Welt, S., Old, L.J., and Vogel, C.-W. (1986) J. Immunol. 136, 2534-2541) and that this protective mechanism can be inhibited by both the free and immobilized anthracycline glycoside doxorubicin (adriamycin) resulting in an enhanced complement susceptibility (Panneerselvam, M., Bredehorst, R., and Vogel, C.-W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9144-9148). In this paper we show that the complement enhancing effect of both free and immobilized doxorubicin is caused by the generation of reactive oxygen species including superoxide anion radical, hydrogen peroxide, and hydroxyl radical. The complement-enhancing effect of the anthracyclines can be completely inhibited by the reactive oxygen scavengers superoxide dismutase, catalase, and dimethyl sulfoxide. Consistent with this observation, 5-iminodaunorubicin, an anthracycline glycoside with an imine-substituted quinone moiety and, therefore, with a significantly reduced ability to form oxygen radicals, did not cause an enhanced-complement susceptibility. The complement-enhancing effect of the anthracycline glycosides could also be inhibited by bivalent metal chelators but was unaffected by sulfhydryl-blocking reagents or glutathione. Our results suggest that the anthracycline glycosides generate in a metal- (most probably iron) dependent reaction superoxide anion radicals with subsequent formation of hydrogen peroxide and hydroxyl radicals. These reactive oxygen species then cause alterations in the melanoma cells resulting in the enhanced complement susceptibility. While the target molecule(s) of the reactive oxygen species responsible for the enhanced complement susceptibility is not known, the data obtained with immobilized doxorubicin suggest that the target molecule(s) is located in the cell membrane.