The overall objective is to obtain new mechanistic insight into how redox activation of anti-cancer antibiotics, such as adriamycin, by endothelial nitric oxide synthase (eNOS) may mediate their cytotoxic effects. This proposal is based on our following recent discoveries: (1) eNOS activates adriamycin to a semiquinone which undergoes redox-cycling to form superoxide. Under these conditions, NO formation is diminished, and (2) the newly developed phosphorylated spin trap, DEP,PO, can be used to detect and quantify superoxide, formed from eNOS, by electron spin resonance (ESR) spectroscopy using a loop-gap resonator. This allows the use of exceedingly small reaction volumes. We hypothesize that eNOS- mediate reductive activation of adriamycin and related anti-tumor analogues causes cardiovascular damage by shifting the balance between O/2 and NO. This mechanism enhances formation of peroxynitrite (ONOO-), a potent oxidant. Superoxide production will be detected by ESR spin- trapping. Nitric oxide will be measured directly by ESR and indirectly by cGMP and nitrite/nitrate measurements. Peroxynitrite formation will be assessed by nitration of protein tyrosines using HPLC and immunohistology. Myofibrillar structure will be examined by immunofluorescence microscopy. The hypothesis will be tested using purified eNOS, heme-deficient eNOS, eNOS-transfected human embryonic kidney (HEK) cells, and myocytes. Specifically, we will determine: (1) the kinetics of redox activation of adriamycin and analogues by purified eNOS; (2) whether peroxynitrite is formed from eNOS-mediated reduction of adriamycin; (3) the effect of adriamycin and related compounds on HEK cells and eNOS-transfected HEK cells; (4) the mechanism of cytotoxicity induced by adriamycin on cardiac myocytes; and (5) the effect of adriamycin treatment on myofibrillar structure. The information obtained from this study of the chemical interactions between adriamycin (and analogues) and eNOS in cellular and cell-free systems will lead to an increased understanding of the free- radical-mediated pathogenesis of anthracycline anti-tumor agents.
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