TYROSINASE-INDUCED PHENOXYL RADICALS OF ETOPOSIDE (VP-16) - INTERACTION WITH REDUCTANTS IN MODEL SYSTEMS, K562 LEUKEMIC-CELL AND NUCLEAR HOMOGENATES

Etoposide (VP-16) is an antitumor drug currently in use for the treatm ent of a number of human cancers. Mechanisms of VP-16 cytotoxicity inv olve DNA breakage secondary to inhibition of DNA topoisomerase II and/ or direct drug-induced DNA strand cleavage. The VP-16 molecule contain s a hindered phenolic group which is crucial for its antitumor activit y because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromoleculer targets. VP-16 phenoxyl rad ical is an essential intermediate in VP-16 oxidative activation and ca n be either converted to oxidation products or reduced by intracellula r reductants to its initial phenolic form. In the present paper we dem onstrate that the tyrosinase-induced VP-16 phenoxyl radical could be r educed by ascorbate, glutathione (GSH) and dihydrolipoic acid. These r eductants caused a transient disappearance of a characteristic VP-16 p henoxyl radical ESR signal which reappeared after depletion of the red uctant. The reductants completely prevented VP-16 oxidation by tyrosin ase during the lag-period as measured by high performance liquid chrom atography; after the lag-period VP-16 oxidation proceeded with the rat e observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR sign al could be observed only after a lag-period whose duration was depend ent on cell concentration; VP-16 oxidation proceeded in cell homogenat es after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-p eriod, which was significantly shorter than that observed for an equiv alent amount of cells. In both cell homogenates and in nuclear homogen ates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance o f the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of t he VP-16 phenoxyl radical with intracellular reductants may be critica l for its metabolic activation and cytotoxic effects.