Inhibition of [formula omitted]-ATPase by phenoxyl radicals of etoposide (VP-16): role of sulfhydryls oxidation

Abstract

In the present work, we studied the effects of phenoxyl radicals, generated by tyrosinase-catalyzed oxidation of a phenolic antitumor drug, Etoposide (VP-16), on a purified dog kidney Na + K + -ATPase by characterizing interactions of VP-16 phenoxyl radicals with the enzyme's SH-groups by ESR and correlating the loss of the enzymatic activity with the oxidation of its SH-groups, and oxidation of VP-16. VP-16/tyrosinase caused inhibition of Na + K + -ATPase which was dependent on the incubation time and concentration of tyrosinase. The inhibition of Na + K + -ATPase was accompanied by a decrease of DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)-titratable SH-groups. In the presence of Na + K + -ATPase, a typical ESR signal of the VP-16 phenoxyl radical could be observed only following a lag period the duration of which was proportional to the concentration of the Na + K + -ATPase added. Our HPLC measurements demonstrated that Na + K + -ATPase protected VP-16 against tyrosinase-catalyzed oxidation. Combined these results suggest that redox-cycling of VP-16/VP-16 phenoxyl radical by SH-groups of Na + K + -ATPase occurred. Ascorbate which is known to reduce the VP-16 phenoxyl radicals, protected the enzyme against inactivation, prevented oxidation of the enzyme's SH-groups. Reduction of VP-16 phenoxyl radicals by ascorbate was directly observed by the semidehydroascorbyl radical signal in the ESR spectra. VP-16 phenoxyl radical-induced oxidation of sulfhydryls and inhibition of the Na + K + -ATPase may be responsible for at least some of its clinical side effects (e.g., cardiotoxicity) which can be prevented by ascorbate.