Inhibition of Na+/K(+)-ATPase by phenoxyl radicals of etoposide (VP-16): role of sulfhydryls oxidation.
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.
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism* / Q000494:pharmacology; D004285:Dogs; D004578:Electron Spin Resonance Spectroscopy; D005047:Etoposide / Q000378:metabolism* / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D010084:Oxidation-Reduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D005609:Free Radicals; D013438:Sulfhydryl Compounds; D005047:Etoposide; D000254:Sodium-Potassium-Exchanging ATPase