Cytochrome b5 increases cytochrome P450 3A4-mediated activation of anticancer drug ellipticine to 13-hydroxyellipticine whose covalent binding to DNA is elevated by sulfotransferases and N,O-acetyltransferases.

Department of Paediatric Haematology and Oncology
 
Abstract
The antineoplastic alkaloid ellipticine is a prodrug, whose pharmacological efficiency is dependent on its cytochrome P450 (P450) – and/or peroxidase-mediated activation in target tissues. The P450 3A4 enzyme oxidizes ellipticine to five metabolites, mainly to 13-hydroxy- and 12-hydroxyellipticine, the metabolites responsible for the formation of ellipticine-13-ylium and ellipticine-12-ylium ions that generate covalent DNA adducts. Cytochrome b(5) alters the ratio of ellipticine metabolites formed by P450 3A4. While the amounts of the detoxication metabolites (7-hydroxy- and 9-hydroxyellipticine) were not changed with added cytochrome b(5), 12-hydroxy- and 13-hydroxyellipticine, and ellipticine N(2)-oxide increased considerably. The P450 3A4-mediated oxidation of ellipticine was significantly changed only by holo-cytochrome b(5), while apo-cytochrome b(5) without heme or Mn-cytochrome b(5) had no such effect. The change in amounts of metabolites resulted in an increased formation of covalent ellipticine-DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. The amounts of 13-hydroxy- and 12-hydroxyellipticine formed by P450 3A4 were similar, but more than 7-foldhigher levels of the adduct were formed by 13-hydroxyellipticine than by 12-hydroxyellipticine. The higher susceptibility of 13-hydroxyellipticine toward heterolytic dissociation to ellipticine-13-ylium in comparison to dissociation of 12-hydroxyellipticine to ellipticine-12-ylium, determined by quantum chemical calculations, explains this phenomenon. The amounts of the 13-hydroxyellipticine-derived DNA adduct significantly increased upon reaction of 13-hydroxyellipticine with either 3'-phosphoadenosine-5'-phosphosulfate or acetyl-CoA catalyzed by human sulfotransferases 1A1, 1A2, 1A3, and 2A1, or N,O-acetyltransferases 1 and 2. The calculated reaction free energies of heterolysis of the sulfate and acetate esters are by 10–17 kcal/mol more favorable than the energy of hydrolysis of 13-hydroxyellipticine, which could explain the experimental data.
 
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