title: 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.
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-fold higher 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.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic. stiborov@natur.cuni.cz
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborová
authors.forename: Radek
authors.initials: R
authors.lastname: Indra
authors.forename: Michaela
authors.initials: M
authors.lastname: Moserová
authors.forename: Věra
authors.initials: V
authors.lastname: Cerná
authors.forename: Martina
authors.initials: M
authors.lastname: Rupertová
authors.forename: Václav
authors.initials: V
authors.lastname: Martínek
authors.forename: Tomáš
authors.initials: T
authors.lastname: Eckschlager
authors.forename: René
authors.initials: R
authors.lastname: Kizek
authors.forename: Eva
authors.initials: E
authors.lastname: Frei
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism* / Q000494:pharmacology; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004611:Ellipticines / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D011355:Prodrugs / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D015238:Sulfotransferases / Q000378:metabolism
chemical_list: C517215:13-hydroxyellipticine; D000972:Antineoplastic Agents, Phytogenic; D004611:Ellipticines; D011355:Prodrugs; D004247:DNA; D015786:Cytochromes b5; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D001191:Arylamine N-Acetyltransferase; D015238:Sulfotransferases