Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N 2 -oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N 2 -oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N 2 -oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N 2 -oxide.
Ellipticine is a potent antineoplastic agent exhibiting the multimodal mechanism of its action. This article reviews the mechanisms of predominant pharmacological and cytotoxic effects of ellipticine and shows the results of our laboratories indicating a novel mechanism of its action. The prevalent mechanisms of ellipticine antitumor, mutagenic and cytotoxic activities were suggested to be intercalation into DNA and inhibition of DNA topoisomerase II activity. We demonstrated a new mode of ellipticine action, formation of covalent DNA adducts mediated by its oxidation with cytochromes P450 (CYP) and peroxidases. The article reports the molecular mechanism of ellipticine oxidation by CYPs and identifies human and rat CYPs responsible for ellipticine metabolic activation and detoxication. It also presents a role of peroxidases (i.e. myeloperoxidase, cyclooxygenases, lactoperoxidase) in ellipticine oxidation leading to ellipticine-DNA adducts. The 9-hydroxy-and 7-hydroxyellipticine metabolites formed by CYPs and the major product of ellipticine oxidation by peroxidases, the dimer, in which the two ellipticine skeletons are connected via N 6 of the pyrrole ring of one ellipticine molecule and C9 in the second one, are the detoxication metabolites. On the contrary, 13-hydroxy-and 12-hydroxyellipticine, produced by ellipticine oxidation with CYPs, the latter one formed also spontaneously from another CYP-and peroxidase-mediated metabolite, ellipticine N 2 -oxide, are metabolites responsible for formation of two ellipticine-derived deoxyguanosine adducts in DNA. The results reviewed here allow us to propose species, two carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, as reactive species generating two major DNA adducts seen in vivo in rats treated with ellipticine. The study forms the basis to further predict the susceptibility of human cancers to ellipticine.
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.
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