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The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies: studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN™) mice and pure enzymes.

Stiborová M, Černá V, Moserová M, Mrízová I, Arlt VM, Frei E - Int J Mol Sci (2014)

Bottom Line: Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP).Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo.We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic. stiborov@natur.cuni.cz.

ABSTRACT
Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

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Ellipticine metabolism by CYPs showing the identified metabolites and those proposed to form DNA adducts. The compounds shown in brackets were not detected under the experimental conditions and/or are not yet structurally characterized. The CYP enzymes predominantly oxidizing ellipticine shown were identified in our previous studies [31,34,35,38,39]. Reactions 1, 2 and 3 lead to ellipticine-13-ylium from 13-hydroxyellipticine, 13-hydroxyellipticine sulfate and 13-hydroxyellipticine acetate, respectively, and rea 1b to ellipticine 12-ylium. ? indicates that the mechanisms of this reaction are not known.
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ijms-16-00284-f003: Ellipticine metabolism by CYPs showing the identified metabolites and those proposed to form DNA adducts. The compounds shown in brackets were not detected under the experimental conditions and/or are not yet structurally characterized. The CYP enzymes predominantly oxidizing ellipticine shown were identified in our previous studies [31,34,35,38,39]. Reactions 1, 2 and 3 lead to ellipticine-13-ylium from 13-hydroxyellipticine, 13-hydroxyellipticine sulfate and 13-hydroxyellipticine acetate, respectively, and rea 1b to ellipticine 12-ylium. ? indicates that the mechanisms of this reaction are not known.

Mentions: Utilizing numerous in vitro systems such as subcellular microsomal fractions and cells in culture expressing CYPs, isolated CYPs reconstituted with other components of the mixed-function-oxidase system [NADPH:CYP reductase (POR), cytochrome b5], and recombinant CYPs, human, rat, rabbit, and mouse CYP enzymes were found to oxidize ellipticine. Ellipticine is oxidized to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine, and ellipticine N2-oxide (Figure 3), and at least two major ellipticine DNA-adducts were generated by these enzymatic systems [3,31,33,34,35,36,38,39,40,48,49]. 7-Hydroxy- and 9-hydroxyellipticine are efficiently excreted by experimental animals and considered to be the detoxification products of ellipticine [52,53]. But 9-hydroxyellipticine is also an efficient inhibitor of Pol-I transcription in vitro with IC50 values in cells in the nanomolar range [29], intercalates into DNA, and inhibits topoisomerase II activity [54,55,56]. It is therefore a pharmacologically important metabolite. 13-Hydroxy- and 12-hydroxyellipticine are the active metabolites, which spontaneously form ellipticine-13-ylium and ellipticine-12-ylium, which reacts with DNA to produce two major deoxyguanosine adducts (see adduct spots 1 and 2 in Figure 2 and the proposed structures of these DNA adducts in Figure 3) [2,3,4,31,32,34,35,36,39,40,49]. In addition, ellipticine N2-oxide is also considered a potent active ellipticine metabolite, since it converts to 12-hydroxyellipticine [31], by the Polonowski rearrangement [57] (Figure 3). All these results suggest that the enzymes activating or detoxifying ellipticine are crucial for its pharmacological effects. Thus, the identification of enzymes necessary for ellipticine metabolism is of great importance.


The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies: studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN™) mice and pure enzymes.

Stiborová M, Černá V, Moserová M, Mrízová I, Arlt VM, Frei E - Int J Mol Sci (2014)

Ellipticine metabolism by CYPs showing the identified metabolites and those proposed to form DNA adducts. The compounds shown in brackets were not detected under the experimental conditions and/or are not yet structurally characterized. The CYP enzymes predominantly oxidizing ellipticine shown were identified in our previous studies [31,34,35,38,39]. Reactions 1, 2 and 3 lead to ellipticine-13-ylium from 13-hydroxyellipticine, 13-hydroxyellipticine sulfate and 13-hydroxyellipticine acetate, respectively, and rea 1b to ellipticine 12-ylium. ? indicates that the mechanisms of this reaction are not known.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307247&req=5

ijms-16-00284-f003: Ellipticine metabolism by CYPs showing the identified metabolites and those proposed to form DNA adducts. The compounds shown in brackets were not detected under the experimental conditions and/or are not yet structurally characterized. The CYP enzymes predominantly oxidizing ellipticine shown were identified in our previous studies [31,34,35,38,39]. Reactions 1, 2 and 3 lead to ellipticine-13-ylium from 13-hydroxyellipticine, 13-hydroxyellipticine sulfate and 13-hydroxyellipticine acetate, respectively, and rea 1b to ellipticine 12-ylium. ? indicates that the mechanisms of this reaction are not known.
Mentions: Utilizing numerous in vitro systems such as subcellular microsomal fractions and cells in culture expressing CYPs, isolated CYPs reconstituted with other components of the mixed-function-oxidase system [NADPH:CYP reductase (POR), cytochrome b5], and recombinant CYPs, human, rat, rabbit, and mouse CYP enzymes were found to oxidize ellipticine. Ellipticine is oxidized to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine, and ellipticine N2-oxide (Figure 3), and at least two major ellipticine DNA-adducts were generated by these enzymatic systems [3,31,33,34,35,36,38,39,40,48,49]. 7-Hydroxy- and 9-hydroxyellipticine are efficiently excreted by experimental animals and considered to be the detoxification products of ellipticine [52,53]. But 9-hydroxyellipticine is also an efficient inhibitor of Pol-I transcription in vitro with IC50 values in cells in the nanomolar range [29], intercalates into DNA, and inhibits topoisomerase II activity [54,55,56]. It is therefore a pharmacologically important metabolite. 13-Hydroxy- and 12-hydroxyellipticine are the active metabolites, which spontaneously form ellipticine-13-ylium and ellipticine-12-ylium, which reacts with DNA to produce two major deoxyguanosine adducts (see adduct spots 1 and 2 in Figure 2 and the proposed structures of these DNA adducts in Figure 3) [2,3,4,31,32,34,35,36,39,40,49]. In addition, ellipticine N2-oxide is also considered a potent active ellipticine metabolite, since it converts to 12-hydroxyellipticine [31], by the Polonowski rearrangement [57] (Figure 3). All these results suggest that the enzymes activating or detoxifying ellipticine are crucial for its pharmacological effects. Thus, the identification of enzymes necessary for ellipticine metabolism is of great importance.

Bottom Line: Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP).Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo.We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic. stiborov@natur.cuni.cz.

ABSTRACT
Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

Show MeSH
Related in: MedlinePlus