Cytochrome b5 plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine

Marie Stiborová; Radek Indra; Eva Frei; Kateřina Kopečková; Heinz H. Schmeiser; Tomáš Eckschlager; Vojtěch Adam; Zbyněk Heger; Volker M. Arlt; Václav Martínek

doi:10.1007/s00706-017-1986-9

Cytochrome b₅ plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine

Marie Stiborová^*, Radek Indra, Eva Frei, Kateřina Kopečková, Heinz H. Schmeiser, Tomáš Eckschlager, Vojtěch Adam, Zbyněk Heger, Volker M. Arlt, Václav Martínek

^*Corresponding author for this work

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Abstract

Abstract: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b₅ reductase and/or cytochrome b₅ in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b₅ reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N²-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the ³²P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b₅ stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b₅ plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b₅ mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b₅ reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b₅ can act as an allosteric modifier of the CYP3A4 oxygenase. Graphical abstract: [Figure not available: see fulltext.]

Original language	English
Pages (from-to)	1983-1991
Journal	Monatshefte für Chemie - Chemical Monthly
Volume	148
Issue number	11
Early online date	4 Jul 2017
DOIs	https://doi.org/10.1007/s00706-017-1986-9
Publication status	Published - Nov 2017

Keywords

Coenzymes
DNA
Enzymes
High pressure liquid chromatography

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Stiborová, M., Indra, R., Frei, E., Kopečková, K., Schmeiser, H. H., Eckschlager, T., Adam, V., Heger, Z., Arlt, V. M., & Martínek, V. (2017). Cytochrome b₅ plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine. Monatshefte für Chemie - Chemical Monthly, 148(11), 1983-1991. https://doi.org/10.1007/s00706-017-1986-9

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title = "Cytochrome b5 plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine",

abstract = "Abstract: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes{\texttrademark} to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes{\texttrademark} generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase. Graphical abstract: [Figure not available: see fulltext.]",

keywords = "Coenzymes, DNA, Enzymes, High pressure liquid chromatography",

author = "Marie Stiborov{\'a} and Radek Indra and Eva Frei and Kate{\v r}ina Kope{\v c}kov{\'a} and Schmeiser, {Heinz H.} and Tom{\'a}{\v s} Eckschlager and Vojt{\v e}ch Adam and Zbyn{\v e}k Heger and Arlt, {Volker M.} and V{\'a}clav Mart{\'i}nek",

year = "2017",

month = nov,

doi = "10.1007/s00706-017-1986-9",

language = "English",

volume = "148",

pages = "1983--1991",

journal = "Monatshefte f{\"u}r Chemie - Chemical Monthly",

issn = "1434-4475",

publisher = "Springer Verlag",

number = "11",

}

Stiborová, M, Indra, R, Frei, E, Kopečková, K, Schmeiser, HH, Eckschlager, T, Adam, V, Heger, Z, Arlt, VM & Martínek, V 2017, 'Cytochrome b₅ plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine', Monatshefte für Chemie - Chemical Monthly, vol. 148, no. 11, pp. 1983-1991. https://doi.org/10.1007/s00706-017-1986-9

TY - JOUR

T1 - Cytochrome b5 plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine

AU - Stiborová, Marie

AU - Indra, Radek

AU - Frei, Eva

AU - Kopečková, Kateřina

AU - Schmeiser, Heinz H.

AU - Eckschlager, Tomáš

AU - Adam, Vojtěch

AU - Heger, Zbyněk

AU - Arlt, Volker M.

AU - Martínek, Václav

PY - 2017/11

Y1 - 2017/11

N2 - Abstract: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase. Graphical abstract: [Figure not available: see fulltext.]

AB - Abstract: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase. Graphical abstract: [Figure not available: see fulltext.]

KW - Coenzymes

KW - DNA

KW - Enzymes

KW - High pressure liquid chromatography

UR - http://www.scopus.com/inward/record.url?scp=85021791759&partnerID=8YFLogxK

U2 - 10.1007/s00706-017-1986-9

DO - 10.1007/s00706-017-1986-9

M3 - Article

AN - SCOPUS:85021791759

SN - 1434-4475

VL - 148

SP - 1983

EP - 1991

JO - Monatshefte für Chemie - Chemical Monthly

JF - Monatshefte für Chemie - Chemical Monthly

IS - 11

ER -

Cytochrome b₅ plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine

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