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Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity.
Chem Biol Interact. 2014 Dec 05; 224:89-99.CB

Abstract

Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently, neferine has attracted attention for its anti-tumor activities. Our study explored its metabolism and cytotoxicity mechanism. Approaches using chemical inhibitors and recombinant human enzymes to characterize the involved enzymes and kinetic studies indicated that the demethylation of neferine by cytochrome P450 (CYP) 2D6 and CYP3A4 fitted a biphasic kinetic profile. Glutathione (GSH) was used as a trapping agent to identify reactive metabolites of neferine, and four novel GSH conjugates were detected with [M+H](+) ions at m/z 902.4, 916.2, 916.1, and 930.4. Based on its structure containing para-methylene phenol and results from a product ion scan, GSH tends to conjugate with C9' after undergoing oxidative metabolism to form the binding site predominated by CYP3A4. Furthermore, the addition of recombinant human GSTA1, GSTT1, and GSTP1 had little effect on the production of the GSH conjugates. In a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, combined with the GSH modulators l-buthionine sulfoximine or N-acetyl-l-cysteine, neferine treatment of MDCK-hCYP3A4 and HepG2 cells revealed that CYP3A4 expression and cellular GSH content could cause an EC50 shift. Metabolic activation mediated by CYP3A4 and GSH depletion significantly enhanced neferine-induced cytotoxicity.

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Authors+Show Affiliations

Shen Q
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Zuo M
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Ma L
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Tian Y
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Wang L
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Jiang H
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Zhou Q
Department of Pharmacy, the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Zhou H
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Electronic address: zhouhui@zju.edu.cn.
Yu L
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Electronic address: yuls@zju.edu.cn.
Zeng S
Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.

MeSH

AcetylcysteineAnimalsAntineoplastic AgentsBenzylisoquinolinesButhionine SulfoximineCytochrome P-450 CYP2C8Cytochrome P-450 CYP2D6Cytochrome P-450 CYP3ACytochrome P-450 CYP3A InhibitorsDogsGlutathioneHep G2 CellsHumansIndolequinonesKetoconazoleKineticsMadin Darby Canine Kidney CellsMicrosomes, LiverTroleandomycin

Pub Type(s)

Journal Article

Language

eng

PubMed ID

25451576

Citation

Shen, Qi, et al. "Demethylation of Neferine in Human Liver Microsomes and Formation of Quinone Methide Metabolites Mediated By CYP3A4 Accentuates Its Cytotoxicity." Chemico-biological Interactions, vol. 224, 2014, pp. 89-99.
Shen Q, Zuo M, Ma L, et al. Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity. Chem Biol Interact. 2014;224:89-99.
Shen, Q., Zuo, M., Ma, L., Tian, Y., Wang, L., Jiang, H., Zhou, Q., Zhou, H., Yu, L., & Zeng, S. (2014). Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity. Chemico-biological Interactions, 224, 89-99. https://doi.org/10.1016/j.cbi.2014.10.014
Shen Q, et al. Demethylation of Neferine in Human Liver Microsomes and Formation of Quinone Methide Metabolites Mediated By CYP3A4 Accentuates Its Cytotoxicity. Chem Biol Interact. 2014 Dec 5;224:89-99. PubMed PMID: 25451576.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity. AU - Shen,Qi, AU - Zuo,Minjuan, AU - Ma,Li, AU - Tian,Ye, AU - Wang,Lu, AU - Jiang,Huidi, AU - Zhou,Quan, AU - Zhou,Hui, AU - Yu,Lushan, AU - Zeng,Su, Y1 - 2014/10/19/ PY - 2014/07/21/received PY - 2014/09/07/revised PY - 2014/10/13/accepted PY - 2014/12/3/entrez PY - 2014/12/3/pubmed PY - 2017/2/14/medline KW - Biphasic kinetics KW - CYP3A4 KW - Cytotoxicity KW - GSH depletion KW - Neferine KW - Quinone methide metabolite SP - 89 EP - 99 JF - Chemico-biological interactions JO - Chem Biol Interact VL - 224 N2 - Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently, neferine has attracted attention for its anti-tumor activities. Our study explored its metabolism and cytotoxicity mechanism. Approaches using chemical inhibitors and recombinant human enzymes to characterize the involved enzymes and kinetic studies indicated that the demethylation of neferine by cytochrome P450 (CYP) 2D6 and CYP3A4 fitted a biphasic kinetic profile. Glutathione (GSH) was used as a trapping agent to identify reactive metabolites of neferine, and four novel GSH conjugates were detected with [M+H](+) ions at m/z 902.4, 916.2, 916.1, and 930.4. Based on its structure containing para-methylene phenol and results from a product ion scan, GSH tends to conjugate with C9' after undergoing oxidative metabolism to form the binding site predominated by CYP3A4. Furthermore, the addition of recombinant human GSTA1, GSTT1, and GSTP1 had little effect on the production of the GSH conjugates. In a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, combined with the GSH modulators l-buthionine sulfoximine or N-acetyl-l-cysteine, neferine treatment of MDCK-hCYP3A4 and HepG2 cells revealed that CYP3A4 expression and cellular GSH content could cause an EC50 shift. Metabolic activation mediated by CYP3A4 and GSH depletion significantly enhanced neferine-induced cytotoxicity. SN - 1872-7786 UR - https://www.unboundmedicine.com/medline/citation/25451576/Demethylation_of_neferine_in_human_liver_microsomes_and_formation_of_quinone_methide_metabolites_mediated_by_CYP3A4_accentuates_its_cytotoxicity_ DB - PRIME DP - Unbound Medicine ER -