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Drug Metabolism and Disposition Fast Forward
First published on June 7, 2006; DOI: 10.1124/dmd.106.009837

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Received for publication February 16, 2006.
Revised May 3, 2006.
Accepted for publication June 5, 2006.

Small Interfering RNA-Mediated Silencing of cytochrome P450 3A4 Gene

Jie Chen 1, Xiao-Xia Yang 2, Min Huang 1, Ze-Ping Hu 2, Ming He 3, Wei Duan 4, Eli Chan 2, Fwu-Shan Sheu 5, Xiao Chen 6, Shu-Feng Zhou 7*

1 Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guang 2 Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 3 Department of Pharmacology, Nanchang University Medical College, Nanchang, 330006, China 4 Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore 5 Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 6 Department of Clinical Pharmacy, 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, 7 National University of Singapore

* Address correspondence to: E-mail: phazsf{at}nus.edu.sg

Abstract

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA level by 65% and protein expression level by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC50 increased from 55-210 mM to >1000 mM. Nifedipine at 5.78, 14.44, and 28.88 µM was significantly (P < 0.01) depleted by about 100%, 40%, and 22%, respectively, in S9 fractions from CHL-3A4 cells, compared to parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study, for the first time, demonstrated the specific suppression of CYP3A4 expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.


Key words: antisense oligonucleotides, CYP3A, cytochrome P450 regulation, human CYP enzymes


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Home page
 Drug Metab. Dispos.Home page
Y.-Z. Pan, W. Gao, and A.-M. Yu
MicroRNAs Regulate CYP3A4 Expression via Direct and Indirect Targeting
Drug Metab. Dispos., October 1, 2009; 37(10): 2112 - 2117.
[Abstract] [Full Text] [PDF]


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