Small interfering RNA-mediated silencing of cytochrome P450 3A4 gene.
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 levels by 65% and protein expression levels 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 to 210 microM to >1000 microM. Nifedipine at 5.78, 14.44, and 28.88 microM was significantly (P < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with 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 demonstrated, for the first time, 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.
Chen
Jie
J
Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, China.
Yang
Xiao-Xia
XX
Huang
Min
M
Hu
Ze-Ping
ZP
He
Ming
M
Duan
Wei
W
Chan
Eli
E
Sheu
Fwu-Shan
FS
Chen
Xiao
X
Zhou
Shu-Feng
SF
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism / Q000633:toxicity; D002121:Calcium Channel Blockers / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003520:Cyclophosphamide / Q000378:metabolism / Q000633:toxicity; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D007069:Ifosfamide / Q000378:metabolism / Q000633:toxicity; D020128:Inhibitory Concentration 50; D009543:Nifedipine / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018906:Antineoplastic Agents, Alkylating; D002121:Calcium Channel Blockers; D009841:Oligonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009543:Nifedipine; D007069:Ifosfamide