ABSTRACT
Purpose
To investigate the roles of the constitutive androstane receptor (CAR) in cyclophosphamide (CPA)- and ifosfamide (IFO)-mediated induction of hepatic drug-metabolizing enzymes (DME).
Methods
Induction of DMEs was evaluated using real-time RT-PCR and Western blotting analysis in human primary hepatocyte (HPH) cultures. Activation of CAR, pregnane X receptor (PXR), and aryl hydrocarbon receptor by CPA and IFO was assessed in cell-based reporter assays in HepG2 cells and/or nuclear translocation assays in HPHs.
Results
CYP2B6 reporter activity was significantly enhanced by CPA and IFO in HepG2 cells co-transfected with CYP2B6 reporter plasmid and a chemical-responsive human CAR variant (CAR1 + A) construct. Real-time RT-PCR and Western blotting analysis in HPHs showed that both CPA and IFO induced the expressions of CYP2B6 and CYP3A4. Notably, treatment of HPHs with CPA but not IFO resulted in significant nuclear accumulation of CAR, which represents the initial step of CAR activation. Further studies in HPHs demonstrated that selective inhibition of PXR by sulforaphane preferentially repressed IFO- over CPA-mediated induction of CYP2B6.
Conclusion
These results provide novel insights into the differential roles of CAR in the regulation of CPA- and IFO-induced DME expression and potential drug-drug interactions.
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Abbreviations
- 3MC:
-
3-methylcholanthrene
- Ad/EYFP-hCAR:
-
adenovirus expressing enhanced yellow fluorescent protein-tagged human CAR
- AhR:
-
aryl hydrocarbon receptor
- CAR:
-
constitutive androstane receptor
- CITCO:
-
6-(4-chlorophenyl) imidazo[2,1-b][1,3]- thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime
- CPA:
-
cyclophosphamide
- CYP:
-
cytochrome P450
- DMSO:
-
dimethyl sulfoxide
- GAPDH:
-
glyceraldehyde-3-phosphate dehydrogenase
- HPH:
-
human primary hepatocytes
- IFO:
-
ifosfamide
- PB:
-
phenobarbital
- PK11195:
-
1-(2-chlorophenyl-Nmethylpropyl)-3-isoquinoline-carboxamide
- PXR:
-
pregnane X receptor
- RIF:
-
rifampicin
- RT-PCR:
-
reverse transcription-polymerase chain reaction
- SFN:
-
sulforaphane
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ACKNOWLEDGMENTS
The authors thank Dr. Masahiko Negishi (National Institute of Environmental and Health Sciences, National Institutes of Health, Research Triangle Park, NC) for providing hCAR expression and UGT1A1 reporter constructs, Dr. Steve Kliewer (University of Texas, Southwestern Medical Center, Dallas, TX) for the pSG5-hPXR expression vector, and Dr. Bryan Goodwin (GlaxoSmithKline, Research Triangle Park, NC) for the CYP3A4 reporter plasmid. This research was supported in part by the National Institute of Health Grant (R01, DK061652) and the Seed Grant from University of Maryland Greenebaum Cancer Center.
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Wang, D., Li, L., Fuhrman, J. et al. The Role of Constitutive Androstane Receptor in Oxazaphosphorine-Mediated Induction of Drug-Metabolizing Enzymes in Human Hepatocytes. Pharm Res 28, 2034–2044 (2011). https://doi.org/10.1007/s11095-011-0429-2
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DOI: https://doi.org/10.1007/s11095-011-0429-2