The Role of Constitutive Androstane Receptor in Oxazaphosphorine-Mediated Induction of Drug-Metabolizing Enzymes in Human Hepatocytes
To investigate the roles of the constitutive androstane receptor (CAR) in cyclophosphamide (CPA)- and ifosfamide (IFO)-mediated induction of hepatic drug-metabolizing enzymes (DME).
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.
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.
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.
KEY WORDSCAR cyclophosphamide CYP2B6 ifosfamide induction
adenovirus expressing enhanced yellow fluorescent protein-tagged human CAR
aryl hydrocarbon receptor
constitutive androstane receptor
6-(4-chlorophenyl) imidazo[2,1-b][1,3]- thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime
human primary hepatocytes
pregnane X receptor
reverse transcription-polymerase chain reaction
- 1.Rao R, Shammo JM, Enschede SH, Porter C, Adler SS, Venugopal P, et al. The combination of fludarabine, cyclophosphamide, and granulocyte-macrophage colony-stimulating factor in the treatment of patients with relapsed chronic lymphocytic leukemia and low-grade Non-Hodgkin‘s lymphoma. Clin Lymphoma. 2005;6:26–30.PubMedCrossRefGoogle Scholar
- 3.Fermand JP, Ravaud P, Chevret S, Divine M, Leblond V, Belanger C, et al. High-dose therapy and autologous peripheral blood stem cell transplantation in multiple myeloma: up-front or rescue treatment? Results of a multicenter sequential randomized clinical trial. Blood. 1998;92:3131–6.PubMedGoogle Scholar
- 5.Bramwell VH, Mouridsen HT, Santoro A, Blackledge G, Somers R, Verweij J, et al. Cyclophosphamide versus ifosfamide: a randomized phase II trial in adult soft-tissue sarcomas. The European Organization for Research and Treatment of Cancer [EORTC], Soft Tissue and Bone Sarcoma Group. Cancer Chemother Pharmacol. 1993;31 Suppl 2:S180–4.PubMedGoogle Scholar
- 24.LeCluyse EL, Alexandre E, Hamilton GA, Viollon-Abadie C, Coon DJ, Jolley S, et al. Isolation and culture of primary human hepatocytes. Meth Mol Biol. 2005;290:207–29.Google Scholar
- 25.Li H, Chen T, Cottrell J, Wang H. Nuclear translocation of adenoviral-enhanced yellow fluorescent protein-tagged-human constitutive androstane receptor (hCAR): a novel tool for screening hCAR activators in human primary hepatocytes. Drug Metab Dispos Biol Fate Chem. 2009;37:1098–106.PubMedCrossRefGoogle Scholar
- 28.Sugatani J, Kojima H, Ueda A, Kakizaki S, Yoshinari K, Gong QH, et al. The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR. Hepatology (Baltimore, Md). 2001;33:1232–8.Google Scholar