Pharmaceutical Research

, Volume 28, Issue 8, pp 2034–2044 | Cite as

The Role of Constitutive Androstane Receptor in Oxazaphosphorine-Mediated Induction of Drug-Metabolizing Enzymes in Human Hepatocytes

  • Duan Wang
  • Linhao Li
  • Jennifer Fuhrman
  • Stephen Ferguson
  • Hongbing Wang
Research Paper

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.

KEY WORDS

CAR cyclophosphamide CYP2B6 ifosfamide induction 

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

REFERENCES

  1. 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
  2. 2.
    Chrystal K, Cheong K, Harper P. Chemotherapy of small cell lung cancer: state of the art. Curr Opin Oncol. 2004;16:136–40.PubMedCrossRefGoogle Scholar
  3. 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
  4. 4.
    Demirer T, Buckner CD, Appelbaum FR, Clift R, Storb R, Myerson D, et al. High-dose busulfan and cyclophosphamide followed by autologous transplantation in patients with advanced breast cancer. Bone Marrow Transplant. 1996;17:769–74.PubMedGoogle Scholar
  5. 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
  6. 6.
    Huang Z, Roy P, Waxman DJ. Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide. Biochem Pharmacol. 2000;59:961–72.PubMedCrossRefGoogle Scholar
  7. 7.
    Boddy AV, English M, Pearson AD, Idle JR, Skinner R. Ifosfamide nephrotoxicity: limited influence of metabolism and mode of administration during repeated therapy in paediatrics. Eur J Cancer. 1996;32A:1179–84.PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang J, Tian Q, Yung Chan S, Chuen Li S, Zhou S, Duan W, et al. Metabolism and transport of oxazaphosphorines and the clinical implications. Drug Metab Rev. 2005;37:611–703.PubMedCrossRefGoogle Scholar
  9. 9.
    Chenand L, Waxman DJ. Cytochrome P450 gene-directed enzyme prodrug therapy (GDEPT) for cancer. Curr Pharm Des. 2002;8:1405–16.CrossRefGoogle Scholar
  10. 10.
    Chen CS, Lin JT, Goss KA, He YA, Halpert JR, Waxman DJ. Activation of the anticancer prodrugs cyclophosphamide and ifosfamide: identification of cytochrome P450 2B enzymes and site-specific mutants with improved enzyme kinetics. Mol Pharmacol. 2004;65:1278–85.PubMedCrossRefGoogle Scholar
  11. 11.
    Chang TK, Weber GF, Crespi CL, Waxman DJ. Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Res. 1993;53:5629–37.PubMedGoogle Scholar
  12. 12.
    Moore MJ, Erlichman C, Thiessen JJ, Bunting PS, Hardy R, Kerr I, et al. Variability in the pharmacokinetics of cyclophosphamide, methotrexate and 5-fluorouracil in women receiving adjuvant treatment for breast cancer. Cancer Chemother Pharmacol. 1994;33:472–6.PubMedCrossRefGoogle Scholar
  13. 13.
    Ren S, Kalhorn TF, McDonald GB, Anasetti C, Appelbaum FR, Slattery JT. Pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients. Clin Pharmacol Ther. 1998;64:289–301.PubMedCrossRefGoogle Scholar
  14. 14.
    Kerbusch T, de Kraker J, Keizer HJ, van Putten JW, Groen HJ, Jansen RL, et al. Clinical pharmacokinetics and pharmacodynamics of ifosfamide and its metabolites. Clin Pharmacokinet. 2001;40:41–62.PubMedCrossRefGoogle Scholar
  15. 15.
    Chang TK, Yu L, Maurel P, Waxman DJ. Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines. Cancer Res. 1997;57:1946–54.PubMedGoogle Scholar
  16. 16.
    Afsharian P, Terelius Y, Hassan Z, Nilsson C, Lundgren S, Hassan M. The effect of repeated administration of cyclophosphamide on cytochrome P450 2B in rats. Clin Cancer Res. 2007;13:4218–24.PubMedCrossRefGoogle Scholar
  17. 17.
    Xie H, Afsharian P, Terelius Y, Mirghani RA, Yasar U, Hagbjork AL, et al. Cyclophosphamide induces mRNA, protein and enzyme activity of cytochrome P450 in rat. Xenobiotica. 2005;35:239–51.PubMedCrossRefGoogle Scholar
  18. 18.
    Harmsen S, Meijerman I, Beijnen JH, Schellens JH. Nuclear receptor mediated induction of cytochrome P450 3A4 by anticancer drugs: a key role for the pregnane X receptor. Cancer Chemother Pharmacol. 2009;64:35–43.PubMedCrossRefGoogle Scholar
  19. 19.
    Lindley C, Hamilton G, McCune JS, Faucette S, Shord SS, Hawke RL, et al. The effect of cyclophosphamide with and without dexamethasone on cytochrome P450 3A4 and 2B6 in human hepatocytes. Drug Metab Dispos Biol Fate Chem. 2002;30:814–22.PubMedCrossRefGoogle Scholar
  20. 20.
    Wang H, LeCluyse EL. Role of orphan nuclear receptors in the regulation of drug-metabolising enzymes. Clin Pharmacokinet. 2003;42:1331–57.PubMedCrossRefGoogle Scholar
  21. 21.
    Moore LB, Parks DJ, Jones SA, Bledsoe RK, Consler TG, Stimmel JB, et al. Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem. 2000;275:15122–7.PubMedCrossRefGoogle Scholar
  22. 22.
    Wang H, Faucette S, Sueyoshi T, Moore R, Ferguson S, Negishi M, et al. A novel distal enhancer module regulated by pregnane X receptor/constitutive androstane receptor is essential for the maximal induction of CYP2B6 gene expression. J Biol Chem. 2003;278:14146–52.PubMedCrossRefGoogle Scholar
  23. 23.
    Chen T, Tompkins LM, Li L, Li H, Kim G, Zheng Y, et al. A single amino acid controls the functional switch of human constitutive androstane receptor (CAR) 1 to the xenobiotic-sensitive splicing variant CAR3. J Pharmacol Exp Ther. 2010;332:106–15.PubMedCrossRefGoogle Scholar
  24. 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. 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
  26. 26.
    LeCluyse EL. Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Eur J Pharm Sci. 2001;13:343–68.PubMedCrossRefGoogle Scholar
  27. 27.
    Kerbusch T, Mathot RA, Keizer HJ, Kaijser GP, Schellens JH, Beijnen JH. Influence of dose and infusion duration on pharmacokinetics of ifosfamide and metabolites. Drug Metab Dispos Biol Fate Chem. 2001;29:967–75.PubMedGoogle Scholar
  28. 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
  29. 29.
    Gardner-Stephen D, Heydel JM, Goyal A, Lu Y, Xie W, Lindblom T, et al. Human PXR variants and their differential effects on the regulation of human UDP-glucuronosyltransferase gene expression. Drug Metab Dispos Biol Fate Chem. 2004;32:340–7.PubMedCrossRefGoogle Scholar
  30. 30.
    Xu L, Li AP, Kaminski DL, Ruh MF. 2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes. Chem Biol Interact. 2000;124:173–89.PubMedCrossRefGoogle Scholar
  31. 31.
    Davarinosand NA, Pollenz RS. Aryl hydrocarbon receptor imported into the nucleus following ligand binding is rapidly degraded via the cytosplasmic proteasome following nuclear export. J Biol Chem. 1999;274:28708–15.CrossRefGoogle Scholar
  32. 32.
    Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K, Negishi M. Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol Cell Biol. 1999;19:6318–22.PubMedGoogle Scholar
  33. 33.
    Zhou C, Poulton EJ, Grun F, Bammler TK, Blumberg B, Thummel KE, et al. The dietary isothiocyanate sulforaphane is an antagonist of the human steroid and xenobiotic nuclear receptor. Mol Pharmacol. 2007;71:220–9.PubMedCrossRefGoogle Scholar
  34. 34.
    Li L, Chen T, Stanton JD, Sueyoshi T, Negishi M, Wang H. The peripheral benzodiazepine receptor ligand 1-(2-chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide is a novel antagonist of human constitutive androstane receptor. Mol Pharmacol. 2008;74:443–53.PubMedCrossRefGoogle Scholar
  35. 35.
    Giraud B, Hebert G, Deroussent A, Veal GJ, Vassal G, Paci A. Oxazaphosphorines: new therapeutic strategies for an old class of drugs. Expert Opin Drug Metab Toxicol. 2010;6:919–38.PubMedCrossRefGoogle Scholar
  36. 36.
    Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, et al. Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. J Biol Chem. 2003;278:17277–83.PubMedCrossRefGoogle Scholar
  37. 37.
    Smirlis D, Muangmoonchai R, Edwards M, Phillips IR, Shephard EA. Orphan receptor promiscuity in the induction of cytochromes p450 by xenobiotics. J Biol Chem. 2001;276:12822–6.PubMedCrossRefGoogle Scholar
  38. 38.
    Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, et al. Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes Dev. 2000;14:3014–23.PubMedCrossRefGoogle Scholar
  39. 39.
    Faucette SR, Sueyoshi T, Smith CM, Negishi M, Lecluyse EL, Wang H. Differential regulation of hepatic CYP2B6 and CYP3A4 genes by constitutive androstane receptor but not pregnane X receptor. J Pharmacol Exp Ther. 2006;317:1200–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Duan Wang
    • 1
  • Linhao Li
    • 1
  • Jennifer Fuhrman
    • 2
  • Stephen Ferguson
    • 3
  • Hongbing Wang
    • 1
  1. 1.Department of Pharmaceutical SciencesUniversity of Maryland School of PharmacyBaltimoreUSA
  2. 2.Department of PathologyUniversity of Maryland School of MedicineBaltimoreUSA
  3. 3.Life Technologies CorporationDurhamUSA

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