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Sensitivity and Resistance to Therapy

Antiepileptic drugs reduce efficacy of methotrexate chemotherapy by downregulation of Reduced folate carrier transport activity

Leukemia volume 23, pages 1087–1097 (2009)Cite this article

Abstract

Concurrent treatment with methotrexate (MTX) and antiepileptic drugs, such as phenobarbital (PB), reduces the efficacy of MTX chemotherapy in childhood acute lymphoblastic leukemia (ALL). This can result from defective Reduced folate carrier (Rfc1)-dependent cellular uptake of MTX. Indeed, we have shown that functional Rfc1 activity is significantly reduced by clinically relevant concentrations of the anticonvulsant drugs PB or carbamazepine in an adequate in vitro model. As PB is known to regulate carrier-associated transport by the nuclear receptor constitutive androstane receptor (CAR), we investigated the involvement of the CAR signaling cascade and the mode of PB-induced downregulation of Rfc1 activity. CAR activation by PB or the CAR agonist 1,4-bis[2-(3,5-dichloro- pyridyloxy)]-benzene resulted in translocation of Ca2+-dependent protein kinase Cα (cPKCα) to the plasma membrane related to significantly elevated PKC activities. In contrast, subcellular localization of Ca2+-independent PKCδ was only marginally altered. Studies on intracellular distribution of the Rfc1 protein indicated that PB-induced activation of cPKCα was associated with carrier internalization from the plasma membrane into the cytosol independent of the Rfc1 phosphorylation status. In conclusion, we identified for the first time the molecular mechanism of this clinically relevant drug resistance in patients with ALL concurrently receiving MTX chemotherapy and antiepileptic drugs.

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References

  1. Matherly LH, Goldman ID . Membrane transport of folates. Vitam Horm 2003; 66: 403–456.

    Article  CAS  Google Scholar 

  2. Relling MV, Pui CH, Sandlund JT, Rivera GK, Hancock ML, Boyett JM et al. Adverse effect of anticonvulsants on efficacy of chemotherapy for acute lymphoblastic leukaemia. Lancet 2000; 356: 285–290.

    Article  CAS  Google Scholar 

  3. Riva M, Landonio G, Defanti CA, Siena S . The effect of anticonvulsant drugs on blood levels of methotrexate. J Neurooncol 2000; 48: 249–250.

    Article  CAS  Google Scholar 

  4. Vecht CJ, Wagner GL, Wilms EB . Interactions between antiepileptic and chemotherapeutic drugs. Semin Oncol 2003; 30: 49–52.

    Article  CAS  Google Scholar 

  5. Ochs JJ, Bowman WP, Pui CH, Abromowitch M, Mason C, Simone JV . Seizures in childhood lymphoblastic leukaemia patients. Lancet 1984; 2: 1422–1424.

    Article  CAS  Google Scholar 

  6. Maytal J, Grossman R, Yusuf FH, Shende AC, Karayalycin G, Lanzkowsky P et al. Prognosis and treatment of seizures in children with acute lymphoblastic leukemia. Epilepsia 1995; 36: 831–836.

    Article  CAS  Google Scholar 

  7. Winick NJ, Bowman WP, Kamen BA, Roach ES, Rollins N, Jacaruso D et al. Unexpected acute neurologic toxicity in the treatment of children with acute lymphoblastic leukemia. J Natl Cancer Inst 1992; 84: 252–256.

    Article  CAS  Google Scholar 

  8. Mahoney Jr DH, Shuster JJ, Nitschke R, Lauer S, Steuber CP, Camitta B . Intensification with intermediate-dose intravenous methotrexate is effective therapy for children with lower-risk B-precursor acute lymphoblastic leukemia: a Pediatric Oncology Group study. J Clin Oncol 2000; 18: 1285–1294.

    Article  CAS  Google Scholar 

  9. Pace A, Bove L, Innocenti P, Pietrangeli A, Carapella CM, Oppido P et al. Epilepsy and gliomas: incidence and treatment in 119 patients. J Exp Clin Cancer Res 1998; 17: 479–482.

    CAS  PubMed  Google Scholar 

  10. Kamen B . Folate and antifolate pharmacology. Semin Oncol 1997; 24: 30–39.

    Google Scholar 

  11. Honscha W, Dötsch KU, Thomsen N, Petzinger E . Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells. Hepatology 2000; 31: 1296–1304.

    Article  CAS  Google Scholar 

  12. Kneuer C, Honscha KU, Honscha W . Rat reduced-folate carrier-1 is localized basolaterally in MDCK kidney epithelial cells and contributes to the secretory transport of methotrexate and fluoresceinated methotrexate. Cell Tissue Res 2005; 320: 517–524.

    Article  CAS  Google Scholar 

  13. Zhang L, Taub JW, Williamson M, Wong SC, Hukku B, Pullen J et al. Reduced folate carrier gene expression in childhood acute lymphoblastic leukemia: relationship to immunophenotype and ploidy. Clin Cancer Res 1998; 4: 2169–2177.

    CAS  PubMed  Google Scholar 

  14. Gorlick R, Goker E, Trippett T, Steinherz P, Elisseyeff Y, Mazumdar M et al. Defective transport is a common mechanism of acquired methotrexate resistance in acute lymphocytic leukemia and is associated with decreased reduced folate carrier expression. Blood 1997; 89: 1013–1018.

    CAS  PubMed  Google Scholar 

  15. Halwachs S, Kneuer C, Honscha W . Downregulation of the reduced folate carrier transport activity by phenobarbital-type cytochrome P450 inducers and protein kinase C activators. Biochem Biophys Acta 2007; 1768: 1671–1679.

    Article  CAS  Google Scholar 

  16. Borst P, Evers R, Kool M, Wijnholds J . A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst 2000; 92: 1295–1302.

    Article  CAS  Google Scholar 

  17. Xu C, Li CY, Kong AN . Induction of phase I, II and III drug metabolism/transport by xenobiotics. Arch Pharm Res 2005; 28: 249–268.

    Article  CAS  Google Scholar 

  18. Tzameli I, Pissios P, Schuetz EG, Moore DD . The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR. Mol Cell Biol 2000; 20: 2951–2958.

    Article  CAS  Google Scholar 

  19. Sueyoshi T, Negishi M . Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu Rev Pharmacol Toxicol 2001; 41: 123–143.

    Article  CAS  Google Scholar 

  20. Siest G, Jeannesson E, Marteau JB, Samara A, Bérangère M, Pfister M et al. Transcription factor and drug-metabolizing enzyme gene expression in lymphocytes from healthy human subjects. Drug Metab Dispos 2008; 36: 182–189.

    Article  CAS  Google Scholar 

  21. Hariparsad N . Hepatic and Extra-Hepatic Induction of Drug Metabolizing Enzymes and Drug Transporters by Antiretrovirals, in the Presence and Absence of Viral Infection [Dissertation Doctor of Philosophy]. University of Cincinnati: Cincinnati, 2006.

    Google Scholar 

  22. Nguyen TT, Dyer DL, Dunnin DD, Rubin SA, Grant KE, Said HM . Human intestinal folate transport: cloning, expression, and distribution of complementary RNA. Gastroenterology 1997; 112: 783–791.

    Article  CAS  Google Scholar 

  23. Carmena D, Sardini A . Lifespan regulation of conventional protein kinase C isotypes. Biochem Soc Trans 2007; 35: 1043–1045.

    Article  CAS  Google Scholar 

  24. Beck JF, Brügger D, Brischwein K, Liu C, Bader P, Niethammer D et al. Anticancer drug-mediated induction of multidrug resistance-associated genes and protein kinase C isozymes in the T-lymphoblastoid cell line CCRF-CEM and in blasts from patients with acute lymphoblastic leukemias. Jpn J Cancer Res 2001; 92: 896–903.

    Article  CAS  Google Scholar 

  25. Balasubramanian N, Advani SH, Zingde SM . Protein kinase C isoforms in normal and leukemic neutrophils: altered levels in leukemic neutrophils and changes during myeloid maturation in chronic myeloid leukemia. Leuk Res 2002; 26: 67–81.

    Article  CAS  Google Scholar 

  26. Alkan S, Huang Q, Ergin M, Denning MF, Nand S, Maududi T et al. Survival role of protein kinase C (PKC) in chronic lymphocytic leukemia and determination of isoform expression pattern and genes altered by PKC inhibition. Am J Hematol 2005; 79: 97–106.

    Article  CAS  Google Scholar 

  27. Jiffar T, Kurinna S, Suck G, Carlson-Bremer D, Ricciardi MR, Konopleva M et al. PKC alpha mediates chemoresistance in acute lymphoblastic leukemia through effects on Bcl2 phosphorylation. Leukemia 2004; 183: 505–512.

    Article  Google Scholar 

  28. Kubitz R, Huth C, Schmitt M, Horbach A, Kullak-Ublick G, Häussinger D . Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells. Hepatology 2001; 34: 340–350.

    Article  CAS  Google Scholar 

  29. Schrader S, Kneuer C, Honscha W . Alternative transcripts of rat slc19a1: defective sorting and inefficient expression of alternative splicing products—an addendum. DNA Seq 2006; 16: 1–6.

    Google Scholar 

  30. Kneuer C, Honscha W . The H(+)-dependent reduced folate carrier 1 of humans and the sodium-dependent methotrexate carrier-1 of the rat are orthologs. FEBS Lett 2004; 566: 83–86.

    Article  CAS  Google Scholar 

  31. Donato MT, Gomez-Lechon MJ, Castell JV . A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal Biochem 1993; 213: 29–33.

    Article  CAS  Google Scholar 

  32. Hodges NJ, Orton TC, Strain AJ, Chipman JK . Potentiation of epidermal growth factor- induced DNA synthesis in rat hepatocytes by phenobarbitone: possible involvement of oxidative stress and kinase activation. Carcinogenesis 2000; 21: 2041–2047.

    Article  CAS  Google Scholar 

  33. Gekeler V, Boer R, Uberall F, Ise W, Schubert C, Utz I et al. Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance. Br J Cancer 1996; 74: 897–905.

    Article  CAS  Google Scholar 

  34. Adams PD, Parker PJ . TPA-induced activation of MAP kinase. FEBS Lett 1991; 290: 77–82.

    Article  CAS  Google Scholar 

  35. Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y et al. Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 1998; 429: 179–182.

    Article  CAS  Google Scholar 

  36. Utesch D, Arand M, Thomas H, Petzinger E, Oesch F . Xenobiotic-metabolizing enzyme activities in hybrid cell lines established by fusion of primary rat liver parenchymal cells with hepatoma cells. Xenobiotica 1992; 22: 1451–1457.

    Article  CAS  Google Scholar 

  37. Halwachs S, Kneuer C, Honscha W . Endogenous expression of liver-specific drug transporters for organic anions in the rat hepatocytoma fusion cell line HPCT-1E3. Eur J Cell Biol 2005; 84: 677–686.

    Article  CAS  Google Scholar 

  38. Newton AC . Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 2001; 101: 2353–2364.

    Article  CAS  Google Scholar 

  39. Martiny-Baron G, Kazanietzs MG, Mischak H, Blumberg PM, Kochs G, Hug H et al. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö6976. J Biol Chem 1993; 268: 9194–9197.

    CAS  PubMed  Google Scholar 

  40. Wolff NA, Thies K, Kuhnke N, Reid G, Friedrich B, Lang F et al. Protein kinase C activation downregulates human organic anion transporter 1-mediated transport through carrier internalization. J Am Soc Nephrol 2003; 8: 1959–1968.

    Article  Google Scholar 

  41. Traub LM, Kornfeld S . The trans-Golgi network: a late secretory sorting station. Curr Opin Cell Biol 1997; 9: 527–533.

    Article  CAS  Google Scholar 

  42. Josting A, Diehl V, Engert A . Methotrexat: Pharmakologie, Toxizität und Rescuemassnahmen bei hochdosierter Behandlung. Der Onkologe 1998; 4: 974–985.

    Article  Google Scholar 

  43. Joannard F, Galisteo M, Corcos L, Guillouzo A, Lagadic-Gossmann D . Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases. Cell Biol Toxicol 2000; 16: 325–337.

    Article  CAS  Google Scholar 

  44. Marc N, Galisteo M, Lagadic-Gossmann D, Fautrel A, Joannard F, Guillouzo A et al. Regulation of phenobarbital induction of the cytochrome P450 2b9/10 genes in primary mouse hepatocyte culture. Involvement of calcium- and cAMP-dependent pathways. Eur J Biochem 2000; 267: 963–970.

    Article  CAS  Google Scholar 

  45. Goodwin B, Moore JT . CAR: detailing new models. Trends Pharmacol Sci 2004; 25: 437–441.

    Article  CAS  Google Scholar 

  46. Aquino A, Hartman K, Knode M, Grant S, Huang K, Niu C et al. Role of protein kinase C in phosphorylation of vinculin in adriamycin-resistant HL-60 leukemic cells. Cancer Res 1988; 48: 3324–3329.

    CAS  PubMed  Google Scholar 

  47. Sugawara S, Kumano N, Saijo S, Numata Y, Sato G, Motomiya M . Protein kinase C activity in human leukemia cell lines with reference to sensitivity to antineoplastic agents. Tohoku J Exp Med 1992; 168: 393–396.

    Article  CAS  Google Scholar 

  48. Kazanietz MG, Areces LB, Bahador A, Mischak H, Goodnight J, Mushinski JF et al. Characterization of ligand and substrate specificity for the calcium-dependent and calcium-independent protein kinase C isozymes. Mol Pharmacol 1993; 44: 298–307.

    CAS  PubMed  Google Scholar 

  49. Jirtle RL, Meyer SA, Brockenbrough JS . Liver tumor promoter phenobarbital: a biphasic modulator of hepatocyte proliferation. Prog Clin Biol Res 1991; 369: 209–216.

    CAS  PubMed  Google Scholar 

  50. Inoue K, Negishi M . Nuclear receptor CAR requires early growth response 1 to activate the human cytochrome P450 2B6 gene. J Biol Chem 2008; 283: 10425–10432.

    Article  CAS  Google Scholar 

  51. Marchant JS, Subramanian VS, Parker I, Said HM . Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in mammalian epithelial cells. J Biol Chem 2002; 277: 33325–33333.

    Article  CAS  Google Scholar 

  52. Kubitz R, Sütfels G, Kühlkamp T, Kölling R, Häussinger D . Trafficking of the bile salt export pump from the Golgi to the canalicular membrane is regulated by the p38 MAP kinase. Gastroenterology 2004; 126: 541–553.

    Article  CAS  Google Scholar 

  53. Westermann P, Knoblich M, Maier O, Lindschau C, Haller H . Protein kinase C bound to the Golgi apparatus supports the formation of constitutive transport vesicles. Biochem J 1996; 320: 651–658.

    Article  CAS  Google Scholar 

  54. Radau B, Otto A, Müller EC, Westermann P . Protein kinase C alpha-dependent phosphorylation of Golgi proteins. Electrophoresis 2000; 21: 2684–2687.

    Article  CAS  Google Scholar 

  55. Buccione R, Bannykh S, Santone I, Baldassarre M, Facchiano F, Bozzi Y et al. Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study. J Biol Chem 1996; 271: 3523–3533.

    Article  CAS  Google Scholar 

  56. Keenan C, Kelleher D . Protein kinase C and the cytoskeleton. Cell Signal 1998; 10: 225–232.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C Lakoma for skillful technical assistance. We also thank E Petzinger (Justus-Liebig-University, Giessen, Germany) for donating the HPCT-1E3 cell line and D Eisinger (University of Munich, Munich, Germany) for generously providing the anti-PKCδ antibody. This work was supported by the Deutsche Forschungsgemeinschaft (HO 2103/2-1).

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Authors and Affiliations

  1. Faculty of Veterinary Medicine, Institute of Pharmacology, Pharmacy and Toxicology, University of Leipzig, Leipzig, Germany

    S Halwachs & W Honscha

  2. Department of Molecular Cell Therapy, Center for Biotechnology and Biomedicine, Faculty of Medicine, University of Leipzig, Leipzig, Germany

    I Schäfer & P Seibel

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  1. S Halwachs
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  2. I Schäfer
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Correspondence to S Halwachs.

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Halwachs, S., Schäfer, I., Seibel, P. et al. Antiepileptic drugs reduce efficacy of methotrexate chemotherapy by downregulation of Reduced folate carrier transport activity. Leukemia 23, 1087–1097 (2009). https://doi.org/10.1038/leu.2009.6

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