title: Antiepileptic drugs reduce the efficacy of methotrexate chemotherapy through accelerated degradation of the reduced folate carrier by the ubiquitin-proteasome pathway.
abstract: OBJECTIVE
  Concurrent treatment with methotrexate (MTX) and enzyme-inducing antiepileptic drugs including phenobarbital (PB) reduces the efficacy of MTX chemotherapy in cancer patients. We have shown that Reduced folate carrier (Rfc1)-mediated uptake of MTX, an essential determinant of MTX chemotherapy, is significantly reduced by PB via protein kinase C (PKC). However, whether PB treatment affects Rfc1 activity through regulation of carrier protein stability and the mechanisms involved remain unclear.
  
  
  RESULTS
  Protein turnover assays using hepatocytoma cells demonstrated that Rfc1 is a long-lived protein that is mainly degraded by the ubiquitin-proteasome proteolytic pathway under basal conditions. Pretreatment with PB significantly reduced Rfc1-mediated MTX uptake and shortened the carrier protein half-life. This effect was abolished by the specific PKC inhibitor Gö6976. Inhibition of proteasomes with MG-132 significantly elevated Rfc1 protein levels and induced colocalization of Rfc1 and ubiquitin particularly in submembranous cellular compartments. Finally, we demonstrated that PB treatment resulted in enhanced levels of Rfc1 polyubiquitin conjugates.
  
  
  CONCLUSIONS
  Our results demonstrate that PB treatment causes downregulation of Rfc1 activity through PKC-dependent accelerated degradation of the Rfc1 protein by the ubiqutin-proteasome pathway. This regulatory mechanism may therefore involve clinically relevant drug resistance in patients concurrently receiving MTX and enzyme-inducing antiepileptic drugs.
authors.affiliation: Institute of Pharmacology, Pharmacy and Toxicology, University of Leipzig, Germany. halwachs @ vetmed.uni-leipzig.de
authors.forename: Sandra
authors.initials: S
authors.lastname: Halwachs
authors.forename: Ingo
authors.initials: I
authors.lastname: Schaefer
authors.forename: Peter
authors.initials: P
authors.lastname: Seibel
authors.forename: Walther
authors.initials: W
authors.lastname: Honscha
mesh_terms: D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D053898:Biosynthetic Pathways; D002460:Cell Line; D000090702:Constitutive Androstane Receptor; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004347:Drug Interactions; D006207:Half-Life; D008727:Methotrexate / Q000493:pharmacokinetics / Q000494:pharmacology*; D010634:Phenobarbital / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011493:Protein Kinase C / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists* / Q000378:metabolism; D058978:Reduced Folate Carrier Protein / Q000378:metabolism*; D051818:Replication Protein C / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D000927:Anticonvulsants; D000964:Antimetabolites, Antineoplastic; D000090702:Constitutive Androstane Receptor; D061988:Proteasome Inhibitors; D011725:Pyridines; D018160:Receptors, Cytoplasmic and Nuclear; D058978:Reduced Folate Carrier Protein; D012097:Repressor Proteins; C113967:Rfc1 protein, rat; D025801:Ubiquitin; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D003577:Cytochrome P-450 Enzyme System; D011493:Protein Kinase C; D046988:Proteasome Endopeptidase Complex; D051818:Replication Protein C; D008727:Methotrexate; D010634:Phenobarbital