Downregulation of the reduced folate carrier transport activity by phenobarbital-type cytochrome P450 inducers and protein kinase C activators.
The sodium dependent reduced folate carrier (Rfc1; Slc19a1) provides the major route for cellular uptake of reduced folates and antifolate drugs such as methotrexate (MTX) into various tissues. Despite its essential role in folate homeostasis and cancer treatment, little is known about Rfc1 regulation. A barbiturate recognition box, which as yet has only been found in the promoter region of xenobiotic metabolizing enzymes, particularly those of the CYP450 enzyme family, was predicted in the 5' untranslated region of rat rfc1 cDNA. We have therefore investigated the regulation of Rfc1 by phenobarbital (PB)-type CYP450 inducers on the functional, transcriptional and translational level in a suitable in vitro model for rat liver. A decrease of >75% in substrate uptake was observed following treatment (48 h) with 1-10 times therapeutic plasma concentrations of PB-type CYP450 inducers like PB, carbamazepine, chlorpromazine, clotrimazole and with 0.1-1 ng/ml of the constitutive androstane receptor agonist TCPOBOP. This was not associated with reduced mRNA and protein levels. Further mechanistic investigations revealed that short-term treatment (2 h) of cells with protein phosphatase 1/2A inhibitor okadaic acid (80.5 ng/ml) and proteinkinase C inducer phorbol 12-myristate 13-acetate (PMA; 0.62 microg/ml) almost abolished Rfc1 mediated MTX uptake. Finally, the reduction in Rfc1 activity caused by PB, TCPOBOP and PMA was reversed by simultaneous incubation with the specific PKC inhibitor bisindolylmaleimide (BIM; 21 ng/ml). These results demonstrate that clinically relevant concentrations of PB-type CYP450 inducers cause a significant PKC-dependent reduction in Rfc1 uptake activity at the posttranscriptional level.
Halwachs
Sandra
S
Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, An den Tierkliniken 15, University of Leipzig, 04103 Leipzig, Germany. halwachs@vetmed.uni-leipzig.de <halwachs@vetmed.uni-leipzig.de>
Kneuer
Carsten
C
Honscha
Walther
W
D000818:Animals; D002220:Carbamazepine / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002746:Chlorpromazine / Q000494:pharmacology; D003022:Clotrimazole / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects*; D020536:Enzyme Activators / Q000494:pharmacology*; D058973:Folate Receptors, GPI-Anchored; D007150:Immunohistochemistry; D019319:Okadaic Acid / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D017931:DNA Primers; D020536:Enzyme Activators; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D019319:Okadaic Acid; D002220:Carbamazepine; D003577:Cytochrome P-450 Enzyme System; D011493:Protein Kinase C; D003022:Clotrimazole; D002746:Chlorpromazine