title: Cytochrome P450 reductase dependent inhibition of cytochrome P450 2B1 activity: Implications for gene directed enzyme prodrug therapy.
abstract: Cytochrome P450 (P450) enzymes are often used in suicide gene cancer therapy strategies to convert an inactive prodrug into its therapeutic active metabolites. However, P450 activity is dependent on electrons supplied by cytochrome P450 reductase (CPR). Since endogenous CPR activity may not be sufficient for optimal P450 activity, the overexpression of additional CPR has been considered to be a valuable approach in gene directed enzyme prodrug therapy (GDEPT). We have analysed a set of cell lines for the effects of CPR on cytochrome P450 isoform 2B1 (CYP2B1) activity. CPR transfected human embryonic kidney 293 (HEK293) cells showed both strong CPR expression in Western blot analysis and 30-fold higher activity in cytochrome c assays as compared to parental HEK293 cells. In contrast, resorufin and 4-hydroxy-ifosfamide assays revealed that CYP2B1 activity was up to 10-fold reduced in CPR/CYP2B1 cotransfected HEK293 cells as compared to cells transfected with the CYP2B1 expression plasmid alone. Determination of ifosfamide-mediated effects on cell viability allowed independent confirmation of the reduction in CYP2B1 activity upon CPR coexpression. Inhibition of CYP2B1 activity by CPR was also observed in CYP2B1/CPR transfected or infected pancreatic tumour cell lines Panc-1 and Pan02, the human breast tumour cell line T47D and the murine embryo fibroblast cell line NIH3T3. A CPR mediated increase in CYP2B1 activity was only observed in the human breast tumour cell line Hs578T. Thus, our data reveal an effect of CPR on CYP2B1 activity dependent on the cell type used and therefore demand a careful evaluation of the therapeutic benefit of combining cytochrome P450 and CPR in respective in vivo models in each individual target tissue to be treated.
authors.affiliation: Austrianova Biotechnology GmbH, Veterinärplatz 1, A-1210 Vienna, Austria.
authors.forename: Johannes
authors.initials: J
authors.lastname: Lengler
authors.forename: Markus
authors.initials: M
authors.lastname: Omann
authors.forename: Dana
authors.initials: D
authors.lastname: Düvier
authors.forename: Harry
authors.initials: H
authors.lastname: Holzmüller
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Gregor
authors.forename: Brian
authors.initials: B
authors.lastname: Salmons
authors.forename: Walter H
authors.initials: WH
authors.lastname: Günzburg
authors.forename: Matthias
authors.initials: M
authors.lastname: Renner
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007069:Ifosfamide / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010078:Oxazines / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011355:Prodrugs / Q000378:metabolism* / Q000627:therapeutic use; D014162:Transfection
chemical_list: D018906:Antineoplastic Agents, Alkylating; D003574:Cytochrome c Group; D010078:Oxazines; D011355:Prodrugs; C014180:resorufin; D019362:Cytochrome P-450 CYP2B1; D009251:NADPH-Ferrihemoprotein Reductase; D007069:Ifosfamide