title: A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein.
abstract: Virus-directed enzyme prodrug therapy (VDEPT) is an emerging strategy against cancer. Our approach is a P450-based VDEPT that consists of using cyclophosphamide (CPA) as a prodrug and a Cytochrome P450 2B6/NADPH cytochrome P450 reductase fusion protein (CYP2B6/RED) as a prodrug-activating enzyme. Due to the heterogenous expression of proteins in tumor cells, basal reductase activity may not be sufficient to supply CYP2B6 with electrons, the fusion protein should enable the expression of both proteins at high levels in tumor cells. CYP/RED fusion proteins have never been previously expressed in mammalian cells, to enable expression the fusion protein was cloned into an adenoviral vector and subsequently several pulmonary tumor cell lines were infected. The CYP2B6/RED fusion protein was detected by Western blot, its mRNA by Northern blot, and its heme incorporation into an active form by spectral analysis. Infection with the fusion gene increased RED activity in microsomes by a factor of 3 compared to the control. After infection and treatment with CPA, in cell lines with low endogenous RED, the fusion protein mediated significantly higher CPA-induced cytotoxicity compared to cells expressing solely CYP2B6. In conclusion, the fusion protein is functional for VDEPT by providing one protein for higher levels of CPA metabolism.
authors.affiliation: INSERM UMRS 490, Molecular Toxicology, Université René Descartes, 45 rue des Saints-Pères, 75006 Paris, France.
authors.forename: Marina
authors.initials: M
authors.lastname: Tychopoulos
authors.forename: Laurent
authors.initials: L
authors.lastname: Corcos
authors.forename: Philippe
authors.initials: P
authors.lastname: Genne
authors.forename: Philippe
authors.initials: P
authors.lastname: Beaune
authors.forename: Isabelle
authors.initials: I
authors.lastname: de Waziers
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism / Q000627:therapeutic use*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D003520:Cyclophosphamide / Q000378:metabolism / Q000627:therapeutic use*; D065702:Cytochrome P-450 CYP2B6; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011355:Prodrugs / Q000627:therapeutic use*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D018906:Antineoplastic Agents, Alkylating; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D003520:Cyclophosphamide; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D009251:NADPH-Ferrihemoprotein Reductase