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. Cancer Gene Therapy (2005) Keywords: VDEPT; P450-based gene therapy; cyclophosphamide; lung cancer G ene-directed enzyme prodrug therapy (GDEPT) 1,2 is an emerging strategy used to improve the selectivity of cancer chemotherapy. This is accomplished through tumor-specific activation of noncytotoxic prodrugs to active drugs by drug-metabolizing enzymes. The general scheme consists of transfection of tumor cells with an enzyme responsible for the bioactivation of a noncytotoxic prodrug and treatment with the prodrug: only cells expressing the transfected drug-metabolizing enzyme can metabolize the prodrug into cytotoxic metabolites, leading to cell death. The use of viral vectors for transgene introduction is more specifically referred to as virusdirected enzyme prodrug therapy (VDEPT). Following promising early results of a P450-based GDEPT strategy, 3 using the rat 9L gliosarcoma cell line, cyclophosphamide (CPA) as a prodrug and CYP2B1 as the prodrug activating enzyme, our approach was to improve the strategy by activating CPA with cytochrome P450 2B6 (CYP2B6) 4,5 or a CYP2B6/NADPH cytochrome P450 reductase (RED) fusion protein in order to avoid the frequent problem of low RED expression in tumor cells. This constructed protein is the fusion of two human proteins namely CYP2B6 and the human RED resulting in a single protein able to transfer electrons from NADPH right through to the heme moiety of the protein to enable metabolism of CYP2B6 substrates. In order to achieve high levels of expression in mammalian cells, the proteins were cloned into adenoviral vectors by homologous recombination.CPA belongs to the oxazaphosphorine class of molecules 6,7 and remains a widely used cytotoxic agent f...

Improvement of Cyclophosphamide Activation by CYP2B6 Mutants: From in Silico to ex Vivo

“…The concept of overexpressing individual forms of prodrug-metabolizing P450 enzymes in tumor cells is now becoming well recognized (McFadyen et al, 2004), in particular the CYP2B6/CPA strategy Jounaidi, 2002;Tychopoulos et al, 2005). However, one of the major drawbacks of this TABLE 1 P450 content and enzyme kinetic analysis of CPA 4-hydroxylation in yeast microsomes expressing wild type CYP2B6 and site-directed mutants P450 content was spectrally determined (Schoene et al, 1972).…”

“…This strategy consists of the introduction of a vector that allows for CYP2B6 expression into tumor cells before CPA treatment. It was first developed by D. J Waxman's group Jounaidi, 2002) and more recently by our laboratory (Tychopoulos et al, 2005). Because of the intratumoral activation of CPA, this approach increases both the sensitivity and selectivity of cancer therapy, resulting in higher efficacy and reduced host tissue toxicity (Braybrooke et al, 2005).…”

Cancer Gene Therapy: The New Targeting Challenge

“…To ensure the production of both CYP2B6 and RED by the same cancer cell, a CYP2B6wt-RED fusion protein having both 4-hydroxylase activity and reductase activity was built. This fusion protein proved more efficient than CYP2B6wt alone for metabolizing CPA in several pulmonary cell lines (Tychopoulos et al, 2005). Recently, we produced a CYP2B6TM-RED fusion protein that is 10 times more efficient than CYP2B6wt-RED in activating CPA (unpublished results from our laboratory).…”

“…Waxman and colleagues and, more recently, by our group (Waxman et al, 1999), (Jounaidi, 2002), (Jounaidi et al, 2006), (Tychopoulos et al, 2005). CYP2B expressed in tumor cells results in the in situ conversion of CPA to cytotoxic metabolites.…”

“…CYP2B expressed in tumor cells results in the in situ conversion of CPA to cytotoxic metabolites. Moreover, the diffusible 4'-OH-CPA metabolite can enter neighboring cells, where it is converted to phosphoramide mustard, leading to the death of nontransfected tumor cells (Wei et al, 1995), (Tychopoulos et al, 2005). This bystander effect plays a major role in the CYP2B-based GDEPT strategy, and several studies of various suicide gene and prodrug combinations have shown that complete eradication of the tumor is possible even when the suicide gene product is expressed by less than 10% of the cells (Portsmouth et al, 2007) In our laboratory, we are developing a GDEPT strategy based on human CYP2B6, the human CYP isoform that preferentially metabolizes CPA (Gervot et al, 1999).…”