Elsevier

Biochemical Pharmacology

Volume 72, Issue 7, 28 September 2006, Pages 893-901
Biochemical Pharmacology

Cytochrome P450 reductase dependent inhibition of cytochrome P450 2B1 activity: Implications for gene directed enzyme prodrug therapy

https://doi.org/10.1016/j.bcp.2006.06.012Get rights and content

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.

Introduction

Cytochrome P450 (P450) enzymes are widely expressed in prokaryotes and eukaryotes. P450 is involved in the metabolic oxidation and reduction of a variety of compounds including xenobiotics used in tumour therapy [1]. Mammalian P450 enzymes are mainly expressed in the liver and only at low levels in other tissues or tumours [2]. Thus, delivery of exogenous P450 genes to tumours is used in gene-directed enzyme prodrug therapy (GDEPT) to enable the hydroxylation of chemotherapeutic prodrugs including cyclophosphamide and ifosfamide (IFO) into their active forms [3], [4].

The flavoprotein NADPH cytochrome P450 reductase (CPR) interacts with P450 as an electron donor and catalyses P450 monooxygenase reactions [5]. Most tested drugs are activated more efficiently in microsomes containing P450 and CPR as compared to P450 alone [6], [7]. Recently, the influence of CPR on P450 has also been shown in transgenic mice, which carry a liver-specific deletion of CPR leading to a reduced P450 activity in this organ [8].

Since the amount of P450 often exceeds that of CPR in microsomal membranes, especially when P450 is overexpressed in GDEPT, the CPR/P450 ratio has been increased by coexpression of CPR to provide an enhanced supply of electrons, thereby enhancing P450 function and thus resulting in a superior therapeutic effect [9]. For instance, the human P450 isoform CYP3A4 was overexpressed in V79 Chinese hamster lung fibroblast cells and CYP3A4 enzymatic activity was measured by the dealkylation and deethylation of 7-alkoxyresorufins [10]. CPR coexpression in these cells stimulated P450 activity 5–10-fold suggesting a lack of endogenous CPR [11]. In other studies the CYP3A4 cDNA was amplified in Chinese hamster ovary cells to obtain high level expression of CYP3A4. Here coexpression of CPR increased CYP3A4 activity 15-fold, although P450/CPR coexpression was accompanied by a decrease in the amount of immunologically and spectrally detectable P450 [12]. CPR overexpression was assumed to increase heme-oxygenase activity, which decreases intracellular heme and thereby P450 protein levels [12].

Coexpression of CPR in P450 mediated GDEPT has been described previously to be effective in 9L rat glioma derived cells, where cell killing upon cyclophosphamide treatment was increased in rat CPR/CYP2B1 coexpressing cell clones as compared to cells expressing CYP2B1 alone [7]. CPR overexpression increased the concentration of toxic derivatives of cyclophosphamide in the presence of CYP2B1 leading to a better therapeutic effect [7].

In this study we evaluated the effect of CPR coexpression on CYP2B1 activity in different cell lines such as NIH3T3 mouse fibroblast cells and HEK293 human embryonal kidney cells, as well as in two human breast cell lines (T47D, Hs578T) and two human pancreatic tumour cell lines (Pan02, Panc-1). Cell lines with and without CYP2B1 and/or CPR expression cassettes were analyzed for the levels of respective protein expression. CPR function analysed in cytochrome c assays and CYP2B1 activity determined in metabolic resorufin, 4-hydroxylation and cell viability assays revealed no or even a dose dependent inhibiting effect of CPR on CYP2B1 activity in the majority of cell lines tested.

Section snippets

Plasmid construction

Construction of plasmids pCMV-CYP2B1 encoding the CYP2B1 gene under control of the immediate early cytomegalovirus (CMV) enhancer/promoter region, as well as the retroviral vector plasmid pPCCMm1, which drives CYP2B1 expression from mouse mammary tumour virus (MMTV) promoter in infected cells have been described elsewhere [13], [14]. CPR cDNA was cloned from rat liver by isolating mRNA using the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and reverse

Expression and function of CPR and CYP2B1

The influence of CPR on CYP2B1 activity in various human cell lines was first investigated in transient cotransfection analyses. For this the CPR coding sequence was isolated using RT-PCR techniques and inserted into an expression plasmid under control of the CMV immediate early enhancer/promoter region to allow constitutive and high level gene expression. Transient transfections of 200 fmol of the resulting plasmid pCMV-CPR into HEK293 cells revealed a CPR-mediated reduction of cytochrome c at

Discussion

Here we describe the effect of CPR on the enzymatic activity of CYP2B1. We observed a CPR dose-dependent inhibition of CYP2B1 enzymatic activity in HEK293 human embryonic kidney cells and Panc-1 pancreatic tumour cells. This observation is in line with corresponding cell viability assays, where HEK293 cells showed better survival after exposure to the prodrug ifosfamide when coexpressing CYP2B1 and CPR than cells expressing CYP2B1 alone. The inhibitory effect of CPR upon CYP2B1 enzymatic

Acknowledgments

We thank Reinhard Ertl for excellent technical support in performing FACS analysis. This project was financed in part by The Austrian Research Promotion Fund programme, FFF Grant No. 804960.

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