Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069

Abstract

Summary P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine Br J Cancer 72: 1144-1150. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069.Keywords: P450 reductase; hypoxia, bioreductive drugs; tirapazamine; RSU 1069; breast cancer It is now well established that regions of low oxygen tension (hypoxia) can exist in many human solid tumours and that this hypoxia can sometimes predispose to failure of some treatments with radiotherapy (Hoeckel et al, 1996). One of the reasons for this failure is likely to be the inherent radiation resistance of hypoxic cells. One strategy being developed to overcome this problem is via the use of drugs that will selectively kill hypoxic cells Stratford, 1986, 1994;Zeman et al, 1986;Kennedy, 1987;Brown and Siim, 1996;Denny et al, 1996). This approach involves the exploitation of various biochemicla processes that, at low oxygen tensions, can result in selective reductive activation of a prodrug to give cytotoxic species (Workman and Stratford, 1993 enzymes. Thus, it has been proposed that hypoxic cells could be more effectively targeted if differences in the levels of the reductases in various cell types were taken into account to direct appropriate agents to particular human tumours based on their enzymology Workman and Stratford, 1993). Patterson et al (1995) examined the possible application of the enzyme-directed approach to the development of tirapazamine. The metabolism of this agent was suggested to be dependent on the presence of P450 reductase (Walton et al, , 1992. In a panel of six breast cancer cell lines with a sixfold range in P450 red...

Oxygen-sensitive enzyme-prodrug gene therapy for the eradication of radiation-resistant solid tumours

“…This flavoenzyme will readily donate single electrons to any prodrug with an appropriate redox potential and we have previously established that constitutive over-expression of P450R confers sensitivity to most classes of bioreductive agents, including nitrocompounds such as RSU1069. 18 In this paper we restrict over-expression of P450R to hypoxic tumour cells by including the HRE from PGK-1 within our therapeutic gene cassette. We have previously demonstrated that PGK-1 HRE-mediated gene expression is activated in tumour cells shown to be radio-resistant in vivo.…”

“…18 To achieve co-expression of both Gene Therapy the human P450R cDNA and enhanced green fluorescent protein (EGFP), under the transcriptional control of the HRE from the PGK-1 gene, we constructed a bicistronic expression cassette employing pCI neo as the vector backbone (Promega, Southampton, UK). A synthetic oligonucleotide containing three copies of the minimal 18 bp PGK-1 HRE sequence TGTCACGTCCTGCACGAC and BglII compatible 5' and 3' ends was cloned in the forward orientation directly upstream of the SV40 minimal promoter (SV40mp) in the pGL3 vector (Promega).…”

“…P450R and pimonidazole adduct presentation was analysed in adjacent sections. The rabbit polyclonal anti-human P450 reductase antibody used 18 was applied at a 1/1000 dilution. Controls were treated with an identical dilution of rabbit serum.…”

Effects of cytokine-induced macrophages on the response of tumor cells to banoxantrone (AQ4N)

“…The latter shares a high degree of sequence homology to NADPH:cytochrome P450 reductase (13). Cytochrome P450 reductase and NOSR have been shown to be involved in the metabolism and activation of Adriamycin (14,15) and certain bioreductive drugs including tirapazamine (16)(17)(18).…”

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

“…On the other hand, there is an increasing body of evidence that the reductive activation of antitumour drugs, for example, mitomycin C, tirapazamine and indoloquinone, could result in covalent binding to DNA (McGuinness et al, 1991;Adams and Stratford, 1994;Bailey et al, 2001) and lead to the important increase in the cytotoxic activity of these compounds against tumour cells (Patterson et al, 1995(Patterson et al, , 1997Chinje et al, 1999;Cowen et al, 2003). Many investigations are also focused on the bioreductive activation of DOX (Bartoszek and Wolf, 1992;Taatjes et al, 1997;Bartoszek, 2002).…”