Non-Nuclear Localized Human NOSII Enhances the Bioactivation and Toxicity of Tirapazamine (SR4233) in Vitro

Abstract

Tirapazamine (TPZ) is the lead member of a class of bioreductive drugs currently in phase II and III clinical trials. TPZ requires metabolic activation to give a cytotoxic free radical species, and this hypoxia-mediated process is carried out by a variety of cellular reductases, including NADPH cytochrome c (P450) reductase (P540R). Nitric-oxide synthase (NOS) is widely expressed in human tumors, and this enzyme consists of an oxidase and a reductase domain, the latter showing striking homology to P450R. Thus, in this article, we have investigated the role of one of the cytosolic isoforms of NOS [inducible NOS (NOSII)] in the bioactivation of this DNA-damaging antitumor agent. To achieve this, we have constitutively overexpressed NOSII in human breast tumor MDA231 cells by employing an optimized expression vector in which the strong human polypeptide chain elongation factor 1␣ promoter drives a bicistronic message encoding the genes for human NOSII and the puromycin-resistant gene (pac). Subcellular localization of NOSII in the stably transfected clones was determined after differential centrifugation and showed that NOSII catalytic activity was exclusively cytosolic as determined by conventional activity assay. This was confirmed by immunostaining followed by fluorescent microscopy studies. The increase in NOSII activity in a series of transfected clones was associated with an increase in TPZ metabolism and toxicity under hypoxic conditions. There was no similar increase in aerobic toxicity. These findings are of significance for two reasons. First, cellular NOSII activity, similar to that seen in human breast cancer, could contribute to TPZ toxicity; second, this will be a result of NOSderived/cytosol-associated TPZ radicals.Tirapazamine 2, SR 4233, Tirazone)] is a promising antitumor agent that has a unique spectrum of activity in that it selectively causes DNA damage in hypoxic tumor cells after one-electron bioreductive activation. Cytotoxicity results from activation by a reductive enzyme(s) that adds an electron to the parent drug to produce radical species that cause DNA single-strand breaks, double-strand breaks (DSBs), and chromosome aberrations (Wang et al

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

“…Tirapazamine is another bioreductive drug selectively activated by iNOS under hypoxic conditions to give highly toxic metabolites (17). Here, we show that coculturing the cancer cells with the iNOS-expressing macrophages did not enhance the toxicity of tirapazamine to cancer cells under both hypoxic and aerobic conditions.…”

“…Here, we show that coculturing the cancer cells with the iNOS-expressing macrophages did not enhance the toxicity of tirapazamine to cancer cells under both hypoxic and aerobic conditions. This is most likely because the tirapazamine radical is short-lived and highly reactive, which prevents the drug from exerting any bystander effects (17,28). However, it also highlights the potential value and utility of AQ4N, which does show these effects.…”

“…iNOSR Activity Following cytokine treatment of the J774.2 cells, the NOSR activity was determined spectrophotometrically in cell lysates as the NADPH-dependent reduction of cytochrome c as described previously (17).…”

“…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).…”

Diflavin Oxidoreductases Activate the Bioreductive Prodrug PR-104A under Hypoxia

“…2C) in an anoxia-specific manner. Two of the enzymes, MTRR and NDOR1, represent novel bioreductive drug-metabolizing enzymes, whereas the POR domain of NOS2A has previously been identified as catalyzing the activation of the nitroaromatic compound 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) (Chandor et al, 2008) and the hypoxic cytotoxin tirapazamine (Chinje et al, 2003). TABLE 1 Frequency and intensity of POR staining in the mixed cancer tumor microarrays Details of the mixed cancer arrays (MA2, MB3, MC2, and CSTB-01) are provided in Supplemental Table S5.…”