Abstract 4558: 6-Nitroquinoline as a metabolically-activated probe for direct fluorescence detection of hypoxia

Abstract

Abstract Hypoxic (oxygen poor) cells in tumors play an important role in cancer biology. For example, the hypoxic environment may select for cells that are incapable of undergoing apoptosis. In addition, there is recent speculation that cancer stem cells that may be responsible for metastases are harbored in the hypoxic niche of tumors. Indeed, tumor hypoxia correlates with poor patient prognosis. Accordingly, detection of tumor hypoxia may soon become an important part of the clinical characterization of human cancers. Fluorescent probes for the direct detection of hypoxia in biological systems could provide a useful complement to the radiochemical imaging and immunohistochemical staining methods that are commonly used in this field. Building on our longstanding interest in metabolically-activated, hypoxia-selective antitumor agents, we set out to develop a probe that would be metabolized under hypoxic conditions into a product with known utility as a fluorescent reporter. We selected the molecule 6-nitroquinoline as a forum to explore the fundament chemistry, biochemistry and biology underlying the development of hypoxia-selective, bioreductively-activated fluorophores. We expected that hypoxia-selective reduction of the nitro group in this probe would generate the fluorophore 6-aminoquinoline. First, we confirmed that 6-nitroquinoline is not fluorescent. We next examined whether enzymatic metabolism converted this probe to a fluorescent product. The xanthine/xanthine oxidase enzyme system was employed for the one-electron reductive activation of the probe. We find that incubation of the probe with xanthine oxidase in the presence of a stoichiometric excess (5 equiv) of the substrate xanthine generates a substantial (approximately 30 fold) increase in fluorescence at 530 nm, as expected for generation of the 6-aminoquinoline product. Mass spectrometric analysis confirms that 6-aminoquinoline is generated under these conditions. In contrast, only a small increase in fluorescence at 530 nm is observed under aerobic conditions. Experiments that varied the amount of xanthine substrate used to “fuel” the reduction reaction revealed the presence of intermediate fluorescent products that generate an emission maximum at about 430 nm. This is likely due to 6-hydroxylamino and dimerized azoxy metabolites. The enzyme NADPH: cytochrome P450 reductase similarly converts 6-nitroquinoline to fluorescent products in a hypoxia-selective manner. In conclusion, we have established that 6-nitroquinoline can serve as a direct-light-up fluorescent probe for the detection of hypoxia and we have characterized the mechanisms by which this agent is converted to fluorescent products. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4558.