Abstract
BackgroundTH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.Methodology/ResultsThe hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm3), significantly delayed tumor growth.Conclusions/SignificanceOur in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.
Highlights
Regions of low oxygen concentration within solid tumors arise due to the structural and functional abnormalities of tumor vasculature [1]
Cell viability assay To evaluate the hypoxia-dependent activation of TH-302, the viability of SCCVII and HT29 cells following incubations with various concentrations of TH-302 under aerobic (21%) and hypoxic (0.1%) conditions was measured using the MTT assay
The growth of both SCCVII and HT29 cells was minimally inhibited under aerobic conditions, similar to cells treated with pimonidazole, a widely used hypoxia marker for immunohistology which has an oxygen sensitive 2-nitroimidazole part but not a cytotoxic Br-IPM effector part (Figure 1, Figure S1 in File S1)
Summary
Regions of low oxygen concentration (hypoxia) within solid tumors arise due to the structural and functional abnormalities of tumor vasculature [1]. It is well recognized that hypoxia is implicated in resistance to radiotherapy and to conventional chemotherapy, both of which are more effective in normoxic regions [6,7,8]. A significant proportion of radiation-induced DNA damage requires oxygen, making radiotherapy less effective in hypoxic regions [9,10]. While the presence of both chronic and acute hypoxia in tumors can limit successful treatment by radiotherapy and/or chemotherapy [12,13], such conditions in turn provide a basis for the use of hypoxia-activated cytotoxins [14]. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302
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