Abstract
Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear. Some studies conclude only nuclear-localized oxidoreductases can give rise to radical-mediated DNA damage and thus cytotoxicity, whereas others identify a broader role for endoplasmic reticulum and cytosolic oxidoreductases, indicating the subcellular location of TPZ radical formation is not a critical requirement for DNA damage. To explore this question in intact cells we engineered MDA-231 breast cancer cells to express the TPZ reductase human NADPH: cytochrome P450 oxidoreductase (POR) harboring various subcellular localization sequences to guide this flavoenzyme to the nucleus, endoplasmic reticulum, cytosol or inner surface of the plasma membrane. We show that all POR variants are functional, with differences in rates of metabolism reflecting enzyme expression levels rather than intracellular TPZ concentration gradients. Under anoxic conditions, POR expression in all subcellular compartments increased the sensitivity of the cells to TPZ, but with a fall in cytotoxicity per unit of metabolism (termed ‘metabolic efficiency’) when POR is expressed further from the nucleus. However, under aerobic conditions a much larger increase in cytotoxicity was observed when POR was directed to the nucleus, indicating very high metabolic efficiency. Consequently, nuclear metabolism results in collapse of hypoxic selectivity of TPZ, which was further magnified to the point of reversing O2 dependence (oxic > hypoxic sensitivity) by employing a DNA-affinic TPZ analogue. This aerobic hypersensitivity phenotype was partially rescued by cellular copper depletion, suggesting the possible involvement of Fenton-like chemistry in generating short-range effects mediated by the hydroxyl radical. In addition, the data suggest that under aerobic conditions reoxidation strictly limits the TPZ radical diffusion range resulting in site-specific cytotoxicity. Collectively these novel findings challenge the purported role of intra-nuclear reductases in orchestrating the hypoxia selectivity of TPZ.
Highlights
Hypoxia is a common pathophysiological feature of solid tumors that plays a central role in cancer progression and resistance to therapy [1,2,3,4]
TPZ has been extensively evaluated in the clinic; encouraging results were obtained from phase
Given that intracellular D is not well-defined, in Figure 1D we modelled higher and lower D values to reflect the wide range of reported values for cytoplasmic microviscosity [36,37]
Summary
Hypoxia is a common pathophysiological feature of solid tumors that plays a central role in cancer progression and resistance to therapy [1,2,3,4]. II clinical trials in non-small cell lung cancer (NSCLC) [16] and advanced head and neck squamous cell carcinoma (HNSCC) [17]. A sub-study of the head and neck trial utilized FMISO PET imaging to identify patients with high levels of tumor hypoxia and showed these patients benefited most from treatment with TPZ [18]. A subsequent Phase III trial in HNSCC showed no evidence for improved survival or local control upon addition of TPZ to chemo-radiotherapy [19]. A lack of efficacy at Phase III was likely due to a number of factors, including an absence of hypoxia pre-screening to target appropriate patient groups [18], challenges with radiotherapy protocol compliance [20], poor tumor penetration of TPZ [21] and the impact of HPV infection on sensitivity to TPZ [22]
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