Radiation enhances the therapeutic effect of Banoxantrone in hypoxic tumour cells with elevated levels of nitric oxide synthase

Banoxantrone (AQ4N) is a prototype hypoxia selective cytotoxin that is activated by haem containing reductases such as inducible nitric oxide synthase (iNOS). In the present study, we evaluate whether elevated levels of iNOS in human tumour cells will improve their sensitivity to AQ4N. Further, we examine the potential of radiation to increase cellular toxicity of AQ4N under normoxic (aerobic) and hypoxic conditions. We employed an expression vector containing the cDNA for human iNOS to transfect human fibrosarcoma HT1080 tumour cells. Alternatively, parental cells were exposed to a cytokine cocktail to induce iNOS gene expression and enzymatic activity. The cells were then treated with AQ4N alone and in combination with radiation in the presence or absence of the iNOS inhibitor N-methyl-Larginine. In parental cells, AQ4N showed little difference in toxicity under hypoxic verses normoxic conditions. Notably, cells with upregulated iNOS activity showed a significant increase in sensitivity to AQ4N, but only under conditions of reduced oxygenation. When these cells were exposed to the combination of AQ4N and radiation, there was much greater cell killing than that observed with either modality alone. In the clinical development of hypoxia selective cytotoxins it is likely they will be used in combination with radiotherapy. In the present study, we demonstrated that AQ4N can selectively kill hypoxic cells via an iNOS-dependent mechanism. This hypoxia-selective effect can be augmented by combining AQ4N with radiation without increasing cytotoxicity to welloxygenated tissues. Collectively, these results suggest that targeting hypoxic tumours with high levels of iNOS with a combination of AQ4N and radiotherapy could be a useful clinical therapeutic strategy.