Abstract
Abstract TH-302 is a hypoxia-activated prodrug currently in clinical trials for the treatment of cancer. TH-302 releases the DNA cross-linking bromo-isophosphoramidate mustard (Br-IPM) under hypoxic conditions and exhibits 400-fold hypoxia-enhanced cytotoxicity in multiple human cancer cell lines in vitro. In this study, we utilized pharmacological tools to dissect the underlying mechanisms of reductase-dependent prodrug activation and DNA repair processes involved in the cellular response to TH-302. The pharmacological modulators used as tools in this study include the flavin reductase inhibitor diphenyliodonium (DPI) chloride, the NAD(P)H:quinone oxidoreductase (NQO1) inhibitor dicoumarol, the PARP inhibitor ABT-888, and the HDAC inhibitor valproic acid (VPA). An in vitro cytotoxicity assay was used as the primary read-out. The effect of the P450 reductase inhibitor DPI chloride on TH-302 activity was assessed in H460, SiHa, and SiHa cells over-expressing P450 reductase (SiHaPOR). The results demonstrated that TH-302 activity was inhibited by DPI. The HAP Tirapazamine exhibited a profile similar to that of TH-302. We investigated the effect of dicoumarol on TH-302-mediated cytotoxicity in H460 cells. Dicoumarol actually enhanced TH-302 activity in a concentration-dependent manner in this cell line. These results are consistent with an interplay between the POR and NQO1 redox systems. The effect of the PARP inhibitor ABT-888 on TH-302 activity was investigated in three human cancer cell lines: H460, A375 and HCT116. TH-302 activity was not affected by the presence of ABT-888. In contrast, the activity of the monoalkylating agent temozolomide was enhanced by ABT-888 in these cancer cell lines. Similarly, the sensitivity of EM9 cells (deficient in XRCC1) exhibited a heighted sensitivity to temozolomide but not to TH-302. Taken together, these results indicate that SSB (single-strand break) DNA repair mechanisms are not involved in the repair of TH-302 lesions. The effect of HDAC inhibitor VPA on TH-302 was investigated in the human prostate cancer cell line DU-145. Inhibition of HDAC has been shown to downregulate HDR (homology dependent DNA repair). Cisplatin was used as a control. Consistent with the published data, VPA enhanced cisplatin-mediated cytotoxicity. Similarly, TH-302 cytotoxicity was also enhanced by VPA. In summary, TH-302 activity can be modulated by pharmacological modulators of both reductases and DNA damage and repair pathways. TH-302 activity can be inhibited by the flavin reductase inhibitor DPI, while the NQO1 inhibitor dicoumoral can enhance TH-302 activity. TH-302 activity was not affected by the PARP inhibitor ABT-888 but was potentiated by the HDAC inhibitor VPA. Taken together, these results support that HDR and not SSB DNA repair mechanisms is involved in repair of TH-302-induced DNA lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2648. doi:10.1158/1538-7445.AM2011-2648
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