ABSTRACT
PR-104, currently in clinical trial, is converted systemically to the dinitrobenzamide nitrogen mustard prodrug PR-104A, which is reduced selectively in hypoxic cells to cytotoxic hydroxylamine (PR-104H) and amine (PR-104M) metabolites. Here, we evaluate the roles of this reductive metabolism, and DNA interstrand cross-links (ICL), in the hypoxic and aerobic cytotoxicity of PR-104. Using a panel of 9 human tumor cell lines, cytotoxicity was determined by clonogenic assay after a 2-hour aerobic or hypoxic exposure to PR-104A. PR-104H and PR-104M were determined by high performance liquid chromatography/mass spectrometry, and ICL with the alkaline comet assay. Under hypoxia, the relationship between ICL and cell killing was similar between cell lines. Under aerobic conditions, there was a similar relationship between ICL and cytotoxicity, except in lines with very low rates of aerobic reduction of PR-104A (A2780, C33A, H1299), which showed an ICL-independent mechanism of PR-104A cytotoxicity. Despite this, in xenografts from the same lines, the frequency of PR-104-induced ICL correlated with clonogenic cell killing (r(2) = 0.747) with greatest activity in the fast aerobic metabolizers. In addition, changing levels of hypoxia in SiHa tumors modified both ICL frequency and tumor growth delay in parallel. We conclude that both aerobic and hypoxic nitroreduction of PR-104A contribute to the monotherapy antitumor activity of PR-104 in human tumor xenografts, and that ICL are responsible for its antitumor activity and represent a broadly applicable biomarker for tumor cell killing by this novel prodrug.
Subject(s)
DNA Damage , DNA, Neoplasm/drug effects , Neoplasms/metabolism , Nitrogen Mustard Compounds/pharmacology , Animals , Cell Death/drug effects , Cell Hypoxia , Cell Line, Tumor , Chlorambucil , Chromatography, Liquid , DNA, Neoplasm/metabolism , Female , HCT116 Cells , HT29 Cells , Humans , Mice , Mice, Nude , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Nitrogen Mustard Compounds/pharmacokinetics , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Tandem Mass Spectrometry , Xenograft Model Antitumor AssaysABSTRACT
Cisplatin is a crucial agent in the treatment of many solid tumors, yet many tumors have either acquired or intrinsic resistance to the drug. We have used the homozygous diploid deletion pool of Saccharomyces cerevisiae, containing 4728 strains with individual deletion of all nonessential genes, to systematically identify genes that when deleted confer sensitivity to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. We found that deletions of genes involved in nucleotide excision repair, recombinational repair, postreplication repair including translesional synthesis, and DNA interstrand cross-link repair resulted in sensitivity to all three of the agents, although with some differences between the platinum drugs and mitomycin C in the spectrum of required translesional polymerases. Putative defective repair of oxidative damage (imp2'Delta strain) also resulted in sensitivity to platinum and oxaliplatin, but not to mitomycin C. Surprisingly in light of their different profiles of clinical activity, cisplatin and oxaliplatin have very similar sensitivity profiles. Finally, we identified three novel genes (PSY1-3, "platinum sensitivity") that, when deleted, demonstrate sensitivity to cisplatin and oxaliplatin, but not to mitomycin C. Our results emphasize the importance of multiple DNA repair pathways responsible for normal cellular resistance to all three of the agents. Also, the similarity of the sensitivity profiles of the platinum agents with that of the known DNA interstrand cross-linking agent mitomycin C, and the importance of the gene PSO2 known to be involved in DNA interstrand cross-link repair strongly suggests that interstrand cross-links are important toxic lesions for cisplatin and oxaliplatin, at least in yeast.
