FdUMP is a 10mer of FdUMP, the thymidylate synthase (TS) inhibitory metabolite of 5-fluorouracil (5-FU). We studied the mechanism by which FdUMP inhibits cell growth and induces apoptosis. COMPARE analysis of the data from the NCI 60 cell line screen revealed unexpected mechanistic similarities for FdUMP with camptothecins (CPTs), suggesting that FdUMP possesses a mechanism of action possibly related to topoisomerase I (top1) inhibition. In this study, we found that FdUMP is much more potent than 5-FU (160-fold and 600-fold, respectively) toward P388 (mouse leukemia) and HCT116 (human colon adenocarcinoma) cells. P388/CPT45 cells that are highly-resistant to CPT as a consequence of top1-deficiency are highly cross-resistant to FdUMP, but not to 5FU. Intense DNA fragmentation following exposure to FdUMP was observed by alkaline elution in P388 and HCT116 cells. Interestingly, FdUMP induced the trapping of top1-DNA complexes both in P388 and HCT116 cells when these complexes were detected with the Immuno Complex of Enzyme (ICE) bioassay. These top1-DNA complexes could only be partially reduced by co-treatment with the broad-spectrum caspase inhibitor Z-VAD.FMK in FdUMP treated HCT116 cells, while Z-VAD.FMK almost had no effect on top1-DNA complexes formation in P388 cells treated with FdUMP. In such case, we propose that the top1-DNA complexes result from trapping of top1 at misincorporation sites (uracil and fluorouracil incorporation) resulting from potent TS inhibition by FdUMP. In conclusion, FdUMP is a markedly more potent antitumor agent than 5-FU. Trapping of top1- DNA complexes in cells by FdUMP could be one of the mechanisms of action. FdUMP mainly targets the DNA pathway, and is mechanistically distinct from 5-FU which targets both RNA and DNA pathways. FdUMP[N] compounds may be more efficacious than 5-FU or alternative FPs in cancer chemotherapy.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]