Cellular studies of anticancer prodrugs
Caruso, Gianna M.
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ABSTRACT The antimetabolite, 5-fluoro-2'-deoxyuridine (5-FdUrd) and two phosphoramidate pronucleotide derivatives, APP-FU and ANP-FU, were evaluated for their growth inhibitory activities in culture using two different mammalian cell lines: the human ovarian cancer cell line A2780 and the mouse fibroblast L TK-(thymidine kinase negative) cell line. Cell culture conditions were optimized and the sulforhodamine B (SRB) assay was chosen to examine the potencies and compare the cell growth inhibitory effects of each agent. The cytotoxicity of 5-FdUrd was assessed in the A2780 cell line, in culture media containing either regular fetal bovine serum (FBA), or dialyzed FBS. The experiments in which regular FBS was used yielded an average IC 50 value of 170 nM. When dialyzed FBS was used, the drug was almost 30-fold more potent with an average IC 50 value of 5.8 nM. The reason for this difference in potency may be due to the presence of thymidine in the un-dialyzed serum. Thymidine may compete with 5-FdUrd for cellular uptake by a nucleoside transporter and for phosphorylation by thymidine kinase. In addition, thymidine may bypass the block of the thymidylate synthase step caused by the drug, accounting for the reduced potency. There was a 2000-fold difference in the potency of 5-FdUrd between the A2780 and the L TK- cell lines, where the IC 50 values were 5.8 nM and 12 μM, respectively. This can be explained by considering that thymidine kinase is responsible for the one-step activation of 5-FdUrd by direct phosphorylation. In it's absence in the L TK- cells, the cytotoxicity is due to 5-FU derived from 5-FdUrd, which requires a multi-step activation pathway. The pronucleotides, APP-FU and ANP-FU are masked phosphate derivatives of 5-FdUrd designed to overcome resistance to the drug by bypassing the thymidine kinase step. The average IC 50 for APP-FU in the A2780 and the L TK- cell line were 1.3 μM and 15 μM, respectively. The 12-fold difference in potency between the two cell lines may be attributed to degradation by hydrolysis to 5-FdUrd outside the cell to a limited extent. Accurate IC 50 -values of ANP-FU could not be determined, because of it's apparent degradation in solution. This can be explained by the strong electron-withdrawing nature of the nitro group, which makes the compound susceptible to rapid hydrolysis. The results suggest that modification of the structures of APP-FU and ANP-FU should be considered to make them more stable to hydrolysis, while maintaining adequate cellular bioavailability. Once this is accomplished their true growth inhibitory effect can be determined and their potential utility in cancer chemotherapy evaluated.