Monitoring of the intracellular activation of 5-fluorouracil to deoxyribonucleotides in HT29 human colon cell line: application to modulation of metabolism and cytotoxicity study

An HPLC method was developed for in vitro detection and monitoring of intra cellular metabolites of [H-3]-5-fluorouracil (FUra). Results showed a prefe rential activation of FUra to ribonucleoside and ribonucleotide derivatives (FURd, FUMP, FUDP and FUTP) in the human colorectal HT29 cell line. We scr eened Various agents so as to determine if they could act as modulators of metabolism and/or toxicity of FUra by reversing the activation pathway of F Ura from ribo- to deoxyribonucleotides, thus enhancing FdUMP formation. Dif ferent drugs (efflux inhibitors, catabolism inhibitors and enzymatic cofact ors) were tested for enhancement of cytotoxicity when associated with FUra. The most promising agents were further studied by assessment of their abil ity to modulate intracellular activation of FUra to enhance thymidylate syn thase (TS) inhibition by FUra and to increase the subsequent induction of a poptosis. 2'-Deoxyinosine (d-Ino), a deoxyribose 1-phosphate donor increasi ng thymidine phosphorylase activity, stood out as the best modulating agent we screened. Results showed an up to 30-fold increase of cytotoxicity alon g with a stronger inhibition of TS when FUra was associated with d-Ino, whi le FUra alone exhibited a lesser effect on TS activity. Besides, HPLC analy sis revealed a complete reversal of the activation pathway of FUra, thus le ading to an intracellular accumulation of deoxyribonucleotides. Assessment of cell cycle distribution showed a marked increase ( + 480 %) of apoptosis in cells exposed to FUra/d-Ino compared to FUra alone. The HPLC method we developed is a convenient tool for assessing to what extent modulators will actually act on the intracellular activation of FUra. This study confirms the potentiality of d-Ino to modulate FUra metabolism in vitro. It proved t o be an agent able to orientate the mechanism of action of FUra towards the inhibition of TS in cells where the normal activation pathway of the drug does not result in the intracellular accumulation of the active metabolite FdUMP. (C) 2000 Editions scientifiques et medicales Elsevier SAS.