title: Monitoring of the intracellular activation of 5-fluorouracil to deoxyribonucleotides in HT29 human colon cell line: application to modulation of metabolism and cytotoxicity study.
abstract: An HPLC method was developed for in vitro detection and monitoring of intracellular metabolites of [3H]-5-fluorouracil (FUra). Results showed a preferential activation of FUra to ribonucleoside and ribonucleotide derivatives (FURd, FUMP, FUDP and FUTP) in the human colorectal HT29 cell line. We screened 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 FUra from ribo- to deoxyribonucleotides, thus enhancing FdUMP formation. Different drugs (efflux inhibitors, catabolism inhibitors and enzymatic cofactors) were tested for enhancement of cytotoxicity when associated with FUra. The most promising agents were further studied by assessment of their ability to modulate intracellular activation of FUra to enhance thymidylate synthase (TS) inhibition by FUra and to increase the subsequent induction of apoptosis. 2'-Deoxyinosine (d-Ino), a deoxyribose 1-phosphate donor increasing thymidine phosphorylase activity, stood out as the best modulating agent we screened. Results showed an up to 30-fold increase of cytotoxicity along with a stronger inhibition of TS when FUra was associated with d-Ino, while FUra alone exhibited a lesser effect on TS activity. Besides, HPLC analysis revealed a complete reversal of the activation pathway of FUra, thus leading 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 to 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.
authors.affiliation: Laboratoire de toxicocinétique et pharmacocinétique, faculté de pharmacie, Marseilles, France. joseph.ciccolini@pharmacie.univ-mrs.fr
authors.forename: J
authors.initials: J
authors.lastname: Ciccolini
authors.forename: L
authors.initials: L
authors.lastname: Peillard
authors.forename: C
authors.initials: C
authors.lastname: Aubert
authors.forename: P
authors.initials: P
authors.lastname: Formento
authors.forename: G
authors.initials: G
authors.lastname: Milano
authors.forename: J
authors.initials: J
authors.lastname: Catalin
mesh_terms: D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002474:Cell-Free System / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003854:Deoxyribonucleotides / Q000378:metabolism*; D005472:Fluorouracil / Q000378:metabolism* / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D007288:Inosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013939:Thymidine Phosphorylase / Q000187:drug effects / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014316:Tritium
chemical_list: D003854:Deoxyribonucleotides; D014316:Tritium; D007288:Inosine; D013940:Thymidylate Synthase; D013939:Thymidine Phosphorylase; C012271:deoxyinosine; D005472:Fluorouracil