Genetic and biochemical modulation of 5-fluorouracil through the overexpression of thymidine kinase: an in-vitro study.
The pro-drug 5-fluorouracil (5-FU) exerts its anti-proliferative action after conversion into cytotoxic metabolites. We previously demonstrated that the anti-cancer action of 5-FU could be enhanced by boosting thymidine phosphorylase (TP) activity in cancer cells, the first step of the DNA pathway, that yields the critical anti-thymidylate synthase (TS) fluorodeoxyuridine monophosphate (FdUMP) metabolite. In the present study, we further studied to what extent 5-FU activity could be optimized by overexpressing cancer cell thymidine kinase (TK), the second step of the DNA pathway, for which controversial data have been published so far. Additionally, screening of biochemical modulators likely to contribute to 5-FU activation was also carried out. TK-overexpressing colorectal cells were obtained after designing vectors harboring viral and human cDNA, and performing stable transfection in the human HT29 cell line. Anti-proliferative assays were subsequently performed so as to evaluate change in cell sensitivity to 5-FU, and metabolism monitoring was carried out to follow drug activation and FdUMP formation after cellular uptake. Finally, TS inhibition was assessed as a pharmacological endpoint. Results showed that overexpression of TK led to a marked desensitization of our model. A negative correlation (r = 0.87) was found between the level of TK activity and 5-FU anti-proliferative action - the higher the activity, the lower the sensitivity. Of the various drugs screened as putative modulators, only those involved in TP activity proved to enhance 5-FU efficacy via optimized FdUMP formation. Conversely, genetically increasing TK activity did not modify 5-FU activation pathway nor subsequent TS inhibition in our model. Therefore, our results indicate that TK is not a limiting step in the production of anti-TS FdUMP and that tumor cells overexpressing TK are likely to resist 5-FU-based chemotherapies.
Fanciullino
Raphaelle
R
Pharmacokinetics Department, School of Pharmacy, Marseille, France.
Evrard
Alexandre
A
Cuq
Pierre
P
Giacometti
Sarah
S
Peillard
Laurent
L
Mercier
Cédric
C
Aubert
Claude
C
Milano
Gérard
G
Ciccolini
Joseph
J
D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000378:metabolism / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D013937:Thymidine Kinase; D005472:Fluorouracil