ISSN:
1432-0843
Keywords:
Key words Irinotecan
;
Deoxynucleotide pools
;
Thymidylate synthase
;
Fluoropyrimidines
;
Colon cancer
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract Purpose: To determine the effect of combined treatment with 7-ethyl-10-hydroxycamptothecin (SN-38, the active metabolite of irinotecan) and 5-fluorouracil/folinic acid (5FU/FA) in vitro using HCT-8 human intestinal adenocarcinoma cells. Methods: Cell survival was examined using colony forming assays. Cell cycle distribution before and after treatment was assessed by flow microfluorimetry. Levels of thymidylate synthase (TS) and topoisomerase I (topo I) in untreated and treated cells were determined by immunoblotting. Changes in deoxynucleotide pools were examined by high-performance liquid chromatography. Results: Clonogenic assays revealed that colony formation was decreased by 50% after a 24-h exposure to 8 ± 2 nM SN-38 or 12 ± 3 μM 5FU, the latter being assayed in the presence of 2 μM FA. When treatment with 5FU/FA was followed by SN-38, the cytotoxicity was similar to that observed with 5FU/FA alone. In contrast, when HCT-8 cells were exposed to both agents simultaneously or to SN-38 followed by 5FU/FA, the cytotoxicity was greater than that of SN-38 or 5FU/FA treatment alone. Investigation of the mechanistic basis for this sequence dependence revealed that SN-38 treatment was associated with a dose- and time-dependent decrease in conversion of [5-3H]-2′-deoxyuridine to [3H]-H2O and thymidylate in intact cells. Immunoblotting failed to reveal any decrease in TS protein that could account for the decreased activity. High-performance liquid chromatography revealed that SN-38 treatment was associated with increased levels of the deoxynucleotide dTTP and decreased levels of dUTP. Flow microfluorimetry revealed that a 24-h treatment with 10 nM SN-38 resulted in accumulation of HCT-8 cells in late S and G2 phases of the cell cycle, with a further increase in the G2 fraction during the 24 h after SN-38 removal. Conclusions: These observations are consistent with a model in which SN-38 sequentially induces diminished DNA synthesis, elevated dTTP pools, inhibition of dUMP synthesis and enhanced toxicity of 5FU/FA. Accordingly, sequencing of irinotecan and 5FU/FA might be important in combining these agents into an effective treatment for colorectal cancer.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002800050835
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