Effect of adding the topoisomerase I poison 7-ethyl-10-hydroxycamptothecin (SN-38) to 5-fluorouracil and folinic acid in HCT-8 cells: elevated dTTP pools and enhanced cytotoxicity

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.