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

Cancer Chemother Pharmacol. 1998;42(5):391-9. doi: 10.1007/s002800050835.

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 microM 5FU, the latter being assayed in the presence of 2 microM 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 wit 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenocarcinoma
  • Antineoplastic Combined Chemotherapy Protocols / pharmacology*
  • Camptothecin / analogs & derivatives
  • Camptothecin / pharmacology
  • Cell Cycle / drug effects
  • Cell Survival / drug effects
  • DNA Topoisomerases, Type I / metabolism
  • Deoxyuracil Nucleotides / metabolism
  • Dose-Response Relationship, Drug
  • Drug Synergism
  • Enzyme Inhibitors / pharmacology*
  • Fluorouracil / pharmacology
  • Humans
  • Intestinal Neoplasms
  • Irinotecan
  • Leucovorin / pharmacology
  • Thymidylate Synthase / antagonists & inhibitors
  • Thymine Nucleotides / metabolism
  • Topoisomerase I Inhibitors*
  • Tumor Cells, Cultured / drug effects

Substances

  • Deoxyuracil Nucleotides
  • Enzyme Inhibitors
  • Thymine Nucleotides
  • Topoisomerase I Inhibitors
  • deoxyuridine triphosphate
  • Irinotecan
  • Thymidylate Synthase
  • DNA Topoisomerases, Type I
  • Leucovorin
  • thymidine 5'-triphosphate
  • Fluorouracil
  • Camptothecin