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.
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.
Authors: Marina Gálvez-Peralta; Nga T Dai; David A Loegering; Karen S Flatten; Stephanie L Safgren; Jill M Wagner; Matthew M Ames; Larry M Karnitz; Scott H Kaufmann Journal: Mol Pharmacol Date: 2008-05-28 Impact factor: 4.436
Authors: Jennifer I Hare; Robert W Neijzen; Malathi Anantha; Nancy Dos Santos; Natashia Harasym; Murray S Webb; Theresa M Allen; Marcel B Bally; Dawn N Waterhouse Journal: PLoS One Date: 2013-04-23 Impact factor: 3.240
Authors: Soon Lee; Se Hoon Park; Do Hyoung Lim; Keon Woo Park; Jeeyun Lee; Joon Oh Park; Young Suk Park; Ho Yeong Lim; Won Ki Kang Journal: Cancer Res Treat Date: 2011-06-30 Impact factor: 4.679
Authors: Shannon K Tomlinson; Susan A Melin; Vetta Higgs; Douglas R White; Paul Savage; Douglas Case; A William Blackstock Journal: BMC Cancer Date: 2002-05-02 Impact factor: 4.430