Combination of 5-fluorouracil and irinotecan on modulation of thymidylate synthase and topoisomerase I expression and cell cycle regulation in human colon cancer LoVo cells: clinical relevance

Abstract

This study was designed to explore the possible interaction of 5-fluorouracil (5-FU) and 7-ethyl-10-hydroxycamptothecin (SN-38) in vitro. Human colon cancer LoVo cells were treated in both a dose- and time-dependent manner using clinically relevant concentrations of and exposure to 5-FU and/or SN-38. The expression of thymidylate synthase (TS), topoisomerase I, and cell cycle kinetics were evaluated by Western blot analysis and flow cytometry, respectively. Cytotoxicity was evaluated by MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) assay. The cytotoxic effects of combination treatment were determined by median effect analysis. Topoisomerase I expression was downregulated following 12 hours of exposure to treatment, and topoisomerase I expression recovered 8 hours after SN-38 was removed. The TS expression was decreased following 24 hours of 5-FU and it remained at reduced levels for > 24 hours after removal of 5-FU. SN-38 induced an arrest at S/G2/M phase, reaching its maximum effect at 12 hours. This cell cycle arrest was reversed 24 hours after SN-38 was removed. 5-FU induced an arrest at the S phase, and maximum arrest occurred at 12 hours and lasted for > 48 hours. After 12 hours of sequential SN-38, LoVo cells were arrested in S phase, thereby potentiating the effect of 5-FU. Cytotoxicity studies confirmed the synergistic interaction between 5-FU and irinotecan. These findings suggest that the proper sequencing of 5-FU/irinotecan depends on regulation of topoisomerase I, and cell cycle kinetics