Pretreatment with insulin enhances anticancer functions of 5-fluorou racil in human esophageal and colonic cancer cells

Aim:To investigate the effects of insulin on enhancing 5-fluorouracil (5-FU) anti-cancer functions and its mechanisms in the human esophageal cancer cell line(Eca 109) and human colonic cancer cell line (Ls-174-t).Methods:The effect ofinsulin/5-FU combination treatment on the growth of Eca 109 and Ls-174-t cellswas evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT) assay.After insulin treatment or insulin/5-FU treatment,cell cycle distri-bution of both cell lines was analyzed by flow cytometry.Western blot assay wasused to assess the expression of caspase-3 and thymidylate synthase (TS).Apoptosis was detected by flow cytometry,DNA fragmentation assay,and termi-nal transferase dUTP nick end labeling assay (TUNEL).Moreover,the changes of5-FU uptake after insulin pretreatment were detected by HPLC assay and Westernblot analysis.Results:We found that insulin enhanced the inhibitory effect of 5-FU on cell proliferation when Eca 109 cells and Ls-174-t cells were pretreated withinsulin for the appropriate time.Insulin increased the cell number of the S phaseand the uptake of 5-FU.Insulin/5-FU treatment enhanced apoptosis of tumor cellsand upregulated the expression of cleaved caspase-3 compared with 5-FU treatment.Moreover,insulin/5-FU treatment induced the changes of free TS and the TSternary complex level compared with 5-FU treatment in Eca 109 and Ls-174-t cells.Conclusion:These data suggest that insulin enhances anticancer functions of 5-FU when it is treated before 5-FU for the appropriate time in human esophagealand colonic cancer cell lines.