Acta Pharmacologica Sinica 2007 May; 28 (5): 721-730; doi: 10.1111/j.1745-7254.2007.00554.x

Aim: To investigate the effects of insulin on enhancing 5-fluorouracil (5-FU) anticancer 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 of insulin/5-FU combination treatment on the growth of Eca 109 and Ls-174-t cells was 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 distribution of both cell lines was analyzed by flow cytometry. Western blot assay was used to assess the expression of caspase-3 and thymidylate synthase (TS). Apoptosis was detected by flow cytometry, DNA fragmentation assay, and terminal transferase dUTP nick end labeling assay (TUNEL). Moreover, the changes of 5-FU uptake after insulin pretreatment were detected by HPLC assay and Western blot 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 with insulin for the appropriate time. Insulin increased the cell number of the S phase and the uptake of 5-FU. Insulin/5-FU treatment enhanced apoptosis of tumor cells and 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 TS ternary 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 esophageal and colonic cancer cell lines.

Keywords: apoptosis; cell cycle; 5-fluorouracil; insulin