Gossypol sensitizes the antitumor activity of 5-FU through down-regulation of thymidylate synthase in human colon carcinoma cells.

5-Fluorouracil (5-FU) is the basic chemotherapeutic agent used to treat colon cancer. However, the sensitivity of colon cancer cells to 5-FU is limited. Gossypol is a polyphenolic extract of cottonseeds. The purpose of this study was to investigate the activities and related mechanism of gossypol alone or in combination with 5-FU against human colon carcinoma cells. The IC50 of gossypol or/and 5-FU in vitro was tested by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the drug interaction was analyzed using the CalcuSyn method. Cell apoptosis was determined using presidium iodide staining and flow cytometric analysis. Western blotting was used to determine the expression of proteins. Transient transfection method was used to silence protein. The IC₅₀ at 48 h of gossypol in colon cancer cells was 26.11 ± 1.04 μmol/L in HT-29 cells, 14.11 ± 1.08 μmol/L in HCT116 cells, and 21.83 ± 1.05 μmol/L in RKO cells. When gossypol was combined with 5-FU, a synergistic cytotoxic effect was observed in HT-29 cells, HCT116 cells, and RKO cells compared with treatment with gossypol or 5-FU alone. The Western blotting results indicated that gossypol down-regulated thymidylate synthase (TS) rather than thymidine phosphorylase protein expression. Furthermore, the mTOR/p70S6K1 signaling pathway was inhibited in gossypol-treated colon cancer cells, and consequently, cyclin D1 expression was decreased, suggesting an additional mechanism of the observed antiproliferative synergistic interactions. All the observation was confirmed by silencing TS and inactivating the mTOR/p70S6K1 signaling pathway by rapamycin, both of which increased the chemo-sensitizing efficacy of 5-FU. These findings suggest that gossypol-mediated down-regulation of TS, cyclin D1, and the mTOR/p70S6K1 signaling pathways enhances the anti-tumor effect of 5-FU. Ultimately, our data exposed a new action for gossypol as an enhancer of 5-FU-induced cell growth suppression.