The Anticancer Effects Of MLN4924 And 5-Fu Synergistically & Smurf1 Inhibitors Alone On Human Colon Cancer Cell Line

ObjectiveTo explore the effects of a combination of MLN4924 and 5-fluorouracil(5-Fu)on the cell viability of human colon cancer cell HCT116 and the relevant mechanisms involved.To explore the effects of Smurf1 inhibitors on the cell viability of human colon cancer cell lines and the relevant mechanisms involved.MethodsCytotoxicity of the agents used singly and in combination was evaluated by cell viability assay.The combination effect of the two drugs was further analyzed using the median-effect method of Chou and Talalay.HCT116 cells were divided into Control group,MLN4924 group,5-Fu group,and Combination group.Cell apoptosis was quantified by flow-cytometry after double-staining of the cells with Annexin V-FITC/PI.The expression levels of Cleaved PARP and Cleaved caspase3 were evaluated by western blotting.MTS assay was used to detect the effect of Smurf1 inhibitors on the cell viability of colon cancer cells.Flow cytometry was used to detect the effects of different concentrations of Smurf1 inhibitors on cell cycle and apoptosis of colon cancer cell lines.Western blotting was used to detect different concentrations of inhibitors on autophagy-related protein expression levels of colon cancer cells..ResultsCell viability assays revealed that MLN4924 or 5-Fu alone inhibited cell viability in a dose-dependent manner.When MLN4924 was combined with 5-Fu,the inhibitory effect was significantly stronger than that of single drug(P<0.001).The combination index(CI)of each combination group was < 1,indicating synergism regarding HCT116 cell viability.The levels of apoptosis induction in HCT116 cells treated with MLN4924 or 5?FU were significantly increased compared with the untreated control cells(P<0.001),and the combined drug effect was significantly better than that of the drug alone(P<0.01).The results of Western blotting analyses revealed an increase in Cleaved PARP and Cleaved caspase3 protein expressions in the combination group compared with control group and single drug group.Compared with normal colonic mucosal epithelial cells,the expression level of Smurf1 in each colon cancer cell line was significantly increased.With the increase of Smurf1 inhibitors concentration,the cell viability of each colon cancer cell line gradually decreased;the cell cycle results of flow cytometry showed that the cell cycle gradually stopped in G1 phase with the increase of Smurf1 inhibitor B06 concentration;B06 has no significant effect on the level of apoptosis in colon cancer cell lines;B06 significantly increased the expression level of autophagy-related protein LC-3 of colon cancer cells in a dose-dependent manner.ConclusionsThe combination of MLN4924 and 5-fluorouracil can significantly inhibit the cell viability and enhance the apoptosis of human colon cancer cell HCT116.The inhibitor of Smurf1 B06 can inhibit the cell viability of colon cancer cells by promoting autophagy-related cell death of colon cancer cells.