Synergistic Effect Of Kaempferol And 5-fluorouracil On Growth Of Colorectal Cancer Cells And Underling Mechanism

Objective:To find out whether Kaempferol combined with 5-FU show synergistic anticancer effect on colorecal cancer cellsand explore the mechanism of synergism from induce apoptosis, inhibit proliferation and inhibit the metobolic enzyme of 5-FU.Methods:1. Combination effect of Kaempferol and 5-FU:MTT assay was conducted to calculated the IC50 of Kaempferol and 5-FU on colorectal cancer cell HCT-8, HCT-116 and HEK-293, and use the IC50 (Kaempferol) divide IC50 (5-FU) to get the combination ratio. Constant ratio to treated CRC cells then calculated CI of different ratio. And detect toxicity of kaempferol combined with 5-FU on HEK-293 cells and select concentration of Kaempferol and 5-FU.2. Mechanism of synergistic effect of Kaempferol and 5-FU:Microscopy and colony formation assay were used to evaluate the cell morphology and survival, respectively. FACS was performed to analyze cell cycle progress. Hoechst staining and AnnexinV/PI staining were used to assess cell apoptosis. Q-PCR and Western-blot were used to determine the mRNA and protein expression of Bcl-2, Bax, TS, DPD and PI3K/Akt signaling pathway.Result:1. Conbination effect of Kaempferol and 5-FU:The IC50 of 5-FU and Kaempferol on HCT-8 cells were 177.78μM,350μM. Constant ratio to treat CRC cells and calculated CI of different ratio. Whenconcentration of5-FU was 50μM and Kaempferol was 100μM, we found Kaempferol combined with 5-FU shows synergistic anticancer effect on HCT-8 cells, which CI was 0.39 (less than 1). Similar situation with HCT-116 cell line, combination index was 0.621.2. Mechanism of synergistic effect of Kaempferol and 5-FU:(1) Kaempferol combined with 5-FU treatment significantly inhibited cancer cell viability and colony formation in HCT-8 cell line compared with single drug treatment. Hoechst staining and Annexin V-PI staining assay indicated that Kaempferol combined with 5-FU treatment significantly induced the apoptosis of HCT-8 cells. Cell cycle indicated that Kaempferol combined 5-FU treatmentsignificantly arrest cell cycle in GO phase. Q-PCR and Western-blot analysis showed that Kaempferol combined with 5-FU treatment significantly down-regulated the expression of Bcl-2, PI3k and p-Akt and up-regulated the Bax compared with 5-FU single treatment group.Conclusion:Kaempferol combined 5-FU show synergistic anticancer effect and alters the expression of Bcl-2, Bax, DPD and TS in colorectal cancer cell HCT-8. These molecular effects lead to the induction of apoptosis, inhibition of cell proliferation and function of TS and DPD, eventually resulting in the synergistic effect of Kaempferal and 5-FU in colorectal cancer cell.