Effect Of Kaempferol On The Biological Behavior Of Human Colon Cancer Via Regulating Mmp1,Mmp2,and Mmp9

Objective: Colon cancer is a common disease threatening human health at present,due to its high incidence,poor prognosis,high mortality characteristics of widespread attention.Kaempferol is a kind of flavonoid compound,widely existing in a variety of plants,with cardioprotective,anti-inflammatory,antioxidant,anti-tumor and other activities.Currently,the treatment of colon cancer includes surgical excision,chemotherapy,radiotherapy,targeted therapy and other therapeutic methods,which are widely used in clinic.However,Chinese medicine,as a unique traditional treatment method,has rarely been found in the treatment of colon cancer.Based on this,this study aims to further explore the anti-tumor effect and related mechanism of kaempferol by exploring the biological behavior of colonic malignant tumor cells and the expression of matrix metalloproteinases(MMPs)1,2,9.Methods: The association and binding pattern between kaferol and target protein were analyzed using HERB database,ALPHAFOLD database of three-dimensional protein structure and TCM system pharmacology database and analysis platform.Human colon cancer cell lines HCT116 and HT29 were successfully constructed by cell culture technology,and then divided into Blank Control group,ordinary control group and Kae group.Cell viability in each group was measured by CCK-8assay,standard curve was drawn and IC50 value(half inhibitory concentration)was calculated.Cell scratch assay,Transwell migration assay,Transwell invasion assay and flow cytometry were used to determine the changes of tumor cell proliferation,migration,invasion,apoptosis and cell cycle after drug intervention.q RT-PCR and Western-Blot assay were used to determine the changes of RNA and protein expression of target genes MMP1,2,9 in tumor cells after drug intervention.Results: CCK-8 results showed that the survival rate of cells decreased gradually with the increase of drug concentration.After being treated with kaempferol for 48 hours,the IC50 value of HCT116 cells was 14.35μM,and that of HT29 cells was 10.31μM.The scratch test results showed that the scratch healing rate of HCT116 cells in Kae group(18.55± 11.74%)was significantly lower than that in Blank group(51.60 ± 10.07%)and Control group(64.33± 6.54%).In HT29 cells,the Blank group(47.44 ±3.07%)and Control group(52.25 ± 8.07%)were higher than Kae group(43.11± 14.37%).Transwell migration experiment showed that the number of penetrating cells in HCT116 cells in Blank group(68.00±2.94)and Control group(60.00±3.74)was higher than that in Kae group(19.00±4.55).In HT29 cells,the Blank group(64.33±11.14)and Control group(61.67±13.27)were higher than Kae group(20.00±2.16).Transwell invasion assay showed that in HCT116 cells,the number of penetrating cells in Blank group(101.00±0.82)and Control group(100.00±1.63)was significantly higher than that in Kae group(71.33±4.19).In HT29 cells,Blank group(112.33±2.37)and Control group(56.67±2.49)were higher than Kae group(23.33±2.05).The results of cell apoptosis experiment showed that the early apoptosis rate was 20.5% in Blank group and 26.1%in Control group,both of which were lower than 43.3% in Kae group.In HT29 cells,the early apoptosis rates of 18.0% in Blank group and 24.3% in Control group were lower than 38.4% in Kae group.The results of flow cytometry showed that the G1 phase Blank group(45.36±3.56%)and Control group(43.59±9.71%)of HCT116 cells were lower than Kae group(65.70±10.86%).S stage Blank group(53.93±6.77%)and Control group(50.97±8.06%)were higher than Kae group(30.41±10.22%).In HT29 cells,G1 phase Blank group(46.31±1.33%)and Control group(46.55±4.15%)were lower than Kae group(59.95±4.31%).S stage Blank group(50.86±7.40%)and Control group(52.26±8.70%)were higher than Kae group(33.31±5.92%).The results of q RT-PCR showed that the expression of MMP1(t= 3.068 P =0.0374)MMP2(t= 3.221 P =0.0080)in Blank group was higher than that in Kae group,while the expression of MMP9(t=1.182 P =0.3026)was not significantly different.The expression of MMP1(t=3.018 P=0.0393),MMP2(t=3.836 P=0.0185)and MMP9(t=4.442P=0.0113)in Control group was higher than that in Kae group.The expression of MMP2(t=3.461 P=0.0258)in Blank group was higher than that in Kae group,while the expression of MMP1(t=1.415 P=0.2301)MMP9(t=2.058 P=0.1087)had no significant difference.Meanwhile,the expressions of MMP1(t=4.905 P=0.0080)MMP2(t=4.887 P=0.0081)MMP9(t=4.239 P=0.0133)in Blank group were higher than those in Kae group.Western-Blot results showed that there was no significant difference in the expression of MMP1(t=1.545 P=0.1971)MMP2(t= 1.228 P =0.2686)MMP9(t=2.331 P=0.0801)in Blank group compared with Kae group.The expression of MMP1(t=3.061 P=0.0058),MMP2(t=3.229 P=0.0320)and MMP9(t=3.029 P=0.0388)in Control group was higher than that in Kae group.The expression of MMP1(t=1.597 P=0.1856)MMP2(t=0.3032P=0.7768)MMP9(t=0.4271 P=0.6913)in Blank group was not significantly different from that in Kae group.The expression of MMP1(t=3.115 P=0.0357),MMP2(t=2.892 P=0.0445)and MMP9(t=2.889P=0.0446)in Control group was higher than that in Kae group.Conclusion: Kaempferol can significantly inhibit the proliferation of colon cancer cells,block cell cycle,induce apoptosis,and effectively inhibit the invasion and migration of colon cancer cells.Meanwhile,kaempferol down-regulates the expression of MMP1,2,9 genes in tumor cells,indicating that the expression level of MMP1,2,9 is closely related to the biological behavior of colon cancer cells.These results also strongly validate the results of our previous bioinformatics analysis.