Experimental study on the inhibition of colon cancer invasion and metastasis by cinobufacini through the regulation of Wnt/β-catenin signaling pathway and EMT
Objective To explore the role of cinobufacini in colon cancer invasion and metastasis and its mecha-nism.Methods The experiment was conducted in the laboratory of the Second Affiliated Hospital of the Air Force Military Medical University from September 2022 to March 2023.Human colon cancer cell line HCT 116 and human normal colon epi-thelial cell line NCM 460 were treated with different concentrations of cinobufacini(0,2.5,5,10,20,40,80,160 mg/L)for treat-ment,and the effects of cinobufacini on the proliferation of colon cancer cells and normal cells were detected by CCK-8 method.HCT 116 cells were randomly divided into the control group and the cinobufacini(80 mg/L)group,and the morpho-logical changes of the cells were observed under the light microscope;apoptosis was detected by Annexin V-FITC/PI double staining;cell migration and invasion were detected by Transwell assay;and metastasis of the cancer cells,EMT,and Wnt/β-catenin related protein expression.Results CCK-8 results showed that the proliferation inhibition rate of HCT 116 cells was gradually increased in a dose and time dependent manner(P＜0.001)after treatment with different concentrations of cinobufacini,and the 24 h semi-inhibitory concentration(IC50)was about 80 mg/L,whereas it did not affect the prolifera-tive ability of normal colonic epithelial cells NCM460.The HCT 116 cells in the control group were in good condition,while the cells in the cinobufacini group showed decreased wall adhesion ability,increased secretion,and wrinkled morphology with a round epithelial cell like phenotype,accompanied by cell membrane rupture of varying degrees;the results of flow cy-tometry showed that the apoptosis rate of the HCT 116 cells was significantly increased compared with that of the treatment at 0 h after cinobufacini treatment for 24,48,and 72 h,respectively(t=17222,17.041,23.651,P＜0.001);and the results of the Transwell experiments showed that the apoptotic rate of HCT 116 cells increased significantly compared with that of the control group.Trans well assay showed that the number of migrating and invading cells of HCT 116 cells in the cinobufacini group was significantly lower compared with the control group(t/P=52.754/＜0.001,7.584/0.002);Western blot results showed that the expression of MMP-2 and MMP-9 in the cells of the cinobufacini group was reduced compared with the control group,the expression of epithelial markers E cadherin was increased,whereas the expression of mesenchymal mark-ers N cadherin and Snail was reduced,and the expression of β-catenin,Wnt3a,c-myc,cyclin Dl and MMP-7 proteins were down regulated,while APC protein expression was up-regulated(t/P=8.908/0.001,29.394/0.001,36.406/0.001,12.860/0.001,14203/0.001,27.862/0.001,20.176/0.001,24.012/0.001,37.245/0.001,33.627/0.001,6.971/0.002).Conclusion Cinobufacini can in-hibit colon cancer cell invasion and metastasis,and its mechanism may be related to the inhibition of EMT and blocking of Wnt/β-catenin signaling pathway.
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