Eriocalyxin B Suppresses Colorectal Cancer Cell Proliferation And Migration Via The Wnt/β-catenin Signaling Pathway

Objective:To investigate the inhibitory effects of Eriocalyxin B(EriB)on the proliferation and migration of colorectal cancer cells.Methods:The proliferation and vitality effect of EriB on the colorectal cancer cells were evaluated by CCK-8 assay and Soft Agar cloning.The apoptosis and cell cycle effects of EriB on the colorectal cancer cells were detected by flow cytometry.The effect of EriB on cell dryness of colorectal cancer cells were detected by Serum free suspension culture.The invasion and migration effect of EriB on colorectal cancer cells were detected by cell invasion and cell scratch method.Western blot was used to detect the expression levels of proteins related to cell cycle,apoptosis and molecular mechanism.The effect of EriB on the growth and proliferation of colon cancer cells was detected in nude mice,The effect of EriB on colon cancer cell metastasis was detected in vivo,The expression of Ki67 was detected by immunohistochemical staining,Synergyfinder was used to analyze the synergistic effect of EriB on 5-FU against colon cancer.Result:Compared with the control group,EriB treatment significantly inhibited the survival of colorectal cancer cells(P<0.05 or P<0.001)and the number of stem cell spheres(P<0.001).The results of flow cytometry showed that compared with the control group,the percentage of S phase and G2 phase in EriB treatment group increased(P<0.001),and the apoptosis increased(P<0.001).The results of cell scratch and Transwell showed that compared with the control group,the migration and invasion ability of EriB treated cells decreased significantly(P<0.05 or P<0.001),Western blot showed that the expression of CD44,Sox2 and Klf4 was down regulated after EriB treatment,The expression of cyclin A and Cdk2 was down regulated,the apoptosis related proteins cl-PARP and cl-caspase-3 were up regulated,the invasion and migration related protein E-cadherin was up regulated,and N-cadherin was down regulated,Wnt/β-catenin pathway related protein,The expressions of β-catenin,TCF4,Klf4 and c-myc were down regulated,EriB treated cells with Wnt/β-catenin pathway activator LiCl and inhibitor XAV939,Western blot showed that LiCl partially reversed down-regulation of β-catenin and target protein expressions(TCF4,cyclin D1 and c-myc),The combination of XAV939 and EriB inhibited β-catenin expression and epithelial-mesenchymal transition(EMT),EriB combined with 5-FU,According to synergyfinder analysis,zip synergy score is greater than+10,which can synergistically inhibit the survival and apoptosis of colorectal cancer cells.Western blot showed that compared with single drug,after combined application,cl-PARP,cl-caspase-3 and E-cadherin were up-regulated and N-cadherin was down regulated.The results of xenograft tumor model in nude mice showed that EriB treatment significantly inhibited tumor growth and reduced the volume of subcutaneous xenograft tumor in nude mice compared with the control group(P<0.001).Immunohistochemical analysis showed that EriB treatment significantly reduced the expression of Ki67 in tumor tissues.The results of nude mouse lung metastasis model showed that all five nude mice in the control group had lung metastasis,while only two of the five nude mice in the EriB treatment group had lung metastasis(P<0.001).Conclusion:EriB could inhibit the proliferation and dry spheroidization of colorectal cancer cells,block the cell cycle in S phase and induce apoptosis.EriB can inhibit cell migration and invasion,and its mechanism is related to the inhibition of β-cateninsignaling pathway.The combination of EriB and 5-FU enhances the ability to induce apoptosis and inhibit cell migration and invasion.EriB could inhibit tumor growth in nude mice,EriB can inhibit colorectal cancer metastasis in vivo,and its mechanism is related to the inhibition of Wnt/β-catenin signaling pathway.