The Role Of Glabridin On Migration Inhibition And Induction Of Apoptosis In Colorectal Cancer Cell And Its Possible Molecular Mechanisms

Objective To explore the effects of glabridin on migration and apoptosis in human colorectal cancer cell lines RKO and HCT116 cells and its possible molecular mechanisms.Methods RKO and/or HCT116 cells were treated by glabirdin,MLCK inhibitor ML-7,ROCK inhibitor H-1152,selective inhibitors of p38 and JNK(SB203580 and SP600125,respectively)and PKC activator PMA.The viability of RKO and HCT116 cells were detected by MTT assay treated with glabridin.Plate colony formation assay was applied to investigate the ability of RKO and HCT116 cells to form colony.Hoechst staining was used to detect the apoptosis of RKO cells.The inhibition of invasion,metastasis by glabridin were detected with wound-healing asssay and transwell assay.Immunofluorescence was used to detect the expression of epithelial markers adhesion molecules E-cadherin and mesenchymal markers adhesion molecules N-cadherin in colorectal cancer cell(RKO and HCT116 cells),and to observe tight junction protein Occludin and ZO-1 expression and location in HCT116 cells.Western blot was performed to examine the proteins associated with apotosis(Bcl-2,Bax,Caspase-3,Caspase-8,Caspase-9)in RKO cells,the tight junctions Occludin and ZO-1 in HCT116 cells,the expression of E-cadherin and N-cadherin and the expression of migration-associated proteins(Rho A,ROCK,MLCK,p-MYPT1,p-MLC),and MAPKs signaling pathway proteins.Results The proliferation,the ability of colony formation,migration and invasion of human colorectal cancer cell lines RKO and HCT116 cells were inhibited significantly after treated with glabrdin.Glabridin induced the apotosis of RKO cells, down-regulated the expression of Bcl-2 and Procaspase 9 and up-regulated the expression of Bax and Caspase3 compared with control group,while caspase 8 expression was constant in all groups.After treated by glabrdin,the phosphorylation of MYPT1 and MLC of RKO and HCT116 cells were reduced,the expression level of Rho A,ROCK1 and ROCK2 were down-regulated in RKO cells,however the expression level of Rho A,ROCK1 and ROCK2 had no change.While after administrated the inhibitor of MLCK,ML-7,or the inhibitor of ROCK,H-1152,the cancer migration were inhibited,and the expression of MLCK,ROCK1 and p-MYPT1 were downregulated in RKO and HCT116 cells,hence we could draw a conclusion that the glabrdin could inhibit the migration of RKO and(or)HCT116 cells by the inhibition of the activity of MLCK and ROCK.Western blot showed that glabrdin could up-regulate the phosphorylation level of p38 and JNK,however no change in ERK in RKO,and glabrdin could up-regulate the phosphorylation level of p38,and down-regulate the phosphorylation level of JNK,however no change in ERK.Western blot analysis showed that glabirdin inhibited the phosphorylation of MYPT1 and MLC as well as SP600125.Glabirdin can induce the expression of E-cadherin,while reducing the expression of N-cadherin in RKO and HCT116 cells;and increase the expression of Occludin and ZO-1 in the membrane of HCT116 cell.Conclusions Glabridin could inhibit the proliferation and migration of RKO and HCT116 cells,induce the apotosis of RKO cells;and further experiments confirmed that the molecular mechanism of glabridin on migration inhibition was by decreasing the phosphorylation level of MLC,increasing the expression of E-cadherin,reducing the expression of N-cadherin,and up-regulating the tight junction-associated proteins ZO-1 and Occludin partly through JNK signal transduction.