The Role And Molecular Mechanisms Of Cytokeratin 18 In BCRP-mediated Multidrug Resistance In Breast Cancer Cells

Background and Objective:Multidrug resistance（MDR）and metastasis are two important factors affecting the success of breast cancer treatment.Over-expression and/or activation of breast cancer resistance protein（BCRP）are the main causes of MDR.Breast cancer cells with MDR have a stronger migration/invasion ability.Epithelial mesenchymal transition（EMT）is closely related to the development of breast cancer,especially in the aspects of MDR,migration,invasion and stem cell characteristics.Down-regulation of cytokeratin 18（CK18）is one of the signs of EMT,which plays an important role in diagnosis and prognosis of breast cancer,however,its role and main molecular mechanisms in BCRP-mediated MDR in breast cancer are not yet understood.In this study,we used the human breast cancer sensitive MCF-7 cells,and resistant MCF-7/MX cells with high expression BCRP induced by mitoxantrone（MX）as models to explore the main mechanisms of abnormal metastasis of MDR cells,aiming to investigate the role and mechanisms of CK18 in BCRP-mediated MDR,migration and invasion in breast cancer cells.Methods:1.MTT test was used to determine the sensitivity of MCF-7 and MCF-7/MX cells to different chemotherapeutic drugs.Western blot was used to determine the expression of drug-resistant proteins,such as BCRP.2.Real time-qPCR and Western blot were applied to examine CK18 mRNA and protein expressions in MCF-7 and MCF-7/MX cells.3.Anti-CK18 shRNA was designed to interfere the CK18 expression in MCF-7 cells,and two monoclonal cell lines with different interference degree and negative control cells were screened and CK18 mRNA and protein expression in above cells was then detected by Real time-qPCR and Western blot.4.MTT test was used to detect the sensitivity of MCF-7,two monoclonal cell lines and negative control cells to diverse chemotherapeutic agents.Western blot was used to detect BCRP expression in above cells.5.Cell Morphological feature was observed via inverted microscope;scratch test,Transwell migration and invasion assay,cell-matrix adhesion assay were performed to detect cell migration,invasion and adhesion ability;RT-PCR and Western blot were used to gauge EMT-related genes and proteins expressions in all above cells.6.RT-PCR and Western blot were used to detect nuclear factor-kappa B p65（NF-κB p65）,Snail,Claudin1 genes and proteins expressions in all above cells.Results:1.The MDR phenotype of MCF-7/MX cells was mediated by BCRP.MTT results showed that MCF-7/MX cells were resistant to diverse chemotherapeutic drugs（P<0.001）,and its BCRP protein expression increased significantly（P<0.001）,compared with MCF-7 cells.2.MCF-7/MX cells conduct EMT in morphology and function.MCF-7 cells showed typical epithelial cell morphology,and MCF-7/MX cells showed interstitial cell phenotype.Compared with the MCF-7 cells,the migration and invasion ability of MCF-7/MX cells increased（P<0.001）,while its cell-matrix adhesion ability and E-cadherin mRNA and protein expression decreased（P<0.001）,and meanwhile N-cadherin mRNA and protein expression,vimentin protein expression enhanced significantly（P<0.001）.3.CK18 was involved in BCRP-mediated MDR in breast cancer.Real time-qPCR and Western blot results showed that CK18 mRNA and protein expressions in MCF-7/MX cells decreased significantly（P<0.001）,compared with MCF-7 cells.After interfering CK18 expression in MCF-7 cells with shRNA,the IC₅₀0 of different chemotherapy drugs and BCRP protein expression in negative control MCF-7/siNC cells were almost no changed（P>0.05）,while that in CK18 down-regulation cells increased remarkabiy（P<0.01）.4.Down-regulation of CK18 promoted EMT in breast cancer MCF-7 cells.MCF-7/siNC cells shaped as epithelial cells;while CK18 down-regulation cells expressed interstitial characteristics.The migration,invasion and cell-matrix adhesion ability of MCF-7/siNC was almost no changed（P>0.05）,while the first two in CK18down-regulation cells increased significantly（P<0.01）,and the last decreased（P<0.001）,compared with MCF-7 cells.In addition,the mRNA and protein of E-cadherin expression in down-regulation cells decreased（P<0.05）,and the expression of vimentin and N-cadherin increased（P<0.01）;but expressions of above molecules in MCF-7/siNC cells were almost no changed（P>0.05）,compared with MCF-7 cells.5.CK18 down-regulation modulated BCRP-mediated MDR in breast cancer cells may through EMT via NF-κB/Snail and NF-κB/Claudin1 pathways.Compared with MCF-7cells,the mRNA and protein expression of NF-κB p65,Snail and Claudin1 in MCF-7/siNC cells were almost no changed（P>0.05）,while that in CK18down-regulation cells enhanced（P<0.05）.Conclusions:1.CK18 is involved in BCRP-mediated MDR of breast cancers.2.CK18 down-regulation modulates BCRP-mediated MDR in breast cancer cells may through EMT via NF-κB/Snail and NF-κB/Claudin1 pathways.