The Research Of The Effect Of Curcumin On The Proliferation And Invasion And Lymphangiogenesis Of Breast Cancer MCF-7 Cell By Reducing The Expression Of Matrix Metalloproteinase-1

Background and ObjectiveNowadays,Breast cancer is the most common malignancy in women,its incidence is about 30%of all malignant tumors of females,secondly the mortality rate in female malignant tumors.Curcumin is a new type of anti-cancer drugs,the scientists found that it can inhibit breast cancer and other malignant tumors.Studies have shown that the membrane-type matrix metalloproteinase-1(MT1-MMP)and breast cancer axillary lymph node metastasis was positively correlated,while the relationship between curcumin and MT1-MMP rarely reported.So we designed MCF-7 cell line to construct MT1-MMP breast cancer cell model,and to study the effects of curcumin on MT1-MMP expression and lymphangiogenesis,and providing a theoretical basis for its application in the treatment of breast cancer.Methods1.Isolation and cultivating the lymphoid endothelial cellsThe conventional circumcision foreskin tissue was harvested whose vascular area was separated,after digestion with trypsin,the endothelial cells cultivate in endothelial cell culture(EBM),with Dil-ac-LDL labeled lymphoid endothelial cells,adding magnetic beads coated human LYVE-1 antibody and the magnetic beads,which can be collected by a magnetic bead collector.The lymphocytes were separated from the magnetic beads by digestion with DNA enzymes,then cultured in the endothelial culture medium of the matrix-coated Petri dishes and cultured in a C02 incubator.2.Establishment of MCF-7 cells who expressing MT1-MMPThe MT1-MMP gene was cloned into eukaryotic expression plasmid pcDNA3.1 and identified by sequencing.LipofectamineTM liposome 2000 was used to transfect into human breast cancer MCF-7 cells.After transfection for 2 days,the MCF-7 clone cells who stably expressing MT1-MMP were selected by G418-containing medium.According to the experimental design the breast cancer MCF-7 cell line was divided into the following three experimental groups:(1)The non-transfected group:MCF-7 in the control group;(2)The blank plasmid group:MCF-7+pcDNA3.1;(3)The transfected group:MCF-7/MT1-MMP group.All the MCF-7 cells were treated with curcumin at different concentrations.3.The protein expression of MT1-MMP was detected by Western Blot in each group.4.The CCK-8 experiment is used to determine the effect of curcumin on MCF-7 cell proliferation,while the effect of curcumin on the invasive ability of MCF-7 cells was tested by Transwell experiment.5..Co-culture of the human breast cancer cell line and lymphoid endothelial cellsThe lymphocytes were cultured in matrigel-coated dishes.After the cells were grown to 80%confluence,then the cells were covered with a layer of M199 medium-free Matrigel.After collagen polymerization,the cells were covered with cell-free matrices(MCF-7)Matrigel,and finally the endothelial cell culture medium was added.And all these cells were treated with curcumin at different concentrations.Statistical analysisAll results in this study were performed by SPSS 21.0,and the measurement data showed as mean±standard deviation(?±s),and One-way analysis of variance was applied to compare data among groups,while SNK test was used to compare data among samples in the same group,and P<0.05 for the statistically significant difference.Results1.Expression of MT1-MMP protein in MCF-7 cellsWestern blot analysis showed that there was no protein expression in untransfected group and blank plasmid group,while MT1-MMP protein was found in MCF-7/MT1-MMP group and MCF-7/MT1-MMP + curcumin group.The expression of MT1-MMP protein in transfected group was higher than that in control group,while was lightened with the curcumin.2.Inhibitory effect of Curcumin on Proliferation and invasion of MCF-7 CellsThe results of CCK-8 and Transwell assay showed that:compared with the non-transfected group and the blank plasmid group,the proliferation of MCF-7 cells was increased and the number of cell invasion was increased in transfected group,which means that the proliferation and invasion abilities of MT1-MMP transfected MCF-7 cells were enhanced obviously.While these were inhibited by curcumin,and the proliferation and invasion abilities of MT1-MMP transfected MCF-7 cells were inhibited by curcumin in a concentration dependent manner.3.Curcumin inhibits the proliferation of of lymphoid endothelial cells and lymphangiogenesis which induced by breast cancer MCF-7 cells in three-dimensional culture.The results showed that each group of breast cancer cells could induce the proliferation of lymphoid endothelial cells and form tubular structure,and the ability of which in the MT1-MMP groups were obvious,while after treated with curcumin,the induction ability of each group was weakened.conclusion1.The overexpression of MT1-MMP of the breast cancer MCF-7 cells increased the proliferation and invasion abilities,while the curcumin can inhibit it.The mechanism that may be related to the inhibition of MT1-MMP.2.The Curcumin can inhibit the proliferation and the formation of lymphoid tubular structure of lymphoid endothelial cells which induced by breast cancer MCF-7 cells.