Salinomycin Reduces Migration And Invasion Of CD44⁺/CD24^-/low Phenotype Breast Cancer Stem Cells By Suppressing MMP-2/9 Expression Through TGFβ1/Smad Pathway

ObjectiveEstablish the model of CD44~+/CD24~-/low/low phenotype breast cancer stem cells(BCSC)by culturing the MCF-7 human breast cancer cell line in serum-free medium(SFM),and analyze its biological characteristics.Purpose to investigate salinomycin could affect migration and invasion of CD44~+/CD24~-/low/low phenotype breast cancer stem cells(BCSC),and to explore its possible mechanism.Methods1.The establishment of CD44~+/CD24~-/low/low phenotype breast cancer stem cells,and analyze its biological characteristics:Culturing MCF-7 cell line by serum-free culture and complete medium,and observing its cell morphology and growth pattern by the inverted microscope.Culturing the microspheres by complete medium,and observing its cell morphology and growth pattern by the inverted microscope.The expression of stem cell surface molecular makers CD24,CD44 was observed by the inverted fluorescent microscope and the flow cytometry(FCM),the proportion of different subpopulation cells were detected by the flow cytometry.Comparing migration and invasion of MCF-7 and BCSC by transwell.2.The possible mechanism of salinomycin affect migration and invasion of CD44~+/CD24~-/low/low phenotype breast cancer stem cells:Culturing CD44~+/CD24~-/low/low subpopulation cells and MCF-7 by salinomycin,detecting their inhibition rate,and observing its cell morphology by the inverted microscope.To select the concentration under IC50 about cell apoptosis of CD44~+/CD24~-/low/low subpopulation cells through MTT.Culturing CD44~+/CD24~-/low/low subpopulation cells by the selecting concentration salino-mycin,to detect migration and invasion of breast cancer stem cells by transwell.The expressions of TGFβ1,Smad2,Smad3,p-Smad2,p-Smad3,MMP-2,MMP-9 were detected by Western blot.Results1.The cell line cultured by serum-free culture grew in the form of microspheres of different sizes suspending in the medium,the cell line cultured by comlete medium grew in the form of monolayer adherent growth.There was no obvious difference in the expression of stem cell surface molecular makers CD44 between the suspended microspheres and MCF-7,but there was distinct decrease in the expression of CD24.The proportion of CD44~+/CD24~-/low/low phenotype cells in the suspended microspheres and the MCF-7 was87.6%±2.7%and 19.28%±2.57%,respectively(P<0.05),the proportion of CD44~+/CD24~+phenotype cells was 11.23%±2.94%and 79.75%±2.92%,respectively(P<0.05).The migration and invasion of BCSC were significantly higher than MCF-7(P<0.05).2.After culturing CD44~+/CD24~-/low/low subpopulation cells by salinomycin,the micr-ophers gradually detached and became fragment.The MTT assay indicated that salin-omycin induced the apoptosis of BCSC and MCF-7 in a dose-dependent manner,its apoptosis significantly intensifed with the increasing of the concentration(P<0.05).The concentration IC50 of salinomycin at 24 h was 7.63±0.07μmol/L.The selecting concen-trations of salinomycin under IC50 were 1,3,5,7μmol/L.The migration and invasion of breast cancer stem cells were significantly reduced with the increasing of the concentration(P<0.05).The expressions of TGFβ1,Smad2,Smad3,p-Smad2,p-Smad3,MMP-2,MMP-9 were significantly decreased with the increasing of the concentration(P<0.05).Conclusions1.Serum-free culture could change the cell morphology and growth pattern of MCF-7,induce the expression of CD24,and increase the migration and invasion.CD44~+/CD24~-/low/low breast cancer stem cells can be effectively enriched by the serum-free culture technology.2.The migration and invasion of CD44~+/CD24~-/low/low breast cancer stem cells could significantly reduce with the increasing of the concentration.Salinomycin maybe reduce migration and invasion of CD44~+/CD24~-/low/low phenotype breast cancer stem cells by suppres-sing MMP-2/9 expression through TGFβ1/Smad pathway.