| ObjectiveEstablish the model of breast cancer stem cells in vitro by culturing the human breastcancer cells MCF-7in serum-free medium(SFM), and analyze their biologicalcharacteristics. Discuss the effect of salinomycin on cell proliferation and apoptosis inhuman breast cancer stem cells, and analyze the molecular mechanism of apoptosis byinhibiting the Wnt/β-catenin signaling pathway in breast cancer stem cells.Methods1. Establishment of the breast cancer stem cell model in vitro: Cultured the breastcancer MCF-7cells in serum-free media(SFM), and observed the changes on cellmorphology and growth mode with inverted microscope. Immunofluorescence stained cellsurface antigens CD44and CD24, and observed the expression of cell surface molecularmarker CD44and CD24using fluorescence microscope. At the same time, analyzed thecell cycle changes and different subsets of cells using flow cytometry, then detected theenrichment efficiency of the breast cancer stem cells.2. Mechanism of breast cancer microsphere cells apoptosis induced by salinomycin:MTT assay was used to detect the effect of salinomycin on the proliferation of breastcancer microsphere cells. Inverted microscope was used to observe apoptotic morphologyand FITC-Annexin V/PI apoptosis detection kit was used to investigate apoptosis in thebreast cancer microsphere cells. PI staining assay was used to determine the effects ofsalinomycin on breast cancer microspheres cell cycle. The levels of proteins involved incell apoptosis were evaluated by Western Blotting assay.Results 1. In the serum-free medium supplemented with growth factors, the suspension cellswere free state and ranged in size microspheres way to grow. Serum-free media couldinduce CD44+/CD24+breast cancer cells to be the CD44+/CD24-/lowcells. Compared to theadherent cells, the number of cells in S phase reduced and the cells in G1phase increased,that is, the cell cycle progression was arrested at G1phase. The expression of CD44wasno significant differences in the cells and the expression of CD24was apparently decreasedin the suspension cells. The proportion of CD44+/CD24-/lowcells proportion in suspensioncells and adherent cells were87.0±0.43%and40.1±1.05%, respectively.2. MTT assay indicated that salinomycin inhibited the proliferation of breast cancermicrosphere cells in a dose-and time-dependent manner, and the inhibition effect wasenhanced as the drug concentration increased. IC50doses of salinomycin at24h was4.97±1.96μM. After salinomycin treatment of breast cancer microsphere cells, mor-phological changes of apoptosis were observed under the inverted microscope, the micro-spheres gradually detached, the cells became edema and some of them were shrinkage intofragments. FITC-Annexin V/PI double-labeled assay showed that the apoptosis ratesignificantly increased as the concentration of salinomycin increased. PI staining assayshowed that the cells apoptosis induced by salinomycin was probably accompanied by cellcycle arrest. Western blotting analysis indicated that, as the drug concentration increased,the levels of cleaved PARP were significantly increased(P<0.05). The expression of Bcl-2was down-regulated after salinomycin treatment, whereas the levels of Bax wereup-regulated(P<0.05). Treatment with salinomycin also could block the Wnt/β-cateninsignaling pathway, and then the related protein levels were decreased(P<0.05).Conclusion1. Serum-free media culture method could make the breast cancer MCF-7cells todedifferentiate and transformed the cell cycle from the S phase to G0/G1phase, thenenriched CD44+/CD24-/lowbreast cancer stem cells.2. Salinomycin inhibited the cancer cells proliferation significantly and inducedcaspase-dependent apoptosis on breast cancer stem cells. The key regulators includedBcl-2, caspase-3, and caspase-9. Inhibitory effect of salinomycin on Wnt/β-cateninsignaling pathway also regulated the apoptotic progression of the breast cancer stem cells. |
