| Background and Objective: Breast cancer is the most commom female malignant tumor, is the leading cause of death in women around the world. Although the current treatment of breast cancer earlier had the very big enhancement, it is not benefiting most of the patients. The finding of doctrine that tumor stem cells are the origin of the tumor and the research of breast cancer stem cell provide new thoughts for the treatment of breast cancer. Breast cancer stem cells are the origin of breast cancer tissues, not only has the ability of infinite proliferation and self-renewal, also has the tumor tissue reconstruction and concurrent chemoradiotherapy resistance characteristics, is also the main causes responsible for failure and recurrence of the tumor radiation and chemotherapy. Recent years research on signaling pathways that related to breast cancer stem cells also provide a new basis for the treatment of breast cancer.Salinomycin is a new kind of antibiotic, originally found in Streptomyces albus, and has particular ring structure, belongs to a new generation of ionophore antibiotics. It can neutralize cation of cell, for the majority of coccidium and all kinds of gram positive bacteria have strong sterilization effect, and it is not easy to produce drug resistance and drug resistance, it is mainly used for the treatment of poultry coccidiosis. In recent years, researchers have found that salinomycin has antitumor effect, and in the aspect of kill breast cancer stem cells was significant than common chemotherapy. Salinomycin inhibit tumor cells through Wnt-βcatenin, ER, MARK and Akt/NF-k B signaling pathway. These signal transduction pathway also play an important role in breast cancer stem cells. The recent study of Notch, Hedgehog signal transduction pathway indicate that abnormal activation of these pathways has a close relationship with the incidence of breast cancer.This experiment mainly provides the basis for salinomycin treatment of breast cancer, especially the relationship between salinomycin inhibit breast cancer stem cells and the Notch, Hedgehog pathway.Methods: Human breast cancer cell line MCF-7 was cultured and treated with Sal in three replicates at concentrations of 0, 0.5, 1, 1.5umol/L and measured after 0, 24, 48, 72, 96 hours. To evaluate the inhibitory rate of cell proliferation, the concentration that inhibits 50% of the cell growth was tested by Cell Counting Assay Kit-8. Colony formation assay was observe the effects of salionmycin on breast cancer cell proliferation ability. Flow cytometry was used to detect the effect of salinomycin on the level cell cycle and apoptosis of breast cancer cells. Tumor cell migration and invasion were analyzed using Scratch assay and transwell assay. Potential target gene of Notch/Hedgehog signaling pathway-Notch1, Hes1, SMO, Gli1, these all tested by Western blotting and RT-PCR assay. Mammosphere formation assay, CD44+CD24-staining, RT-PCR and Western blotting, these were all used in analyzing the impact of salinomycin on breast cancer stem cells.Results: 1, The CCK8 assay verified: Sal depresses the growth of the MCF-7 cells, and in a dose-and time-dependent. Lower concentration(<0.5umol/L) of salinomycin inhibitory effect is not obvious, and the higher concentrations(>1umol/L)were significantly enhance its inhibition. The IC50 of 48 h is 1umol/L. The colony formation assay shows that Sal inhibited the formation ability of colony in dose dependent. Flow cytometry results show that salinomycin prevented MCF-7 cells from G1 to S phase period, reduced the percent of cells in S period(0, 0.5, 1.0,1.5μmol/L with the S-phase percentage 45.21%,33.23%,24.39%,11.85%)(P<0.05). The results of flow cytometry analysis of Annexin-V and propidium iodide(PI) stain showed a concentrations dependent enhancement of cell apoptosis by increasing concentration of Sal, compared with the control group(0umol/L,0.5umol/L,1umol/L,1.5umol/L with the percentage of apoptosis 1.5%,5.65%,17.71%,20.41%). 2, Flow cytometry and the mammosphere formation assay results show that salinomycin not only reduced CD44+CD24-cell population from 1.2% to 0.18% in human breast cancer cells, but also dereased the size and the number of the mammospheres, RT-PCR and Western blot analysis revealed that the salinomycin suppressed the expression of ALDH1 m RNA and protein levels in breast cancer cells in dose dependent. 3, The transwell chamber and scratch assay revealed that salinomycin inhibits the ability of invasion and migration of MCF-7 cells in a dose dependent. 4, By Western blotting and RT-PCR assay, we revealed that the expression of the key signaling molecules(Notch1,Hes1,SMO,Gli1) in the Hh and Notch were significantly downregulated compared to control.Conclusion: 1, Salinomycin depresses the proliferation of breast cancer cell, Cause the stagnation of the cell cycle, accelerates the levels of the apoptosis in MCF-7 cells 2, Salinomycin can significantly restrains the breast cancer stem cells 3, Sal inhibits breast cancer stem cells and the potential mechanism is inhibition of Notch/Hh signaling. |
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