| Objective:To detect the enrichment of breast cancer stem cells induced by TGF-β1, and the effect on the metastasis, invasion, cell cycle regulation, drug resistance of breast cancer stem cells; To detect the killing effect of salinomycin on breast cancer stem cells and the effect of reversing epirubicin and docetacel resistance.Methods:The breast cancer stem cells from MCF7/ADM were enriched by mammosphere method cultured with or without TGF-β1 (lOng/ml) which labeled with SC and SC-T respectively. MCF7, MCF7/ADM, MDA-MB-231, SKBR3 cultured with or without TGF-β1 (10ng/ml) were labeled with ALDEFLOUR kit, the proportion of ALDH1 positive cells in four cell lines and SC, SC-T cells were detected with flow cytometry (n=3); Expression of p21, MMP1, SATB1, Vimentin, E-cadherin protein were detected with Q-RT-PCR and Western blot; The cell cycle of the six cells were detected with flow cytometry; The invasion ability of MCF7/ADM,SC and SC-T cells were detected with Transwell. The cell self-renewal ability of the three cells were detected with mammosphere formation method. The cell cycle of the three cells were detected with flow cytometry (n=3); The drug sensitivity of the three cells were detected with cell counting kit 8 (CCK8) method. The growth inhibition of salinomycin combined with epirubicin and/or docetaxel were detected with CCK8 method (n=3).Results:The proportion of ALDH1 positive cells in MCF7, MCF7/ADM, MDA-MB-231, SKBR3 were 1.49%,8.38%,1.53%,4.23%; The proportion of ALDH1 positive cells in MCF7, MCF7/ADM, SKBR3 which were cultured with TGF-β1 were 3.83%,10.06%, 6.32% respectively (n=3), which were obviously higher than the cell cultured without TGF-β1 (n=3, p＜0.05)); Compared with cells cultured without TGF-β1, there is G1 phase cell cycle arrest when MCF7, MCF7/ADM, MDA-MB-231, SKBR3 cells cultured with TGF-β1 (n=3, p＜0.05). The expression of p21 in the cell lines were significant higher than the cells cultured without with TGF-β1 (n=3,P＜0.05). TGF-β1 up-regulated the expression of p21, SATB1, MMP1, while down-regulated the expression of E-cadherin. TGF-β1 enhanced the mammosphere formation ability (p＜0.001), invasion ability of breast cancer stem cells (p＜0.05) and G1 phase arrest (p＜0.001). The IC50 value of epirubicin (EPI) and docetaxel (DOX) in MCF7 were 0.4ug/ml,0.2ug/ml respectively (n=3); the IC50 value of EPI and DOX in MCF7/ADM cell were 20ug/ml,0.6ug/ml respectively (n=3); while the IC50 value of EPI and DOX in SC cell were 50ug/ml, lug/ml respectively (n=3); the IC50 value of EPI and DOX in SC-T cell were 120ug/ml, 10ug/ml respectively (n=3). The IC50 value of salinomycin (SAL) in MCF7/ADM, SC, SC-T cells were 3±0.2ug/ml,8±0.5ug/ml, 9±0.2umol/ml respectively; The growth inhibition on SC and SC-T cells were lower than MCF7/ADM, with EPI, DOX and SAL respectively. Combined with two of EPI, DOX and SAL, the better growth inhibition effect was arised when DOX combined with SAL, p＜0.01; Combined with all three drugs, the growth inhibition were more than 70% on MCF7/ADM, SC and SC-T cells, p＞0.1.Conclusion:1. TGF-β1 could increase the proportion of breast cancer stem cells, and could up-regulate the expression of p21 mRNA and help the cells arrest in G0/G1 phase as well.2. TGF-β1 could up-regulate the expression of p21,MMP,SATB1,Vimentin, while down-regulate the expression of E-cadherin.3. TGF-β1 could enhance the enrichment of breast cancer stem cells. TGF-β1 could boost self-renewal and invasion ability of breast cancer stem cell, and induce the chemotherapy resistance, promote the expression of breast cancer stem cell marker.4. Salinomycin could kill breast cancer stem cells effectively. Salinomycin sensitizes breast cancer cells and breast cancer stem cells to the effect of epirubicin and docetaxel. |
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