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  • After counting the mammospheres in

    2020-08-12

    After counting, the mammospheres in the different groups were transferred from ultra-low adherent 6-well culture plates to ordinary petri dishes and cultured in SSM for 4 days, respectively. The mam-mospheres were able to form monolayer Driselase which indicated that they had differentiation abilities. The number of differentiated cells de-creased following treatment with curcumin and Cisplatin when com-pared with the DMSO group. These results indicate that curcumin may effectively inhibit the differentiation of mammospheres derived from MCF-7 and MDA-MB-231 cells (Fig. 4A and B).
    Curcumin reduces the CD44+CD24− cell subpopulation of MCF-7 and MDA-MB-231 mammospheres
    mRNA expression is regulated by curcumin in mammospheres
    The mRNA levels of EMT markers, including E-cadherin, N-cad-herin, β-catenin, Vimentin, and Fibronectin were examined by RT-qPCR assay. Compared with the adherent cells, MDA-MB-231 cells derived from the mammosphere culture exhibited elevated mRNA levels of mesenchymal markers [N-cadherin (∼2.76-fold), β-catenin (∼1.57-fold), Vimentin (∼2.08-fold), and Fibronectin (∼2.74-fold)]. These results indicated there may have been a mesenchymal-like state in the SFM culture derived cells. After MDA-MB-231 mammospheres were treated with curcumin (30 µM), treatment reduced the mRNA levels of
    Vimentin, Fibronectin, and β-catenin in MDA-MB-231 mammospheres, while the E-cadherin mRNA level was up-regulated, suggesting that curcumin markedly inhibited the growth of mammospheres which may be related to the regulation of the EMT process (Fig. 6).
    Protein expression of stem cell genes are affected by curcumin
    To test the effect of curcumin on the protein level of stem cell genes, the present study detected Oct4, Nanog, and Sox2 levels using western blot analysis. The protein levels of Oct4, Nanog, and Sox2 were up-regulated in MCF-7 and MDA-MB-231 mammospheres. Curcumin (30 µM) decreased these proteins levels in both adherent cells and the mammospheres of MCF-7 and MDA-MB-231 cells (Fig. 7A and B). These results indicate that curcumin may inhibit stem cell properties in mammospheres.
    Discussion
    Curcumin is a polyphenolic compound with multiple pharmacolo-gical activities (Mimeault and Batra, 2011). Many studies have focused on its chemopreventive or anti-tumor effects on breast cancer (Beatrice 
    et al., 2007; Wang et al., 2014; Karunagaran et al., 2005). Recently, studies have reported that curcumin may have the potential efficacy to have anti-invasion and anti-migration activities in breast cancer (Charpentier et al., 2014; Kakarala et al., 2010; Chung et al., 2015). However, the underlying mechanism is unclear. Therefore, it is relevant to further determine if curcumin could affect CSC-like properties, the EMT process and to uncover the mechanisms of its anti-metastasis ef-fects.
    MCF-7 and MDA-MB-231 cells were selected to explore the potential anti-metastasis effect of treatment with curcumin according to their different characteristics (Zhang et al., al.,2012). Firstly, the present study carried out CCK-8 and colony formation ability assays with MCF-7 and MDA-MB-231 adherent cells. Curcumin was revealed to retard MCF-7 and MDA-MB-231 cell viability and proliferation in a dose- and time-dependent manner when compared with the untreated or DMSO treated control groups. When the concentration of curcumin reached 20 µM, it exerted an inhibitory effect on MCF-7 and MDA-MB-231 cells. Overall MDA-MB-231 cells were more sensitive to curcumin than MCF-7 cells.
    After this, the present study investigated the tumor invasive and migratory abilities of curcumin treated MDA-MB-231 cells with high
    Fig. 6. mRNA expression is regulated by curcumin in MDA-MB-231 mammospheres. mRNA level of the EMT marker genes (E-Cad, N-Cad, β-cat, Vim and FN1) in MDA-MB-231 mammospheres treated with curcumin (30 µM) and DMSO relative to MDA-MB-231 adherent cells (DMSO) as determined by reverse transcription-quantitative polymerase chain reaction. β-actin mRNA was used to normalize the variability in template loading. *p < 0.05 vs. DMSO in MDA-MB-231 MS. E-Cad, E-Cadherin; N-Cad, N-cadherin; β-cat, β-catenin; Vim, Vimentin; FN1, fibronectin; Cur, curcumin.
    metastatic potential. Transwell chamber and wound-healing assays were employed to determine cell invasive and migratory abilities. When cells were treated with curcumin (15, 20, 25, and 30 μM) for 24 h, there was a significant reduction in cell motility and invasion ability which illustrated that curcumin had anti-metastasis effects on high metastatic breast cancer cells.
    Moreover, a growing body of evidence has revealed that CSCs are the main source of recurrence and metastasis in a variety of human malignant tumors (He et al., 2015; Sampieri and Fodde, 2012). When the breast cancer adherent cells were cultured in SFM supplemented with EGF, bFGF, and B27 in ultra-low attachment plates, the subset of BCSCs survived and grew in the serum-free suspension conditions. These highly differentiated cells could not tolerate the SFM conditions and only a small proportion of non-differentiated cells could survive and proliferate in the form of suspension mammospheres. Therefore, the method of sphere culture has been widely used for the enrichment of CSCs. The special stem-like phenotype (CD44+ CD24−/low) would be increased in breast cancer mammospheres, which was also supported by our data.