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  • br The present study employed the tumor

    2020-08-12


    The present study employed the tumor sphere formation culture for three generations to characterize the CSCs of MCF-7 and MDA-MB-
    231 cells. Cells with CD44+CD24−/low surface markers were highly enriched for stem-like mammospheres when compared with the same type of adherent cells. Flow cytometry analysis revealed that the percentage of BODIPY-Cholesterol with CD44+CD24− in tumor spheres increased markedly in MCF-7 (from 37.9% to 63.6%) and MDA-MB-231 cells (from 94.2% to 98.2%). The mRNA level of mesenchymal marker genes (N-cadherin, Vimentin, Fibronectin and β-catenin) and the protein expression of stem cell genes (Oct4, Nanog, and Sox2) were significantly higher in mammospheres when compared with MDA-MB-
    231 adherent cells which suggested that their stemness character and cell motility elevated correspondingly, also confirming that spheroid culture conditions induced stem-like properties and mesenchymal transition. These results provide new opportunities to achieve multi-cellular spheroids and may be acceptable for the study of metastatic processes in in vitro models. 
    When curcumin (15, 20, 25, and 30 µM) was used to treat the suspension spheres of both MCF-7 and MDA-MB-231 cells, the number of spheroids was markedly decreased. The results demonstrated that curcumin could inhibit the proliferation of suspension stem-like spheres. Furthermore, spheres of the third generation in the control group (treated with DMSO) cultured in SFM were prepared via re-petitive pipetting to generate a unicellular suspension and seeded in plates with SSM. Cells could still adhere to the plate walls and grow into a monolayer. After the adherent cells were digested with trypsin and cultured in SFM, mammospheres could form again. It was re-vealed that differentiation ability was not weakened in the spheres passaged in the control group. But when mammospheres were treated with curcumin, their induced differentiation ability was reduced in a concentration-dependent manner. Furthermore, the results revealed that the stem-like surface marker CD44+CD24− cell subpopulation was decreased following curcumin (15, 20, and 30 µM) treatment in both MCF-7 and MDA-MB-231 mammospheres, and curcumin at 30 µM had a stronger inhibiting ability than curcumin at 15 or 20 µM. The RT-qPCR assay demonstrated that curcumin (30 µM) could in-crease the expression of the epithelial marker E-cadherin, and de-crease the expression of mesenchymal markers in MDA-MB-231 mammospheres at the mRNA level. The protein expression of these stem cell genes were reduced treated with curcumin in both adherent cells and mammospheres in MCF-7 and MDA-MB-231 as determined by western blot analysis.
    The results of the present study demonstrated that curcumin can inhibit cell migration and invasion which may be correlated with cell resistance to CSC-like characters and the EMT process. To the best of our knowledge, the efficacy of curcumin in targeting mammospheres by inhibiting stem-like properties and regulating the EMT process has been reported for the first time. These data indicate that curcumin could function as a type of anti-metastasis agent for breast cancer and thus may be advantageous in preventing and treating tumor recurrence and metastasis.
    Acknowledgments
    The authors would like to thank the laboratory staff at Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation for performing FCM. We also want to express our sincere thanks to Dr. Yugang Wang for revising the paper.
    Funding
    Conflict of interest
    We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant fi-nancial support for this work that could have influenced its outcome.
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