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  • br Statistical analysis br Statistical analyses were


    Statistical analysis
    Statistical analyses were performed using Student's t-test in Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA).
    Fig. 1. Cell viability and colony formation ability are inhibited by cur-cumin. Cell viability was determined by a CCK8 assay in (A) MCF-7 and
    The results were presented as the mean ± standard deviation of tri-plicate experiments and p < 0.05 was considered to indicate a statis-tically significant difference.
    Effects of curcumin on cell viability
    To assess the effects of curcumin on cancer cell viability, MCF-7 and MDA-MB-231 ABT-263 (Navitoclax) were grown in 96-well plates and treated with 
    curcumin (10, 15, 20, 25, 30, 35, and 40 µM). The well-known cancer inhibitor Cisplatin (3 µg/ml) was used as the positive control. Cell viability was determined by a CCK8 assay and the results were ex-pressed as the percentage relative to control (cells exposed to DMSO). The results demonstrated that in comparison with the cells treated with DMSO only, treatment with curcumin reduced the cell viability. Treatment with curcumin at 20 µM (Cur20) for 24 and 48 h exerted an inhibitory effect on the cell viability of both types of tumor cells (Fig. 1A and B). Moreover, treatment with curcumin at 15 µM (Cur15) for 24 and 48 h had marked inhibitory effects on the cell viability of
    Fig. 2. Curcumin suppresses MDA-MB-
    MDA-MB-231 cells, with greater effects observed in MDA-MB-231 cells when compared with MCF-7 cells. In addition, Cisplatin exhibited great inhibitory effects in both types of breast cancer cells. MDA-MB-231 cells were more sensitive when compared with MCF-7 cells. These results indicate that curcumin has significant inhibitory effects on breast cancer cells. 
    Colony formation ability is inhibited by curcumin
    In order to assess the survival and proliferative ability of the MCF-7 and MDA-MB-231 cell lines following treatment with curcumin (15, 20, 25, and 30 µM), colony formation assays were conducted. Representative stained colony plates are presented in Fig. 1C; also
    Fig. 3. Mammosphere formation ability is affected by curcumin. (A) Mammosphere formation in the MCF-7 cell line was observed. (B) The number of MCF-7 mammospheres. (C) Mammosphere formation in the MDA-MB-231 cell line was observed. (D) The number of MDA-MB-231 mammospheres. The number of multi-cellular tumor spheres (>20 cells) was counted in each well. p < 0.01 and p < 0.001 vs. DMSO. Cur, curcumin.
    depicted in Fig. 1D is a plot showing the colony formation rate for MCF-7 and MDA-MB-231 cells. MDA-MB-231 and MCF-7 cell colony num-bers were reduced following treatment with curcumin when compared with the DMSO control groups. Cisplatin also exhibited similar in-hibitory effects with curcumin at 15 µM (Cur15) in MCF-7 cells and curcumin at 30 µM (Cur30) in MDA-MB-231 cells (Fig. 1D). In MDA-MB-231 cells, the colony numbers were lower when compared to MCF-7 cells.
    Curcumin suppresses the MDA-MB-231 cell invasion and migration
    MDA-MB-231 is a highly metastatic cancer cell line. To study the effects of curcumin on cell motility, transwell chamber assays were employed to determine the effect of curcumin on the invasion cap-ability of MDA-MB-231 cells. Cells treated with curcumin (15, 20, 25, and 30 µM) exhibited a dose-dependent decline in the number of 
    invading cells (Fig. 2A and B). Scratch wound assays also revealed that curcumin impaired wound closure when cells were treated for 24 h (Fig. 2C and D). The consistent results showed that curcumin strongly suppressed the motility of MDA-MB-231 cells, and indicated that cur-cumin had anti-invasion and anti-migration effects on cancer cells.
    Mammosphere formation and differentiation abilities are affected by curcumin
    Mammosphere culture with SFM has been widely used for the en-richment of BCSCs. The present study cultured mammospheres of MCF-7 and MDA-MB-231 cells using SFM for 7 days, then the mammospheres were digested and passaged to the second generation for another 7 days. The third generation of mammospheres were treated with DMSO, Cisplatin and curcumin (15, 20, 25, and 30 µM) for 4 days, respectively. The number of multi-cellular tumor spheres (>20 cells) was counted.
    Fig. 4. Mammosphere differentiation capability is affected by curcumin. (A) In the MCF-7 and MDA-MB-231 cell lines, including their adherent cells, mammospheres and differentiated cells were observed. (B) The differentiated cells of MCF-7 and MDA-MB-231 mammospheres treated with Cisplatin, curcumin (15, 20, 25, and 30 µM) and DMSO were also observed. Cur, curcumin.
    Curcumin was revealed to reduce the number and size of both MCF-7 and MDA-MB-231-derived mammospheres. This suggested that cur-cumin may effectively inhibit mammosphere formation in the two types of breast cancer cells investigated (Fig. 3).