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  • br In the present study we validated the


    In the present study, we validated the anti-proliferative and colony formation inhibiting activities of curcumin in the breast cancer cell
    lines, MCF-7 and MDA-MB-231. Curcumin suppressed the migration and invasion of MDA-MB-231 cells. The CD44+CD24−/low subpopula-
    tion was elevated in mammospheres when compared with adherent cells. Further study showed that curcumin inhibited mammosphere formation and differentiation abilities. Moreover, curcumin down-regulated the mRNA expression of Vimentin, Fibronectin, and β-ca-tenin, and up-regulated E-cadherin mRNA expression levels. Western blot analysis revealed that curcumin decreased the protein expression of stem cell genes including Oct4, Nanog and Sox2. Our results suggest that curcumin may inhibit cell migration and invasion by resisting CSC-like characters and the EMT process. These data indicate that curcumin may function as a type of anti-metastasis agent for breast cancer.
    Materials and methods
    Cell culture and curcumin treatment
    Human breast cancer cell lines MCF-7 (less flexible, non-metastatic, and epithelium-like breast cancer cell line with ER positive) and MDA-MB-231 (more flexible, metastatic, aggressive, and mesenchymal-like breast cancer cell line with ER/PR/HER2 negative) were chosen as the two representative cell lines which were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were maintained in a monolayer culture in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in an incubator at 37°C in 5% CO2. Curcumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; purity >99.5%) was added to the culture separately to treat the adherent cells and mammospheres. Cisplatin (Sigma-Aldrich; Merck KGaA) was included as a positive control and cells treated with dimethyl sulfoxide (DMSO) only were used as a control (vehicle solvent). Each assay was repeated at least three times and statistically significant differences were ana-lyzed.
    Cell viability
    Cell viability in cells treated with curcumin was determined by Cell Counting Kit-8 (CCK8) assay. MCF-7 and MDA-MB-231 cells obtained from the exponential phase of growth were separately digested with 0.25% trypsin-ethylene diamine tetraacetic Anidulafungin (EDTA) (Gibco; Thermo Fisher Scientific, Inc.) and viable single cell suspensions were Anidulafungin collected by centrifugation at 800 rpm for 5 min. The suspended cells were seeded into 96-well plates at a density of 3000 cells/well. Three replicates were set for each group. After treatment with DMSO, Cisplatin and varying concentrations of curcumin for 24 and 48 h, 10 μl CCK-8 solution (EnoGeneCell Counting Kit-8; EnoGene Biotech Co., Ltd., New York, NY, USA; cat. no. EG20170721) was added to each well, and cultured for 3 h at 37°C in an incubator. The absorbance of each well was measured using a PerkinElmer EnSpire Reader at 450 nm (Perkin Elmer, Inc., Waltham, MA, USA). The cell viability of the treated cells was expressed relative to that of the cells treated with DMSO only (relative viability). All values are presented as the mean ± standard deviation of at least triplicate samples.
    Colony formation assay
    MCF-7 and MDA-MB-231 cells were harvested at 24 h after treat-ment with Cisplatin and curcumin. All of the cells were resuspended in DMEM supplemented with 10% FBS and seeded in 6-well plates at a density of 400 cells/well. After 14 days of culture under standard conditions, the colonies on the plates were fixed with 4%
    paraformaldehyde for 30 min and stained with 0.1% Gimsa for 30 min. The clones were photographed and cell colonies greater than 100 cells were counted.
    Transwell assay
    The MDA-MB-231 cell line was chosen for the cell invasion assay due to its high metastatic potential. Cell invasion was assessed by using the Matrigel invasion system. Transwell chambers (8 μm pore size, polycarbonate filters, 6.5 mm diameter; Corning Costar) in 24-well plates were coated with 100 μl of polymerized Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). To assess the level of invasion in the presence of curcumin, cells were placed into the upper chamber with medium that contained Cisplatin and curcumin, respectively. After incubation at 37°C for 48 h, the invaded cells in the lower chamber were obtained by staining and counted.
    Scratch wound healing assay
    MDA-MB-231 cells ( 5 × 105 cells/well) were seeded in growth medium in 6-well plates. After 24 h to reach complete confluency, a scratch wound was created with a 10 μl pipette tip on confluent cells in the center of the plate as the starting point. Cells were treated with Cisplatin and curcumin separately. Migration and cell movement throughout the wound area was observed with an inverted optical microscope (Carl Zeiss AG, Oberkochen, Germany) and imaged using a camera (SonyCyber-shot) attached to the microscope at x100 magnifi-cation at 12 and 24 h. The “healing” of this wound gap by cell migration and growth towards the center of the gap was quantitated using ima-ging software (ImageJ; National Institutes of Health, Bethesda, MD, USA). The gap size at the starting point was set to 100% and the per-centage of wound closure was calculated at 12 and 24 h after image analysis.