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  • br Corresponding author Department of Biochemistry and


    ∗ Corresponding author. Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene Street, Baltimore, MD 21201, USA
    cause SUM159 does not express estrogen receptor, it is clear that the inhibition of constitutive p-STAT3 (Y705) level and cell proliferation by
    bazedoxifene is not through modulation of the estrogen receptor target. Previous studies indicate that IL-6/GP130/STAT3 is one of the major cancer proliferation pathways in TNBC [11]. We also observed that bazedoxifene induced apoptosis and suppressed tumor growth of pan-creatic cancer cells; bazedoxifene combined with paclitaxel or gemci-tabine synergistically inhibited cell viability and cell migration of pancreatic cancer, rhabdomyosarcoma and medulloblastoma [12–14].
    Paclitaxel is considered one of the first line drugs in the treatment of breast cancer [15]. However, paclitaxel can cause Cell Counting Kit-8 side effects [15]. The main purpose of this study was to investigate the effect of improving drug efficacy and reducing the amount of paclitaxel dosage by com-bined administration with bazedoxifene, which may help reduce the side effects of paclitaxel.
    2. Materials and methods
    2.1. Cell lines and reagents
    Human estrogen receptor positive breast cancer cell lines MCF7, T47D, and BT474, human TNBC cell lines MDA-MB-231 and MDA-MB-468 and mouse TNBC 4T1 Cell Counting Kit-8 were purchased from ATCC (American Type Culture Collection, Manassas, VA). All cell lines were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/L glucose L-glu-tamine and sodium pyruvate (DMEM, Mediatech Inc. A Corning Subsidiary, Manassas, VA), except for T47D, which was cultured in RPMI-1640 medium (Mediatech Inc. A Corning Subsidiary, Manassas, VA), and all media were supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin/ streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt) and maintained in a humidified 37 °C incubator with 5% CO2.
    Reagents used in the present study were as follows: BZA/CEE was from Acesys Pharmatech (Fairfield, NJ, USA) and paclitaxel was from LC Laboratories (Woburn, MA). According to the instructions from the manufacturers, these drugs were dissolved in sterile dimethyl sulfoxide (DMSO, Sigma-Aldrich; Merck KGaA) to make a 20 mM stock solution and then stored at −20 °C. Additional reagents were 3-(4, 5-di-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA), crystal violet (Sigma-Aldrich; Merck KGaA), N, N-dimethylformamide (Fisher Scientific; Thermo Fisher Scientific, Inc., Waltham, MA), and lysis buffer (Cell Signaling Technology, Inc. Danvers, MA).
    2.2. MTT cell viability assay
    MCF7, T47D, BT474, MDA-MB-231, MDA-MB-468, and 4T1 breast cancer cells were seeded in 96-well microtiter plates in triplicate at a density of 3000 cells per well and grown overnight at 37 °C. The fol-lowing day, the cells were treated with bazedoxifene as indicated and incubated for 48 h. MCF7, MDA-MB-231 and 4T1 cells were also treated with bazedoxifene or paclitaxel or their combination as indicated and incubated for 48 h. Then, 25 μL MTT was added to each well and in-cubated for 4 h, followed by the addition of 150 μL N, N-di-methylformamide solubilization solution. The plates were placed on a shaker at room temperature overnight to allow complete lysis of the cells and read at 595 nm the following day. Experiments and data processing were performed as described previously [16]. Half-maximal inhibitory concentrations (IC50) were determined using Sigma Plot 9.0 Software (Systat Software, Inc., San Jose, CA). Combination index (CI) was performed using data obtained from MTT assay with CompuSyn software [17]. The CI values indicate a synergistic effect when < 1, an antagonistic effect when > 1, and an additive effect when equal to 1.  Cancer Letters 448 (2019) 11–19
    MCF7, BT474, MDA-MB-231, MDA-MB-468, and 4T1 breast cancer cells were seeded in 96-well plates in triplicate at a density of 5000 cells per well and incubated overnight at 37 °C. The next day, cells were treated with bazedoxifene, paclitaxel or their combination as indicated and incubated for 5 h. The caspase-3/7 activity was detected using Caspase-3/7 Fluorescence Assay Kit (Cayman, Ann Arbor, MI) ac-cording to the manufacturer's instructions.