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  • br Materials and methods br Cell culture and

    2020-08-18


    2. Materials and methods
    2.1. Cell culture and cell proliferation assay
    All cell lines used in this study were originally obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). DLD-1, HCT-116 (human colon cancer), MDA-MB-231 (human breast cancer), A375P (human melanoma), AsPC-1 (human pancreas cancer), and DU145 (human prostate cancer) cells were maintained in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA). CFPAC-1 (human pancreatic cancer), PC-3 (human prostate cancer), B16BL6 (mouse melanoma), and HFF (human fibroblast) cells were maintained in Dulbecco’s mod-ified Eagle’s medium (Gibco). MCF-10A (human mammary epithelial) cells were maintained in DMEM-F12 (Gibco). All culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS;  Biochemical Pharmacology 163 (2019) 46–59
    Gibco), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cell cultures were maintained in a 37 °C incubator under a humidified 1905453-18-0 with 5% CO2.
    The cells were seeded in 96-well plates in RPMI 1640 medium containing 10% FBS. After 24 h, the wells were replenished with fresh complete medium containing either a test compound or 0.1% DMSO. After incubation for 24–72 h, the cell proliferation reagent WST-1 (Roche Diagnostics, Indianapolis, IN, USA) was added to each well. The amount of WST-1 formazan produced was measured at 450 nm using an ELISA Reader (Bio-Rad, Hercules, CA, USA).
    2.2. Cell migration assay
    The cell migration assay was performed using Transwell inserts with an 8.0 μm pore size (BD Biosciences, San Jose, CA, USA). The cells (5 × 104 cells in 0.2 ml of serum-free medium) were added to the upper chamber. Various concentrations of chemicals in 0.5 ml of medium with 10% FBS were applied to the lower chamber. After 6–24 h, the migrated cells attached to the lower surface were stained with crystal violet (500 μl of 5 mg/mL crystal violet dissolved in 20% methanol) (Sigma-Aldrich) and allowed to incubate for 10 min. The membrane was then washed several times with PBS, and the cells that penetrated the filter were counted under a microscope (Nikon Eclipse TE300; Nikon, Tokyo, Japan).
    To identify small molecules that inhibit cancer cell migration, we used the previously established PRL-3-overexpressing DLD-1 (DLD-1 (PRL-3)) cells in our laboratory. We tested 719 compounds in pheno-type screening from existing drug library proprietary to our institute and commercially available drugs. DLD-1 (PRL-3) cells were plated in 24-well plates at a density of 5 × 104 cells. Compounds were tested at 10 μg/mL against the cells, and we found 16 potential compounds with more than 50% inhibition. The selected compounds were tested in four serial dilutions ranging from 1 μg/mL to 10 μg/mL and we finally se-lected benproperine, which inhibited the cell migration in a dose-de-pendent manner without having cytotoxic effects (data not shown).
    2.3. In vivo orthotopic xenograft assay and in vivo metastasis assay
    All animal work was performed in accordance with a protocol ap-proved by the Institutional Animal Care and Use Committee. For the in vivo orthotopic xenograft assay, AsPC-1 cells (9 × 105 cells/mouse) that stably expressed luciferase were directly injected into the pancreas of female BALB/c nude mice (6-week-old; Nara Biotech., Seoul, Republic of Korea). Benp was administered 5 days per week (50 mg/kg, oral gavage) starting on day 1. Tumor growth was monitored for 4 weeks by bioluminescence imaging of luciferase activity using a Photon Imager (Biospace Lab, Paris, France). For the bioluminescence images, the mice were injected intraperitoneally with D-luciferin (100 mg/kg in PBS; Gold Bio, St. Louis, MO, USA) 10 min before in vivo imaging and an-aesthetized with 2% isoflurane (Sigma-Aldrich). On day 27, the mice were sacrificed and examined for tumor growth in major organs in-cluding the pancreas, spleen, liver, kidney, and colon. Photon flux was measured by drawing a rectangular region (16 cm2) in the mouse ab-dominal region. The data are expressed as total photon flux (photons/s/ sr).
    For the lung metastasis assays, AsPC-1 cells (1 × 106 cells/mouse) that stably expressed luciferase were injected into the lateral tail vein of mice. The mice were imaged for luciferase activity immediately after the tail vein injection to confirm that the cancer cells were successfully xenografted. Benp was administered 5 days per week (50 mg/kg, oral gavage) starting on day 1 (the day after cell injection). For the liver metastasis assays, colon cancer cells (HCT-116 and DLD-1) that stably expressed luciferase were injected into the spleens of the mice (2 × 106 cells/mouse). On day 4, the spleen of each mouse was surgi-cally removed and the increase in metastasis was monitored by biolu-minescence imaging for luciferase activity using a Photon Imager