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  • br Drug affinity responsive target

    2020-08-18


    2.9. Drug affinity responsive target stability (DARTS)
    Cells at 70–80% confluence were cultured in 100 mm2 culture plates. After washing once with ice-cold PBS, the 387334-31-8 were treated with ice-cold M-PER lysis buffer (Thermo Fisher Scientific Inc.) supple-mented with a protease inhibitor cocktail, 1 mM Na3VO4 (Sigma-Aldrich), and 1 mM NaF (Sigma-Aldrich). After collecting the cells with a scraper, the cells were incubated for 10 min at 4 °C. The cell lysates were centrifuged at 13,000 rpm for 10 min and the supernatant was diluted with M-PER lysis buffer to a final protein concentration of 2 mg/ ml. The protein lysates were mixed with 10× TNC buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 100 mM CaCl2) (Sigma-Aldrich). The lysates in 1× TNC buffer were split into 1.5 ml tubes and incubated with DMSO or Benp (100 μM) for 1 h at room temperature. Following the incubation, each sample was split into 50 μl of aliquots (50 μg in proteins) and proteolyzed in various concentrations of pronase (25, 50, 125, 250, and 500 ng) (Roche Diagnostics, 10165921001) for 10 min at room temperature. After 10 min, 2 μl of ice-cold 20× protease inhibitor cocktail was added to stop proteolysis and the samples were im-mediately placed on ice. Digestion was further stopped by adding 5× sample loading dye and boiling at 95 °C for 10 min. An equal portion of each sample was then loaded onto SDS-PAGE gels for Western blotting.
    2.10. Cellular thermal shift assay (CETSA)
    For CETSA with cell lysates, AsPC-1 cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.2% NP-40, 5% gly-cerol, 1.5 mM MgCl2, 25 mM NaF, 1 mM Na3VO4, and 1× protease inhibitor cocktail). After centrifugation, the lysates were incubated with DMSO or various concentrations of Benp (from 1 nM to 1 mM) for 1 h at room temperature. The lysates were aliquoted into 0.2 ml PCR tubes and heated for 5 min at the indicated temperature in a PCR machine (Applied Biosystems, Inc., Foster City, CA, USA). The precipitated proteins were separated from the soluble fraction by centrifugation and equal portions of the supernatants were loaded onto SDS-PAGE gels for Western blotting.
    2.11. Knockdown and overexpression of target genes
    Small interfering RNAs (siRNAs) against each gene were pre-in-cubated for 20 min in serum-free Opti-MEM medium (Gibco) containing
    A
    C
    DMSO
    Benp
    D
    Migrated cells (% of control)
    B
    viability
    Benproperine
    CFPAC-1
    E
    control)
    cells *
    Migrated **
    Invaded cells (% of control) 
    Fig. 1. Inhibition of cancer cell migration and invasion by Benproperine. (A) Structure of benproperine (Benp). (B) Dose-dependent changes in cell viability for 24 h, as measured using WST-1 (n = 3). (C and D) Representative images and quantification of the migrated cells that were treated with DMSO or Benp (10 μM) for 24 h (n = 3). Scale bars, 100 μm. (E) Cell migration assay of the indicated cells that were treated with various concentrations of Benp for 24 h (n = 3). (F) Cell invasion assay of the indicated cells that were treated with various concentrations of Benp for 24 h (n = 3). The data represent means ± s.d.; comparisons were performed with t-tests (two groups); *P < 0.05, **P < 0.01, ***P < 0.001.
    the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA). RPMI 1640 or DMEM medium containing 10% FBS was added 6 h after incubation. After 48 h, the transfected cells were collected and used for the experiments described below. The sequences 
    A Vehicle Benp (50 mg/kg)
    Pancreas
    Spleen
    Liver
    Kidney
    Colon
    B
    Tumor weight (mg) 
    C
    % of initial body weight 
    50 Vehicleecontrol
    Days
    D
    Day
    photons/s/sr
    Benp
    Benp
    Days 
    E
    Day
    Benp
    Days 
    F
    Benp
    Benp
    flux
    Photon 50
    Days
    Fig. 2. Inhibition of cancer cell metastasis by Benproperine. (A) Suppression of pancreatic tumor growth and metastasis by Benp in an orthotopic mouse model. Bioluminescence images of diverse organs from luciferase-expressing AsPC-1 cells including the pancreas, spleen, liver, kidney, and colon (n = 8 per group). (B) Quantification of photon flux in primary and metastatic tumor on day 27 (left) and the pancreatic tumors weight on day 27 (right). V.C, Vehicle. (C) Evaluation of the body weight of each mouse (n = 10 per group) at the indicated time points. (D) Inhibition of pancreatic cancer cell metastasis to the lungs by Benp in the lung metastasis model. Top, Representative bioluminescence images from luciferase-expressing AsPC-1 cells in the whole body (n = 6 per group). Bottom, Quantification of photon flux in the lungs at the indicated time points. (E and F) Inhibition of colon cancer cell metastasis to the liver by Benp in the liver metastasis model. Top, Representative bioluminescence images from luciferase-expressing HCT-116 (E) and DLD-1 (F) cells in the whole body (n = 8 per group). Bottom, Quantification of photon flux in the livers at the indicated time points. The color scale indicates radiance (×102 photons/s/sr). The data represent means ± s.d.; comparisons were performed with t-tests (two groups); *P < 0.05, **P < 0.01.