In vitro activity: In vitro studies indicate that E7050 potently inhibits phosphorylation of both c-Met and VEGFR-2. E7050 also potently represses the growth of both c-met amplified tumor cells and endothelial cells stimulated with either HGF or VEGF. E7050 circumvents resistance to all of the reversible, irreversible, and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevents the emergence of gefitinib-resistant HCC827 cells induced by continuous exposure to HGF.

Kinase Assay: The phosphorylation status of c-Met and VEGFR-2 is detected by Western blot analysis. For c-Met, MKN45 cells are incubated with a serial dilution of E7050 in complete medium at 37 °C for 2 h. For VEGFR-2, HUVEC are starved with human endothelial serum free medium containing 0.5% FBS for 24 h. Subsequently HUVEC are incubated with a serial dilution of E7050 for 1 h and then incubated with 20 ng/mL of human VEGF for 5 min. Cells are lysed by lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA [pH 8.0], 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride 1 mM sodium orthovanadate, 10 μg/mL aprotinin, 50 μg/mL leupeptin, and 1 μg/mL pepstatin A). The resected tumor samples are homogenized with lysis buffer containing 25 mM β-glycerophosphate and 0.5% (v/v) phosphatase inhibitor cocktail 2 at 4 °C. Cellular debris is removed by centrifugation at 17 860g for 20 min at 4 °C. Aliquots of the supernatants containing 5-20 μg of protein are subjected to SDS-PAGE under reducing conditions. The proteins are then transferred onto PVDF membranes, blocked with TBS containing 0.05% Tween-20 and either 5% skim milk or 5% BSA. The membranes are probed with the following antibodies: anti-c-Met polyclonal antibody (C-28) and anti-VEGFR-2 polyclonal antibody (C-20); mouse anti-phosphotyrosine clone 4G10; and anti-VEGFR-2 polyclonal antibody, anti-phospho-VEGFR-2 (Tyr996) polyclonal antibody, and anti-phospho-c-Met (Tyr1234/1235) polyclonal antibody. Detection is performed using a Super Signal enhanced chemiluminescence kit. Immunoreactive bands are visualized by chemiluminescence with an Image Master-VDS-CL detection system. The intensity of each band is measured by using an image analyzer. 

Cell Assay: Cells (1–3 × 103 cells/100 μL/well, MKN45, EBC-1, Hs746T, SNU-5, A549, SNU-1 and MKN74) are seeded on 96-well culture plates with various concentrations of E7050 and cultured for 3 days. Then, 10 μL of WST-8 reagent is added to each well, and absorbance is measured at 450 nm compared with a reference measurement at 660 nm using a MTP-500 microplate reader. HUVEC (2 × 103 cells/well) are cultured for 3 days in medium containing HGF (30 ng/mL), VEGF (20 ng/mL), or basic fibroblast growth factor (bFGF) (20 ng/mL) together with serially diluted E7050.