NPS-1034

Cell experiment:

To perform the MTT assay, cells (0.5×104/well) are plated in 96-well sterile plastic plates and allowed to attach overnight. Cells are exposed to varying doses of NPS-1034 in medium containing 1% FBS. After 72 hours, 15 μL of MTT solution (5 mg/mL) is added to each well and plates are incubated for 4 hours. Crystalline formazan is solubilized with 100 μL of a 10% (w/v) SDS solution for 24 hours. Absorbance at 595 nm is read spectrophotometrically using a microplate reader[1].

Animal experiment:

Female severe combined immunodeficiency (SCID) mice (17 to 20 g, 6 weeks of age) are used. Tumors are grown by implanting 5×106 cells in Matrigel into the mouse flanks. Treatment of 5 mice per group is started when the tumors have reached a volume of 50 to 100 mm3 with vehicle control or NPS-1034 (10 mg/kg, 5 days a week). NPS-1034 is administered orally. Treatment is stopped at the indicated day and mice are followed-up for tumor recurrence. To measure tumor size, the length (L) and width (W) of the tumor are measured with calipers, and tumor volume (TV) is calculated as TV=(L×W2)/2. Immunohistochemical staining is performed using a specific primary antibody, the EnVision Plus staining kit, and the APO-Direct terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay kit, according to the suppliers' instructions. Quantitative analysis of section staining is performed by counting immunopositive cells in 5 arbitrarily selected fields at ×40 magnification[1].

References:

[1]. Rho JK, et al. MET and AXL inhibitor NPS-1034 exerts efficacy against lung cancer cells resistant to EGFR kinase inhibitors because of MET or AXL activation. Cancer Res. 2014 Jan 1;74(1):253-62.
[2]. Shin JS, et al. NPS-1034, a novel MET inhibitor, inhibits the activated MET receptor and its constitutively active mutants. Invest New Drugs. 2014 Jun;32(3):389-99.