FIGURE 5.

Inhibition of cellular FGFR phosphorylation by ARQ 069 in Kato III gastric carcinoma cells. A, Kato III human gastric carcinoma cells were plated and treated for 2 h with the indicated concentrations of ARQ 069 and ARQ 068. Ten minutes after stimulation by keratinocyte growth factor, cells were harvested, lysed, and subjected to SDS-PAGE and Western blotting using an antibody to phosphorylated FGFR. An antibody to β-actin served as a loading control. Because this cell line is known to harbor predominantly FGFR2, with a very low abundance of FGFR1, FGFR3, and FGFR4 (data not shown), the signal generated by the anti-phosphorylated FGFR is considered to be largely phosphorylated FGFR2 (24). ARQ 069 inhibited FGFR phosphorylation with an IC₅₀ value of 9.7 μm, whereas ARQ 068 completely failed to inhibit FGFR phosphorylation at concentrations up to 60 μm. B, anti-proliferative activity of FGFR inhibitors in Kato III cells. Kato III gastric carcinoma cells were seeded in 96-well plates overnight (2,000 cells/well) in culture medium with 10% FBS. The next day, cells were treated with different concentrations of the indicated compounds for 72 h at 37 °C. Cells were then incubated and stained for 4 h with methane thiosulfonate (MTS) reagent (final concentration of 0.5 mg/ml) (Promega) per well, lysed, and color development was quantitated by spectrophotometry at λ = 450 nm. The concentration of kinase inhibitor required to inhibit 50% of cell growth (GI₅₀) was calculated using ExcelFit^TM. Although ARQ 069 showed anti-proliferative activity in this FGFR2-dependent cell line, ARQ 068 was a much weaker inhibitor.