Nilotinib antitumor activity in GIST xenograft models.

<p>(A) Immunohistochemistry staining for KIT in xenograft lines established from human GISTs: GK1X, GK2X and GK3X. (B) Tumor tissue fragments (∼5 mm³) were transplanted s.c. into the backs of BALB/cSlc-<i>nu/nu</i> mice that were randomized into 3 groups (n = 6–8). Doses of 40 mg/kg/day of imatinib, nilotinib or pure water (control) were administered by oral gavage daily for 28 days. Tumor size was measured every two to three days. (C) Tumor growth inhibition (TGI) on the day of evaluation was calculated as the ratio of tumor volume on the evaluation day to that on day 1.</p

Quantitative phosphorylation analysis.

<p>Parental (GK1C and GK3C; red histograms) or imatinib-resistant GIST cell lines (GK1C-IR and GK3C-IR, blue histograms) were fixed and stained with anti phospho-KIT (Tyr719), anti phospho-PDGFRA (Tyr754), anti phospho-SRC (Tyr416), anti phospho-AKT (Ser473) and anti phospho-ERK1/2 (Thr202/Tyr204). Finally, cells were detected with Alexa Fluor 488 donkey anti-rabbit IgG antibody (Isotype control was reacted only with the secondary antibody). The MFI (mean of fluorescence intensity) values were calculated by FlowJo. GK1C: p-KIT = 3.21, p-PDGFRA = 10.3, p-SRC = 7.19, p-AKT = 20.3, p-ERK1/2 = 37.8. GK1C-IR: p-KIT = 3.30, p-PDGFRA = 12.8, p-SRC = 9.35, p-AKT = 20.5, p-ERK1/2 = 94.4. GK3C: p-KIT = 2.65, p-PDGFRA = 7.29, p-SRC = 5.35, p-AKT = 19.5, p-ERK1/2 = 32.2. GK3C-IR: p-KIT = 3.89, p-PDGFRA = 9.82, p-SRC = 8.31, p-AKT = 21.3, p-ERK1/2 = 115.</p

Antitumor activity of nilotinib on GIST and imatinib-resistant GIST cells.

<p>Cells were maintained in supplemented medium for 12 h, and then incubated with nilotinib (0∼100 µM) for 72 h. Cell viability was determined by comparing treated cells with the untreated control. Data are means of triplicates from a representative experiment.</p

Establishment of imatinib-resistant GIST cell lines.

<p>(A, B) Immunohistochemical assay of KIT expression in GK1C-IR and GK3C-IR as determined by staining with DAB (magnification, 400x). (C, D) GK1C and GK1C-IR cells, GK3C and GK3C-IR cells, 2.5×10³ cells (per well) were seeded into 96-well microplates in triplicate 12 h before treatment, and then exposed to different concentrations (0∼100 µM) of imatinib for 72 h. The percentage of cellular proliferation was gauged using the WST-8 method. Imatinib-resistant (IR) cells showed resistance to imatinib with IC₅₀ of 11.74±0.17 µM (<i>p<0.001</i>) or 41.37±1.07 µM (<i>p<0.001</i>). Data are presented as means ± SD and evaluated using Student's <i>t</i> test.</p