Chu et al: Imatinib mesylate (Gleevec) inhibits head and neck squamous cell carcinoma siRNA or Imatinib at 6ÌM for 24 hours. Scramble siRNA was used as a negative control. After incubation with Imatinib or Akt siRNA, cells were harvested and centrifuged at 2,000 rpm. Protein was extracted from cells using lysis buffer containing protease inhibitor and was separated using 10% NuPAGE® Novex Bis-Tris Gels and blotted onto a nitrocellulose membrane. The membrane was probed using anti-p-Akt antibody (1:100; Cell signaling Technology), anti-Akt1 antibody (1:100; Santa Cruz Biotechnology), and anti-!-actin antibody (1:5,000; SigmaAldrich Inc.), followed by 1-hour room temperature incubation with the following secondary antibodies: anti-mouse antibody (1:5,000) for p-Akt; anti-goat antibody (1:10,000) for Akt1; and anti-mouse antibody for actin (1:2,000). Positive protein signals were visualized with the enhanced chemiluminescence detection system.
By Western Blot analysis, human HNSCC cell lines, UMSCC10B, HN12, and HN30, were shown to express PDGFR-", PDGFR-!, c-Kit, and c-Abl (Figure 1). Exposure of HNSCC cells to 6 µM Imatinib for 24 hours has no apparent effect on the quantitative expressions of these PTKs.
B. Inhibition of cell growth by Imatinib at clinically relevant concentrations The effect of Imatinib on three HNSCC cell lines was measured by means of clonogenic survival analysis. A near-complete growth inhibition in HN12 and HN30 cells was observed when treated with 3 µM Imatinib, whereas UMSCC10B cells exhibited a similar response at a concentration of 10 µM (Figure 2).
III. Results A. Expression of TKs in HNSCC cell lines
Figure 1. Western blot analysis of PDGFR-", PDGFR-!, c-Kit, and cAbl in UMSCC10B, HN12, and HN30 cells. The protein tyrosine kinases were present in all three cell lines, but their expression was not changed after 24 hours of Imatinib treatment at 6 µM.
Figure 2. Clonogenic assay in three head and neck squamous cell carcinoma (HNSCC) cell lines. Each cells line was exposed to control (DMSO) or increasing concentration of Imatinib (0, 0.5, 1, 3, 6, and 10 µM) and stained eight days after seeding. The darker staining spots represented viable cells. The viability of UMSCC10B cell ( A) was grossly inhibited only at the dose of 10 µM Imatinib. Cell viability of HN12 cells (B) and HN30 (C) cells were markedly inhibited at the dose of 3 µM Imatinib. Each assay condition was performed in triplicate.
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