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Cancer Therapy: Preclinical

Dasatinib (BMS-354825) Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non–Small Cell Lung Cancer Cells

Faye M. Johnson, Babita Saigal, Moshe Talpaz and Nicholas J. Donato
Faye M. Johnson
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Babita Saigal
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Moshe Talpaz
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Nicholas J. Donato
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DOI: 10.1158/1078-0432.CCR-05-0757 Published October 2005
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    Fig. 1.

    Representative cytotoxicity using trypan blue exclusion after treatment with BMS-354825 for 72 hours at the given concentrations in Tu167 (A), JMAR (B), and H322 (C) cells in vitro. Columns, percentage of control cells (treated with DMSO alone) with cells that excluded trypan blue (alive) in white and cells that stained with trypan blue (dead) in black.

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    Fig. 2.

    Effect of BMS-354825 on the cell cycle. Cells (A, Tu167; B, JMAR; C, H322; D, A549) were treated for 0, 24, or 48 hours with 100 nmol/L BMS-354825, fixed, stained with propidium iodide, and analyzed by FACScan. Three separate experiments were done and combined. Bars, SD. There was no significant effect on control cells grown in parallel (data not shown).

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    Fig. 3.

    Effect of BMS-354825 on apoptosis. Cells were treated for 24 hours with 100 nmol/L BMS-354825, stained with Annexin V and propidium iodide, and analyzed by FACScan. Cells were then classified as alive with no apoptosis (i.e., propidium iodide negative, Annexin V negative), dead (i.e., propidium iodide positive), or apoptotic (i.e., Annexin V positive).

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    Fig. 4.

    Effect of BMS-354825 on cell morphology. Tu167 cells were plated on either collagen I (A) or laminin (B) for 24 hours and then treated with 100 nmol/L BMS-354825 for 24 hours. Serial photographs were taken, and representative fields are shown.

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    Fig. 5.

    Effect of BMS-354825 on cell migration. Cells were plated to confluence on tissue culture plastic. A single scratch was made in the confluent monolayer. The scratch was monitored and photographed every 6 hours (representative data, A). Cells that migrated into the scratch after 6 hours (12 hours for H322) were counted (B). The data represent four independent experiments; bars, SD. In all cases, when treated samples are compared with corresponding controls, P < 0.05.

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    Fig. 6.

    Effect of BMS-354825 on cell invasion. Cells were plated onto Matrigel-coated filters in a modified Boyden chamber. Cells were allowed to attach and spread for 16 hours before the addition of 10 nmol/L (for HNSCC cells) or 50 nmol/L (for NSCLC cells) BMS-354825 to the upper chamber and conditioned medium to the lower chamber. After 24 hours, cells still in the upper chamber were removed and cells that had invaded through the Matrigel were stained and counted. In parallel, cells treated identically were assessed for viability and cell number with trypan blue. The average number of cells per field is expressed as a percentage of the control after normalizing for cell number. The concentration of BMS-354825 was lowered in this experiment to minimize its effect on the absolute cell number. In all cases, when treated samples are compared with corresponding controls, P < 0.05.

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    Fig. 7.

    Baseline Src expression and inhibition by BMS-354825. Western blot analysis of untreated cells revealed that all cell lines expressed Src and activated Src (A). A dose-response effect of BMS-354825 on Src activation was observed in all cell lines by Western blot after treatment for 15 minutes. Src activation was inhibited partially by 10 nmol/L BMS-354825 and maximally at doses >100 nmol/L (two representative cell lines, B).

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    Fig. 8.

    The effect of BMS-354825 on cell signaling. Cells were treated with 100 nmol/L (A, B, and D) or 500 nmol/L (C) BMS-354825 for the indicated times, lysed, and analyzed by Western blotting (WB) or immunoprecipitation (IP) with the indicated antibodies. Cell lines are as indicated; Tu167 cells were used in (D).

Tables

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  • Table 1.

    Head and neck squamous cell carcinoma and NSCLC cell lines and their dasatinib IC50

    Cell lineCell typeGenetic backgroundIC50 (nmol/L)
    686LNHNSCC, lymph node, base tongue primaryWt p5330
    Tu167HNSCC, floor of mouth primaryMutant p5350
    MDA1986HNSCC, lymph node, base tongue primaryWt p5360
    Tu159HNSCC, tongue primaryND90
    JMARHNSCC, anoikis-resistant Tu167Mutant p53250
    Tu686HNSCC, base tongue primaryMutant p53540
    LN158HNSCC, lymph node from tonsil primaryND1,600
    358BHNSCC, buccal mucosal tumorND2,100
    H322NSCLC, adenocarcinomaMutant p53, wt K-ras, wt EGFR80
    H460NSCLC, large cellWt p53, wt K-ras, wt EGFR1,800
    H226NSCLC, squamous cellMutant p53, wt K-ras, wt EGFR10,000
    A549NSCLC, adenocarcinomaWt p53, mutant K-ras, wt EGFR10,000
    • Abbreviations: ND, not determined; wt, wild type.

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Clinical Cancer Research: 11 (19)
October 2005
Volume 11, Issue 19
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Dasatinib (BMS-354825) Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non–Small Cell Lung Cancer Cells
Faye M. Johnson, Babita Saigal, Moshe Talpaz and Nicholas J. Donato
Clin Cancer Res October 1 2005 (11) (19) 6924-6932; DOI: 10.1158/1078-0432.CCR-05-0757

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Dasatinib (BMS-354825) Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non–Small Cell Lung Cancer Cells
Faye M. Johnson, Babita Saigal, Moshe Talpaz and Nicholas J. Donato
Clin Cancer Res October 1 2005 (11) (19) 6924-6932; DOI: 10.1158/1078-0432.CCR-05-0757
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