Inhibitory effect of a combined treatment of docetaxel with an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), ZD1839 and a cyclooxygenase-2 (Cox-2) inhibitor, celecoxib, on head and neck squamous cell carcinoma (HNSCC) cells

In our previous study, we observed synergistic or additive growth inhibitory effects of combined treatment of ZD1839 (Z) and celecoxib (C) on HNSCC in vitro (Proc. AACR 44:940a, 2003 (#4729)). Since single agent of Z or C had shown to enhance tumor response to cytotoxic agents, we postulated that the addition of docetaxel (D) to Z and C might show further increase of antitumor activity. To elucidate the effect of the triple drug combination of D, Z and C in the treatment of HNSCC, we performed cell growth inhibition and apoptosis assays in HNSCC in vitro. We first evaluated the growth inhibition rate of this combination in five HNSCC cell lines (Tu177, Tu212, Tu686, 686LN, and 886LN) using a colorimetric method. We have carefully selected concentrations for each agent that achieve 10 ∼40% of growth inhibition for each cell line when used as single agents and used them for the combined treatment (Tu177, D (0.75nM), Z (0.5uM), C (25uM); Tu212, D (0.5nM), Z (0.5uM), C (20uM); Tu686, D (0.75nM), Z (0.1uM), C (25uM); 686LN, D (0.5nM), Z (5uM), C (25uM); 886LN, D (0.75nM), Z (10uM), C (25uM)). We treated each cell line with D, Z, C, or the combinations of DZ, DC and ZC or the triple combination of DZC for 72 hours. We analyzed data for D and ZC vs. DZC assuming a multiplicative model. Synergistic growth inhibition (defined by p<0.007, two-sided test with Bonferroni adjustment) was observed in three (Tu177, Tu212, 886LN) of five cell lines (Table). A multiplicative growth inhibitory effect was observed in the other two cell lines (Tu686 and 686LN). Apoptosis assay (Tunnel) of 886LN using the same concentrations as the growth inhibition assay illustrated that the DZC combination induced a higher rate of apoptosis than the ZC combination. To clarify the underlying mechanisms of the observed synergistic or multiplicative effects, we performed western blot analysis for signaling associated protein markers. The DZC combination showed a further decrease of phosphorylations of EGFR and ERK than the single or double combinations. These results indicate that an addition of D to ZC combination appears to show further cell growth inhibition through the augmentation of apoptosis and further blocking of EGFR and Cox-2-mediated pathways. The other signaling pathway related molecular markers will be also reported. (Supported by Aventis Pharmaceuticals and NIH U01 CA101244).