Although combined treatment with docetaxel (D) and cisplatin (P) has shown antitumor activity in preclinical and clinical studies, clinical outcome of therapy with DP combination is far from being satisfactory in SCCHN. We hypothesize that the addition of Iressa (I) to DP combination may enhance cytotoxic effects. Therefore, we tested the cell inhibitory effects of combination of I and DP, focusing on treatment sequence and the underlying mechanisms. We tested the growth inhibitory effect of single (D, P, or I), double (DP, DI, or PI) and triple drug combination (DPI) (D 0.08nM∼20nM; P 0.2uM∼50uM; I 0.1uM∼30uM) in four SCCHN cell lines (Tu177, Tu212, Sqccy1 and 886LN) using sulforhodamine B assay. In all experiments, cells were exposed to the each drug treatment for 72 hrs. We assessed cell growth inhibitory effects using the method of Chou and Talalay [combination index (C.I.) indicated: synergy, statistically significantly less than 1.0; antagonism, statistically significantly greater than 1.0; additivity, not statistically significantly different from 1.0]. Two cell lines (Tu212 and Sqccy1) were used to elucidate the sequence dependent effect. Cells were simultaneously treated with D, P and I (DPI) or sequentially with DP followed by I, 12hr later (DP→I), or vice versa (I→DP). Apoptosis was measured by flow cytometry. Relevant protein markers were evaluated by Western blot. The inhibitory effects of I addition to DP were sequence dependent. Simultaneous treatment with DPI showed synergistic effects in 3 of 4 cell lines [Tu177, C.I. = 0.51∼0.63 (p<0.01); Tu212, C.I. =0.29∼0.52(p<0.001); Sqccy1, C.I. =0.49∼0.54 (p<0.04)] and additive effect in 1 cell line [886LN, C.I. = 1.0∼1.13] at multiple effect levels (IC25∼IC75). DP followed by I (DP→I) gave similar synergistic effects with simultaneous DPI treatment in both Tu212 and Sqccy1 cell lines. However, I followed by DP combination (I→DP) gave an antagonistic effect (C.I. = 1.42∼1.53) in Tu212 and a less synergistic effect (C.I=0.66∼0.71) in Sqccy1 cell line. Apoptosis induction was also sequence dependent; DPI or DP→I treatment showed significantly higher augmentation of apoptosis than I→DP (p=0.03). The observed synergistic inhibitory effects of DPI or DP→I were associated with decreased expression of Bcl-xl, Bcl-2 and survivin, reduced activation of AKT and ERK1/2 pathways and augmentation of p38 activation. We conclude that I treatment simultaneously with DP or DP followed by I synergistically enhances cell growth inhibition in SCCHN and are associated with promotion of apoptosis and inactivation of EGFR down stream signaling molecules. (Supported by Aventis Pharmaceuticals and NIH U01 CA101244).
[Proc Amer Assoc Cancer Res, Volume 46, 2005]