Combined targeted inhibition of EGFR tyrosine kinase activity and MEK-1 in human colon cancer cells

Abstract

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Background Both high expression of the EGFR and abnormal activation of downstream signaling, including the Ras/Raf MAP kinase cascade, have been demonstrated in uncontrolled cell proliferation in colorectal cancer (CRC) and other human tumors. For these reasons, the targeted blockade of the EGFR-driven autocrine pathway by small-molecule inhibitors has been proposed as a means of preventing human tumor growth. In this study, using human CRC cell lines, we assessed the in vitro antitumor activities of the combination of ZD6474, an orally active agent that selectively inhibits VEGFR/EGFR tyrosine kinase activity, and UO126, a selective inhibitor of the MEK-1 component of the MAPK pathway. Materials and Methods The antiproliferative effects of 48- and 72-h exposure to ZD6474 and UO126 (as single and combined agents) against human HCT 116 (p53 wt; ras mu) and HT-29 (p53 mu; ras wt) CRC cell lines were determined in separate experiments using a standard MTT assay. Combination effects were analyzed by determining combination index (CI) values using isobolographic methods. Cell-cycle distribution and apoptosis were both quantitated by flow cytometry. Target proteins and phosphorylation of downstream effectors of EGFR were assessed by both Western blot and flow cytometric intracellular staining techniques. Results In both HT29 and HCT116 cells, MTT studies showed that combined drug exposure (0.5μM UO126 and 5μM ZD6474) for either 48 or 72 h resulted in potent synergy (CI values <0.1) for inhibiting proliferation. HT29 cells were notably more sensitive to the combined drug effects on proliferation than HCT116 cells. Cell apoptosis was increased by approximately 3-fold and 1.5-fold by UO126 and ZD6474, respectively, after 72-h exposure, and the increase was more than additive (>6-fold) with combined drug exposure. Combination drug treatment for 72 h also resulted in a marked decrease in expression of the antiapoptotic protein, Bcl-xL. Following 72-h exposure to combination drug treatment, at concentrations that inhibited proliferation and increased apoptosis, there was an increase (∼20%) in the number of HT29 cells in G1, with no obvious effects observed in HCT116 cells. Conclusions These in vitro data indicate that combined targeting of the EGFR and MEK-1 with ZD6474 and UO126, respectively, results in a synergistic inhibition of cell proliferation and induction of apoptosis in human CRC cells. The synergy most likely results from the complementary mechanisms of action of these drugs. In vivo studies in mice are ongoing to investigate combined targeting with these inhibitors in xenograft models of CRC.