Potentiation of the antitumor activity of bortezomib, a proteasome inhibitor, by the combination with epidermal growth factor receptor tyrosine kinase inhibitors in human cancer cell lines.

The proteasome is involved in the housekeeping of cellular proteins. It also plays a pivotal role in the turnover of several regulatory proteins, including key intracellular transduction molecules which are induced by activated cell membrane growth factor receptors. Cancer cells are more sensitive than normal cells to proteasome inhibition, indicating that the proteasome could be a valuable target for cancer therapy. In this study, we tested the effect of bortezomib (Velcade®), a specific proteasome inhibitor, alone and in combination with gefitinib, a selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), with ZD6474, a dual EGFR and vascular endothelial growth factor TKI and with cetuximab, a blocking anti-EGFR chimeric human-mouse monoclonal antibody. The antiproliferative activity of bortezomib alone (range doses 0.1nM - 100 nM) and in combination with the three EGFR-TKIs was evaluated in human lung (A549, GLC-82, Calu3), colon (GEO, HCT-15, HCT-116, HT-29), pancreatic (MiaPaca2), and esophageal (KYSE-30) cancer cell lines containing functional TGF-EGFR autocrine pathway, by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and soft agar colony formation assays. Combination effects were analyzed according to the Chou and Talalay method. Cell cycle distribution and apoptosis were quantified by flow cytometry in A549, Calu-3, HCT15, HCT116, after 6 h, 12 h, 24 h and 48 h of treatment with bortezomib (25 nM), alone and in combination with ZD6474 (0.5µM) and cetuximab (1µg/ml). Effects on protein expression were evaluated using western blotting techniques in the same cell lines, after 24 h of treatment with bortezomib alone and in combination with ZD6474 and cetuximab at the same doses. Bortezomib determined a dose-dependent growth inhibition in the nine-cancer cell line panel (IC50 values, range 6-42 nM), showing a significant synergistic antiproliferative effect alone and in combination with either gefitinib, cetuximab, or ZD6474 (Combination Index values, range 0.10-0.55). This effect was accompanied by a significant induction in apoptosis by the combined treatment in the four treated cancer cell lines. Western blot analyses demonstrated that in HCT-116 cell line bortezomib induced a reduction of total and phosphorylated (P)-EGFR expression, and an increase of P-MAPK and p27 expression, without changes of total and P-Akt, MAPK, Bcl-2, Bcl-xL, and p21 expression. In contrast, the combined treatment with bortezomib and each EGFR inhibitor caused an efficient suppression of P-EGFR, P-MAPK and P-Akt levels with a parallel significant increase of p27 protein expression. Our results provide the rationale to test the combination of a proteasome inhibitor with an EGFR-TKI in vivo experiments as multitargeted therapy for human cancers.