Radiosensitization of Ras-mutated human tumor cells in vitro by the specific EGF receptor antagonist BIBX1382BS.

Abstract

To investigate the cellular and molecular consequences of antagonizing radiation-induced EGFR-activation in vitro. The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS on radiation sensitivity was determined after single- and fractionated-dose irradiation in human cell lines of bronchial carcinoma (A549), breast adeno-carcinoma (MDA-MB-231), pharyngeal squamous-cell carcinoma (FaDu), squamous-cell carcinoma of cervix (HTB-35) as well as normal (HSF-7) and transformed (HH4-DED) human skin fibroblasts. Applying immuno-precipitation and western blotting pattern of radiation-dependent activation of different components of EGFR-signaling after pre-treatment with and without BIBX1382BS or other tyrosine kinase inhibitors was analyzed. Autophosphorylation of EGFR which occurred 1-5 min after irradiation (IR, 2 Gy) or treatment with EGF (100 ng/ml) could be inhibited in all cells tested by pre-treatment with BIBX1382BS for 30 min. Combination of drug treatment with fractionated irradiation (4x2 Gy) led to a strong radiosensitizing effect in Ras-mutated A549 and MDA-MB-231 cells, but not in normal Ras presenting cell lines FaDu and HTB-35 or normal and transformed human skin fibroblasts. Both BIBX1382BS as well as the PI3 kinase inhibitor LY294002 led to a blockage (for A549 cells) or reduction (for FaDu cells) of radiation-induced P-AKT. In contrast to FaDu cells, treatment of A549 cells with LY294002 resulted in a significant decrease of post-irradiation survival of A549 cells. Furthermore, only in Ras-mutated cells, but not in normal Ras cells clonogenic survival and phosphorylation of AKT was sensitive to pre-treatment with TGF-alpha-neutralizing antibody indicating an important role of TGF-alpha in regulating radiation-induced EGFR signaling. Enhancement of radiation sensitivity by the specific EGFR-tyrosine kinase inhibitor BIBX1382BS is not generally achieved in human tumor cells, but depends most likely on the Ras genotype of the cell lines tested.