Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways

<p>Abstract</p> <p>Background</p> <p>Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro.</p> <p>Methods</p> <p>Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis.</p> <p>Results</p> <p>In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis.</p> <p>Conclusion</p> <p>Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.</p

213Bi-anti-EGFR radioimmunoconjugates and X-ray irradiation trigger different cell death pathways in squamous cell carcinoma cells

Introduction: Treatment of patients with squamous cell carcinoma of head and neck is hampered by resistance of tumor cells to irradiation. Additional therapies enhancing the effect of X-ray irradiation could be beneficial. Antibodies targeting EGFR have been shown to improve the efficacy of radiation therapy. Therefore, we analyzed cytotoxicity of 213Bi-anti-EGFR immunoconjugates in combination with X-ray irradiation. Methods: The monoclonal anti-EGFR antibody matuzumab was coupled to CHX-A”-DTPA forming stable complexes with 213Bi. Cytotoxicity of X-ray radiation, of treatment with 213Bi-anti-EGFR-MAb or of a combined treatment regimen was assayed using cell proliferation and colony formation assays in UD-SCC5 cells. Key proteins of cell-cycle arrest and cell death were examined by Western blot analysis. Cell cycle analysis was performed by flow cytometry. DNA double-strand breaks were detected via γH2AX and quantified using Definiens™ software. Results: Irradiation with X-rays or treatment with 213Bi-anti-EGFR-MAb resulted in LD50 values of 12 Gy or 130 kBq/ml, respectively. Treatment with 37 kBq/ml of 213Bi-anti-EGFR-MAb or 2 Gy of X-rays had only little effect on colony formation of UD-SCC5 cells. In contrast, a combined treatment regimen (37 kBq/ml plus 2 Gy) significantly decreased colony formation and enhanced the formation of DNA double-strand breaks. As revealed by flow cytometry, radiation treatments caused accumulation of cells in the G0/G1 phase. Both treatment with 213Bi-anti-EGFR immunoconugates and application of the combined treatment regimen increased expression of genes involved in cell-cycle arrest and induction of apoptosis like p21/Waf, GADD45, Puma and Bax. Activation of these genes could rarely be observed after X-ray irradiation of cells. Conclusions: 213Bi-anti-EGFR-MAb enhances cytotoxicity of X-ray irradiation in UD-SCC5 cells most probably due to effective induction of DNA double-strand breaks. Upregulation of key proteins of cell-cycle arrest and cell death almost exclusively is due to 213Bi-anti-EGFR-MAb, thus demonstrating the cytotoxicity of α-emitters.JRC.E.5-Nuclear chemistr