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.

PICKHARD Anja;  PIONTEK Guido;  SEIDL C.;  KOPPING Samuel;  BLECHERT B.;  MISSLBECK Martin;  BROCKHOFF Gero;  BRUCHERTSEIFER Frank;  MORGENSTERN Alfred;  ESSLER M.; 

2014-01-16

ELSEVIER SCIENCE INC

JRC83180

0969-8051,   

http://www.sciencedirect.com/science/article/pii/S0969805113002096,    https://publications.jrc.ec.europa.eu/repository/handle/JRC83180,   

10.1016/j.nucmedbio.2013.09.010,