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

IntroductionTreatment 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 may 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.MethodsThe 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 monoclonal antibodies (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.ResultsIrradiation with X-rays or treatment with ²¹³Bi-anti-EGFR-MAb resulted in median lethal dose (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 immunoconjugates and application of the combined treatment regimen triggered activation of genes of signaling pathways involved in cell-cycle arrest and induction of apoptosis like p21/Waf, GADD45, Puma and Bax, which were only marginally modulated by X-ray irradiation of cells.Conclusions²¹³Bi-anti-EGFR-MAb enhances cytotoxicity of X-ray irradiation in UD-SCC5 cells most probably due to effective induction of DNA double-strand breaks. Induction of genes involved in cell-cycle arrest and cell death is almost exclusively due to ²¹³Bi-anti-EGFR-MAb and seems to be independent of p53 function.