title: ²¹³Bi-anti-EGFR radioimmunoconjugates and X-ray irradiation trigger different cell death pathways in squamous cell carcinoma cells.
abstract: BACKGROUND
  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 may be beneficial. Antibodies targeting EGFR have been shown to improve the efficacy of radiation therapy. Therefore, we analyzed cytotoxicity of (213)Bi-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 (213)Bi. Cytotoxicity of X-ray radiation, of treatment with (213)Bi-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.
  
  
  RESULTS
  Irradiation with X-rays or treatment with (213)Bi-anti-EGFR-MAb resulted in median lethal dose (LD50) values of 12 Gy or 130 kBq/mL, respectively. Treatment with 37 kBq/mL of (213)Bi-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 (213)Bi-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.
  
  
  CONCLUSIONS
  (213)Bi-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 (213)Bi-anti-EGFR-MAb and seems to be independent of p53 function.
authors.affiliation: Department of Otolaryngology Head and Neck Surgery, Technische Universität München, Munich, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Pickhard
authors.forename: Guido
authors.initials: G
authors.lastname: Piontek
authors.forename: Christof
authors.initials: C
authors.lastname: Seidl
authors.forename: Samuel
authors.initials: S
authors.lastname: Kopping
authors.forename: Birgit
authors.initials: B
authors.lastname: Blechert
authors.forename: Martin
authors.initials: M
authors.lastname: Mißlbeck
authors.forename: Gero
authors.initials: G
authors.lastname: Brockhoff
authors.forename: Frank
authors.initials: F
authors.lastname: Bruchertseifer
authors.forename: Alfred
authors.initials: A
authors.lastname: Morgenstern
authors.forename: Markus
authors.initials: M
authors.lastname: Essler
mesh_terms: D000512:Alpha Particles / Q000627:therapeutic use; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D017209:Apoptosis / Q000528:radiation effects*; D001729:Bismuth / Q000627:therapeutic use*; D002294:Carcinoma, Squamous Cell / Q000473:pathology* / Q000532:radiotherapy; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000276:immunology*; D059585:G1 Phase Cell Cycle Checkpoints / Q000528:radiation effects; D006258:Head and Neck Neoplasms / Q000473:pathology / Q000532:radiotherapy; D006801:Humans; D018796:Immunoconjugates / Q000627:therapeutic use*; D018499:Linear Energy Transfer; D016499:Radioimmunotherapy; D011868:Radioisotopes / Q000627:therapeutic use*; D015398:Signal Transduction / Q000528:radiation effects; D016019:Survival Analysis; D014964:X-Ray Therapy
chemical_list: D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D011868:Radioisotopes; C512478:EGFR protein, human; D066246:ErbB Receptors; D001729:Bismuth
keywords: Apoptosis; Cell-cycle arrest; Cytotoxicity; Head and neck squamous cell carcinoma; Radioimmunotherapy; UD-SCC5 cells; α-emitter (213)Bi