Cell death triggered by alpha-emitting 213Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death

Purpose: Radioimmunotherapy with alpha-particle-emitting nuclides, such as Bi-213, is a promising concept for the elimination of small tumour nodules or single disseminated tumour cells. The aim of this study was to investigate cellular damage and the mode of cell death triggered by Bi-213-immunoconjugates. Methods: Human gastric cancer cells (HSC45-M2) expressing d9-E-cadherin were incubated with different levels of activity of Bi-213-d9MAb targeting d9-E-cadherin and Bi-213-d8MAb, which does not bind to d9-E-cadherin. Micronucleated (M) cells, abnormal (A) cells and apoptotic (A) [(MAA)] cells were scored microscopically in the MAA assay following fluorescent staining of nuclei and cytoplasm. Chromosomal aberrations were analysed microscopically following Giemsa staining. The effect of z-VAD-fmk, known to inhibit apoptosis, on the prevention of cell death was investigated following treatment of HSC45-M2 cells with sorbitol as well as Bi-213-d9MAb. Activation of caspase 3 after incubation of HSC45-M2 cells with both sorbitol and Bi-213-d9MAb was analysed via Western blotting. Results: Following incubation of HSC45-M2 human gastric cancer cells expressing d9-E-cadherin with Bi-213-d9MAb the number of cells killed increased proportional to the applied activity concentration. Microscopically visible effects of alpha-irradiation of HSC45-M2 cells were formation of micronuclei and severe chromosomal aberrations. Preferential induction of these lesions with specific Bi-213-d9MAb compared with unspecific Bi-213-d8MAb (not targeting d9-E-cadherin) was not observed if the number of floating, i.e. unbound Bi-213-immunoconjugates per cell exceeded 2x10(-4), most likely due to intense crossfire. In contrast to sorbitol-induced cell death, cell death triggered by Bi-213-immunoconjugates was independent of caspase 3 activation and could not be inhibited by z-VAD-fmk, known to suppress the apoptotic pathway. Conclusion: Bi-213-immunoconjugates seem to induce a mode of cell death different from apoptosis in HSC45-M2 cells.