Induction of oxidative stress and apoptosis in myeloma cells by the aziridine-containing agent imexon

Imexon is an iminopyrrolidone derivative that has selective antitumor activ ity in multiple myeloma, The exact mechanism of imexon action is unknown. I n human 8226 myeloma cells, the cytotoxicity of imexon was schedule-depende nt, and long exposures (greater than or equal to 48 hr) to low concentratio ns of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also demonstrated by HPLC studies that in hum an 8226 myeloma cells, imexon depletes cellular stores of cysteine and glut athione. Oxidative stress in 8226 cells exposed to imexon was detected by i mmunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxygua nosine (8-OHdG), followed by confocal microscopy. These images showed incre ased levels of 8-OHdG in the cytoplasm of cells treated with different conc entrations of imexon at 8, 16, and 48 hr. Interestingly, 8-OHdG staining wa s not observed in the nuclei of imexon-treated cells, in contrast to the di ffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to im exon showed classic morphologic features of apoptosis upon electron microsc opy, and increased levels of phosphatidylserine exposure, detected as Annex in-V binding, on the cell surface. To prevent depletion of thiols, 8226 mye loma cells exposed to imexon were treated with N-acetylcysteine (NAC). Simu ltaneous, as well as sequential, treatment with NAC before imexon exposure resulted in protection of myeloma cells against imexon-induced cytotoxicity . Conversely, the glutathione synthesis inhibitor buthionine sulfoximine in creased imexon cytotoxicity. These data suggest that imexon perturbs cellul ar thiols and induces oxidative stress leading to apoptosis in human myelom a cells. (C) 2000 Elsevier Science Inc.