Induction of oxidative stress and apoptosis in myeloma cells by the aziridine-containing agent imexon.
Imexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule-dependent, and long exposures (> or = 48 hr) to low concentrations 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 human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione. Oxidative stress in 8226 cells exposed to imexon was detected by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG), followed by confocal microscopy. These images showed increased levels of 8-OHdG in the cytoplasm of cells treated with different concentrations of imexon at 8, 16, and 48 hr. Interestingly, 8-OHdG staining was not observed in the nuclei of imexon-treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis upon electron microscopy, and increased levels of phosphatidylserine exposure, detected as Annexin-V binding, on the cell surface. To prevent depletion of thiols, 8226 myeloma cells exposed to imexon were treated with N-acetylcysteine (NAC). Simultaneous, 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 increased imexon cytotoxicity. These data suggest that imexon perturbs cellular thiols and induces oxidative stress leading to apoptosis in human myeloma cells.
Arizona Cancer Center, The University of Arizona, Tucson 85724, USA.
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Dvorakova
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Payne
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Tome
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Briehl
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McClure
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Dorr
D000478:Alkylation; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D006588:Hexanones / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013945:Thymoma / Q000473:pathology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D006588:Hexanones; D019253:Proto-Oncogene Proteins c-bcl-2; D013438:Sulfhydryl Compounds; C014919:4-imino-1,3-diazabicyclo(3.1.0)hexan-2-one; D004247:DNA; D005978:Glutathione; D003545:Cysteine