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.