Role of zinc ions in ricin-induced apoptosis in U937 cells

Abstract

We found that treatment of U937 cells with ZnCl 2 resulted in marked inhibition of ricin-induced DNA fragmentation and nuclear morphological change. Zn 2+ also completely inhibited the activation of caspase-3-, caspase-6-, and caspase-9-like proteases in ricin-treated cells, while no significant effect of Zn 2+ on these protease activities was observed when added directly to the lysate of ricin-treated cells, suggesting that Zn 2+ blocks the process of the activation of these caspases rather than the direct inhibition of the already activated enzymes. Fluorescence microscopic observation with Zn 2+ specific fluorescent probe dansylaminoethyl-cyclen suggested that there was a substantial increase in probe-detectable Zn 2+ in ricin-treated cells. Since the differences in the total Zn 2+ contents between ricin-treated and -untreated cells as measured with an atomic absorption spectrophotometer were too small to explain the increase in probe fluorescence in ricin-treated cells, it was suggested that release of Zn 2+ from intracellular stores or metalloproteins may occur rather than enhanced uptake from the medium. The Zn 2+ probe fluorescence change was observed prior to the depletion of intracellular glutathione. Carbobenzoxy-Asp-1-yl-[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH 2-DCB), a caspase family protease inhibitor, prevented ricin-induced increase in Zn 2+ probe fluorescence. These results suggest that redistribution of intracellular Zn 2+ occurs during ricin-induced apoptosis as early apoptotic event, and exogenously added Zn 2+ may prevent such intracellular Zn 2+ redistribution resulting in the inhibition of apoptosis.