Studies on the mechanism of apoptosis induced by selenium compounds in promyelocytic leukemia cells

Abstract

In the present study, biochemical and molecular changes during cell death induced by selenite and Se-methylselenocysteine (MSC) were studied in human promyelocytic leukemia (HL-60) cells. Treatment of selenite and MSC decreased cell viability in a dose-dependent manner measured by WST-1. Lactate dehydrogenase leakage assay indicated that selenite caused more extensive membrane damage than MSC. Morphological observations revealed that selenite-treated cells showed mainly necrotic morphologies and MSC-treated cells exhibited apoptotic morphologies. Although both selenite and MSC induced apoptotic DNA fragmentation in a dose-dependent manner, flow cytometric analysis of apoptosis revealed that apoptotic population was much higher in MSC-treated cells. Caspase-3, -8, and -9 were activated by both selenite and MSC, but the magnitude of their activation was much smaller in selenite-treated cells than in MSC-treated cells. On the other hand, the extent of cytochrome c release was much higher in selenite-treated cells, which implies that selenite caused extensive mitochondrial damage. Caspase inhibitors, z-DEVD-fmk, z-LEHD-fmk, and z-IETD-fmk all significantly recovered the decreased viability by the treatment with MSC, while they had no effects in case of selenite-treated cells. Caspase-3 activity was completely inhibited by z-IETD-fmk, whereas inhibition by z-LEHD-fmk was partial. These results suggest that caspase activation cascade mediated by caspase-8 is required for MSC-induced cell death while selenite-induced cell death occurred irrespective of caspase activation. Intracellular calcium level was increased by selenite and MSC. Calcium channel blocker $Ni^{2+}$ could significantly lower the intracellular calcium level and increase the viability in selenite-treated cells, which indicates that calcium influx is important in selenite-induced cell death. Further investigations on DNA strand breaks suggested that selenite caused caspase-3-independent DNA double and s...