The Effects of Divalant Cation on the Idarubicin-Induced Apoptosis

Choi, Du Young; Oh, Man Tak; Oh, Yeon Geun; Kim, Jong Duck; Park, Rae Kil

Abstract

PURPOSE: Zinc ion is critical for the functional and structural integrity of eukaryotic cells and participate in the regulation of apoptosis. In general, zinc inhibits a nuclear endonuclease, thereby causing inhibition of apoptosis. Recent studies have pointed to a role for a family of caspase proteases that act upstream of endonuclease. The widely used chemotherapeutic agents exert effects by inducing of apoptosis in sensitive tumor cells. In this study, we investigated the effects of zinc ion and other divalent cation on the idarubicin (IDA)-induced apoptosis of HL-60 cells. In addition, to determine whether Zn inhibits an event upstream of endonuclease activation, we analysed the activity of caspase-3, 9 and proteolytic cleavage of procaspase-3 and PARP [poly (ADP-ribose) polymerase].
METHODS
HL-60 cells were cultured in RPMI 1640 and treated with various doses and time periods of IDA with or without pretreatment of ZnCl2, CaCl2 and MgCl2. Cell viability was measured by trypan blue staining. For detection of apoptotic death, cells were stained with Hoechst dye and observed under fluorescence microscopy. The activities of caspase-3 and caspase-9 were measured by the proteolytic cleavages of Ac- DEVD-AMC and Ac-LEHD-AFC as flurogenic substrates, respectively. The proteolytic cleavages of procaspase-3 and PARP were analyzed by Western blotting using anti- caspase-3 and anti-PARP antibody, respectively.
RESULTS
IDA induced the apoptotic death of HL-60 cells in a dose and time dependent manner, which was characterized by increasing chromatin condensation and DNA fragmentation. Pretreatment of HL-60 cells with ZnCl2 caused potent inhibition of IDA-induced apoptosis. Consistent with apoptotic death of HL-60 cells, IDA induced the catalytic activation of caspase-3 and caspase-9. After pretreatment of ZnCl2, the activation of caspase- 3 and the proteolysis of PARP induced by IDA were potently inhibited. But, after pretreatment of CaCl2 and MgCl2, there were no significant changes of IDA-induced apoptosis and proteases activity.
CONCLUSION
Zinc ion suppressed the IDA-induced apoptosis via the inhibitions of caspase-9 and caspase-3. But calcium and magnesium ions didn't affect the IDA-induced apoptosis.