Aim: To study the relationship between Daxx expression and the antiapoptotic effects of probucol in THP-1 macrophage.
Materials and methods: Apoptosis of THP-1 derived macrophages was induced by exposure to oxidized low density lipoprotein (oxLDL). The development of apoptosis was determined by flow cytometry analysis and nucleic acid-binding dye acridin orange. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and indirect immunofluorescence were used to evaluate the expression of Daxx and caspase-3 at both mRNA and protein level.
Results: As expected, THP-1 macrophages exposed to 100 mg/l oxLDL for 48 h exhibited typical morphologic changes of apoptosis, including condensed chromatin and shrunken nucleus. oxLDL treatment markedly increased Daxx expression in a time- and dose-dependent manner, and facilitated Daxx translocation from cytoplasm to nucleus. The percentage of cells with Daxx in nuclei was significantly increased from 8 to 59%. Treatment with probucol (50 micromol/l) for 4 h prior to exposure to oxLDL significantly inhibited Daxx expression and THP-1 macrophage apoptosis by 61.3%. Furthermore, oxLDL enhanced caspase-3 expression with increased mRNA and protein levels, but without obvious change in translocation of caspase-3 (the cells with nuclear Daxx: 14 vs 8%). In contrast, probucol attenuated oxLDL-stimulated caspase-3 expression in THP-1 macrophages.
Conclusion: OxLDL-induced apoptosis of THP-1 macrophage is associated with Daxx up-regulation; while inhibition of apoptosis by probucol is related to decreased Daxx expression and nuclear translocation.