Effect of toosendanin on proliferation and apoptosis of K562 cells
AIM: To study the effects of proliferation and apoptosis on K562 cells induced by toosendanin. METHODS: The growth inhibition rate of K.562 cells and Peripheral Blood Mononuclear Cell (PBMC) was measured by MTY. Morphology of K562 cells and PBMC was detected by wright's stain. The ultrastructure changes of K562 ceUs were analyzed by transmission electron microscope. DNA fragmentation and the percentage of apoptoticcells were examined by Sepharose electrophoresis and flow cytometry (FCM), respectively. The activities of Caspase-8, Caspase-9 and Caspase-3 were analyzed by spectrophotometry. The expression of Bcl-xl, Bax and Fas was investigated by immunocytochemistry. RESULTS: Toosendanin presented the striking proliferation inhibition, as well as apoptosis induction potency on K.562 cells in vitro in a time-and dose-dependent manner, with IC_50 value for 72 h being (52.13 ±0.82) nmoL/L(P <0.01 ). The morphological features of apoptosis were observed by light microscopy and transmission electron microscopy in cell shrinkage, even appearance of apoptosis body. The morphology and cell growth inhibition of PBMC had no significant changes. After Toosendanin treatment at 10, 30 and 50 nmoL/L for 72 h, the apoptosis rate of 1(562 cells was 9.66%, 26.06% and 52.70%, respectively(P <0.01 ). A typical ladder pattern was identified in DNA electrophoresis. The enzyme activity changes of Caspase-3, Caspase-8 and Caspase-9 were detected by spectrophotometry. Immunocytochemistry technique showed that Toosendanin could significantly down-regulate Bcl-xl protein expression and up-regulate Bax and Fas proteins expression ( P < 0.05). CONCLUSION: Toosendanin was a potent inhibitor of proliferation of K562 ceils without toxicity to PBMC. The mechanisms of apoptosis-induced of Toosendanin may be mediated by mitochondrion and death-receptor dual pathways.
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