Mechanism of euphorbiasteroid inducing apoptosis of HL-60 cells

OBJECTIVE To investigate the effect of euphorbiasteroid on inducing the apoptosis of HL-60 cells and identify the role of Bcl-2/Bax signaling pathway in this process.METHODS HL-60 cells were treated with low,middleand high dose of euphorbiasteroid in vitro for 24 hand after that cell counting Kit-8was used to detect cell proliferation.The morphology of HL-60 cells was observed under light and fluorescent microscopy.The early cell apoptosis was detected using flow cytometry with AnnexinⅤ/PI double staining.The expressions of Bcl-2,Bax,Caspase-9,Caspase-3mRNA were analyzed by RT-PCR.The activity of Caspase-9,3was examined by chromatometry,ELISA detect Caspase-9,Caspase-3activity of protein.RESULTS HL-60 cell proliferation was inhibited significantly by euphorbiasteroid administration(F=42.97,P0.001).Comparison between different concentrations,the difference was statistically significant,P0.05.Early cell apoptosis rate of HL-60 cells was enhanced markedly after euphorbiasteroid administration(F=56.74,P0.001),after 10,20,40μg/mL euphorbiasteroid treatment for 24 h,the HL-60 cells early apoptosis rate respectively were(23.4±3.1)%,(35.7±4.3)%,(53.2±3.9)%,and cells presented typical apoptosis morphological changes.Bax mRNA transcription level increased significantly,the Bcl-2mRNA transcription level decreased significantly(F=53.45,P0.001)after euphorbiasteroid administration in a dose-dependent manner.Caspase-9,Caspase-3mRNA were up-regulated in the transcriptional level,and a compound dose dependent(F=34.21,P0.001),semen euphorbiae sterol effect on the HL-60 cells after 24hCaspase-9,3protein activity significantly increased(F=54.33,P0.001),with the increase of concentration of semen euphorbiae sterol the protein activity increased.CONCLUSION Euphorbiasteroid induces HL-60 cells to apoptosis via promoting Bcl-2/Bax apoptotic signaling pathway in a dose-dependent manner.