Objective: To investigate the expression of mitochondria-mediated apoptosis pathway related genes bcl-2, Bax, survivin and Smac/DIABLO in bufalin induced HL-60 cell apoptosis.
Methods: Cell viability was determined by trypan blue exclusion, apoptosis by morphology and flow cytometry, expressions of Bcl-2, Bax, Survivin, Smac/DIABLO and caspase-3 protein by Western blot, and expression of survivin mRNA by RT-PCR.
Results: Proliferation of HL-60 cells was inhibited by bufalin and the IC(50) at 24, 48 and 72 h were 25.8, 8.0 and 2.1 nmol/L, respectively. Apoptosis was induced when the cells were treated with bufalin at concentrations of 50.0 nmol/L and higher. Compared with the control, HL-60 cells treated with bufalin at 50.0 nmol/L for 6, 12, 24 and 48 h showed decrease of Bcl-2 protein expression to 88.6%, 53.3%, 19.2% and 9.5%, Bcl-2/Bax ratio (control 2.0) to 1.7, 1.1, 0.4 and 0.2, Survivin protein expression to 75.2%, 54.8%, 37.5% and 20.3%, and survivin mRNA to 85.7%, 39.4%, 12.5%and 0%, respectively. The expression of Smac/DIABLO protein was downregulated to 77.5% (12 h), 21.2% (24 h) and 15.3% (48 h) in mitochondrial fraction and upregulated to 1.4-(12h), 2.0-(24 h) and 3.5- folds (48 h) in cytosolic fraction, respectively. The active subunits of caspase-3 were displayed after treatment for 8 h till 48 h.
Conclusion: Apoptosis induced by bufalin is related to downregulation of expressions of bcl-2 and survivin, decrease of Bcl-2/Bax ratio, mitochondrial release of Smac/DIABLO, and activation of caspase-3. The mitochondria-mediated apoptotic pathway may be involved in the apoptosis induced by bufalin in HL-60 cells.