Induction of apoptosis by biaminotetradecanoic acid via activating caspase-dependent mitochondrial signaling in HL-60 cells

OBJECTIVE To explore mechanisms of apoptosis induced by biaminotetradecanoic acid(D-82) on HL-60 cells via caspase-dependent mitochondrial pathways.METHODS HL-60 cells were cultured with D-82 1.5,3 and 6 mg·L-1 for 6 h.The cell survival rate was detected by trypan blue staining.The apoptosis rate was evaluated by acridine orange(AO) /ethidium bromide(EB) staining.DNA fragments were observed by agarose gel electrophoresis.The mitochondrial membrane potential was detected by flow cytometry following rhodamine 123 fluorescence staining.The activity of caspase 9 and caspase 3 were detected by Colorimetric Assay Kit.The expression of Bcl-2,Bax,cytoplasmic cytochrome c(Cyt c) and mitochondrial Cyt c were detected by Western blotting.RESULTS The concentration and duration of D-82 were related to HL-60 cell survival.After HL-60 cells incubated with D-82 1.5,3 and 6 mg·L-1 for 6 h,the cell survival rate was(81 ± 11) %,(70 ±9) % and(56 ±11) %,respectively.After HL-60 cells treated with D-82 6 mg·L-1 for 12 and 24 h,the survival rate further decreased to(36 ±7) % and(24 ±9) %.Moreover,the demonstrating typical morphological transformation and DNA ladder were detected,and cell apoptosis rates increased to(18 ±4) %,(40 ± 11) % and(75 ± 11) % from(2 ± 2) %(n = 3,P 0.05).Compared with normal control group,the mean fluorescence intensity of rhodamine decreased from 24 ± 5 to 20 ± 5 and 12 ±4(n =3,P 0.05) in D-82 3 and 6 mg·L-1 treatment group,which demonstrated while the potential loss of mitochondrial membrane.Caspase 3 activity increased by 39% and 125% caspase 9 activity increased by 61% and 145%.Cyt c in mitochondria decreased by 22% and 38%(P 0.05,P 0.01),and in cytoplasm increased by 22% and 65%(P 0.05) in D-82 3 and 6 mg·L-1 groups;Bax expression increased by 45% and 116%(P 0.05,P 0.01) and Bcl-2 expression decreased by 27% and 40%(P 0.01).CONCLUSION D-82 induces apoptosis via activating caspase-dependent mitochondrial signal transduction pathways in HL-60 cells.