HL-60 Cells In Dose Research Articles

Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway

Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities including anti-tumor. Here, for the first time, we studied the molecular mechanism of Juglone-induced apoptosis in human leukemia HL-60 cells. In the present study, HL-60 cells were incubated with Juglone at various concentrations. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry. Expression of Bcl-2 and Bax mRNA was determined by quantitative polymerase chain reaction (qPCR). The results showed that Juglone inhibits the growth of human leukemia HL-60 cells in dose- and time-dependent manner. Topical morphological changes of apoptotic body formation after Juglone treatment were observed by Hoechst 33342 staining. The percentages of Annexin V-FITC-positive/PI negative cells were 7.81%, 35.46%, 49.11% and 66.02% with the concentrations of Juglone (0, 0.5, 1.0 and 1.5 μg/ml). Juglone could induce the mitochondrial membrane potential (△ Ψm) loss, which preceded release of cytochrome c (Cyt c), Smac and apoptosis inducing factor (AIF) to cell cytoplasm. A marked increased of Bax mRNA and protein appeared with Juglone treatment, while an evidently decreased of Bcl-2 mRNA and protein appeared at the same time. These events paralleled with activation of caspase-9, -3 and PARP cleavage. And the apoptosis induced by Juglone was blocked by z-LEHD-fmk, a caspase-9 inhibitor. Those results of our studies demonstrated that Juglone-induced mitochondrial dysfunction in HL-60 cells trigger events responsible for mitochondrial-dependent apoptosis pathways and the elevated ratio of Bax/Bcl-2 was also probably involved in this effect.

Cyclic adenosine monophosphate inhibits quinolone alkaloid evocarpine-induced apoptosis via activation of protein kinase A in human leukaemic HL-60 cells.

Evocarpine, an isoquinolone alkaloid isolated from the fruit of Evodia rutaecarpa, was found to induce apoptotic cell death in promyelocytic leukaemia HL-60 cells in dose- and time-dependent manners. We investigated the involvement of protein kinase A during the evocarpine-induced apoptotic cell death. Evocarpine-induced apoptosis was markedly inhibited by treatment of the cells with dibutyryl-cyclic adenosine monophosphate. Similar results were obtained with other commonly used cyclic adenosine monophosphate analogues, chlorophenylthio-cyclic adenosine monophosphate and the intracellular cyclic adenosine monophosphate-elevating agent, forskolin. In contrast, pretreatment of HL-60 cells with KT5720, an inhibitor of cyclic adenosine monophosphate-dependent protein kinase A, abrogated the protective effects of cyclic adenosine monophosphate analogues and forskolin on evocrpine-induced apoptosis. These findings suggest that cyclic adenosine monophosphate-dependent activation of protein kinase A plays a crucial role in protecting HL-60 cells from the evocarpine-induced apoptotic cell death.