Kayeassamin A Isolated from the Flower of Mammea siamensis Triggers Apoptosis by Activating Caspase-3/-8 in HL-60 Human Leukemia Cells

Background

Mammea siamensiŝ(Miq.) T. Anders. is used as a medicinal plant in Thailand and has several traditional therapeutic properties. In a previous study, we isolated eight compounds from the ˚ower of M. siamensis and demonstrated that kayeassamin A~(KA) exhibited potent antiproliferative activity against human leukemia and stomach cancer cell lines.

Objective

In this study, we investigated the effect of KA on cell viability and apoptotic mechanisms in HL-60 human leukemia cells.

Materials and Methods

Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Nuclear morphology and DNA fragmentation were observed using Hoechst 33258 staining and agarose gel electrophoresis, respectively. The sub-G1 phase of cells was analyzed by ˚ow cytometry after the cellular DNA had been stained with propidium iodide. The protein levels of poly~ (ADP-ribose) polymerase~ (PARP) and caspases were determined by Western blotting.

Results

KA exhibited a signi˛cant cytotoxic effect in a dose-~ and time-dependent manner, and induced chromatin condensation, DNA fragmentation, and sub-G1 phase DNA content, known as molecular events associated with the induction of apoptosis. In addition, KA strongly induced the activation of PARP and caspase-3 and~-8, with weak caspase-9 activation. Furthermore, KA-induced DNA fragmentation was abolished by pretreatment with z-VAD-FMK (a broad caspase inhibitor), z-DEVD-FMK (a caspase-3 inhibitor), and z-IETD-FMK~ (a caspase-8 inhibitor), but not by z-LEHD-FMK (a caspase-9 inhibitor) pretreatment.

Conclusion

These results indicate that KA triggers apoptotic cell death by activation of caspase-3 and~-8 in HL-60~cells.