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

Background: Mammea siamensis (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 flower 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 flow 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 significant 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.