CCY-1a-E2 induces G2/M phase arrest and apoptotic cell death in HL-60 leukemia cells through cyclin-dependent kinase 1 signaling and the mitochondria-dependent caspase pathway

Our previous study demonstrated that 2-[(3-methoxybenzyl)oxy]benzaldehyde (CCY-1a-E2) is a potent compound that acts against multiple human leukemia cell lines. CCY-1a-E2 was also shown to have efficacious anti-leukemic activity in vivo. However, the molecular mechanism of action of CCY-1a-E2 attributed to its anticancer effect remains poorly understood. In the present study, CCY-1a-E2 suppressed cell viability in multiple leukemia cell lines (HL-60, K562, KG-1 and KG-1a) via inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. CCY-1a-E2 exhibited a marked toxic effect on HL-60 cells and displayed low cytotoxicity in normal human peripheral blood mono-nuclear cells (PBMCs). Results from flow cytometric analysis indicated that CCY-1a-E2 promoted G2/M phase arrest and promoted apoptosis in the HL-60 cells. CCY-1a-E2 treatment upregulated cyclin B, cyclin-dependent kinase 1 (CDK1), cell division cycle 25C (cdc25C) and p21 protein expression. CCY-1a-E2 caused apoptotic cell death and DNA fragmentation as determined by 4,6-diamidino-2-phenylindole (DAPI) staining and DNA gel electrophoresis. Elevated activities of caspase-8, -9 and -3 were observed during CCY-1a-E2-induced cell apoptosis; their specific inhibitors were found to block CCY-1a-E2-induced apoptosis, respectively. Moreover, CCY-1a-E2 time-dependently disrupted the mitochondrial membrane potential (m), and it enhanced the protein levels of Fas/CD95, cytochrome c, Bax, cleaved PARP, as well as attenuated Bcl-2 expression in the HL-60 cells. Our results provide direct evidence that supports the future potential therapeutic application of CCY-1a-E2 in leukemia.