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The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G2-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G2- arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G1 cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G2-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G2-checkpoint activation, which caused not only G2-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondriadependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δψm loss, and caspase cascade activation.

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