5-Hydroxy-3,6,7,8,3‘,4‘-hexamethoxyflavone Induces Apoptosis through Reactive Oxygen Species Production, Growth Arrest and DNA Damage-Inducible Gene 153 Expression, and Caspase Activation in Human Leukemia Cells

This study examined the growth inhibitory effects of structurally related polymethoxylated flavones in human cancer cells. Here, we report that 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) induces growth inhibition of human cancer cells and induction of apoptosis in HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of 5-OH-HxMF-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that 5-OH-HxMF induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bax was found in 5-OH-HxMF-treated HL-60 cells. In addition, a caspase-independent pathway indicated by endonuclease G also contributed to apoptosis caused by 5-OH-HxMF. Antioxidants suppress 5-OH-HxMF-induced apoptosis. 5-OH-HxMF markedly enhanced growth arrest DNA damage-inducible gene 153 (GADD153) protein in a time-dependent manner. N-acetylcysteine (NAC) and catalase prevented up-regulation of GADD153 expression caused by 5-OH-HxMF. These findings suggest that 5-OH-HxMF creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in HL-60 cells. Meanwhile, ROS were proven an important inducer in this apoptotic process. The C-5 hydroxyl on the ring of 5-OH-HxMF was found to be essential for the antiproliferative and apoptosis-inducing activity. Our study identified the novel mechanisms of 5-OH-HxMF-induced apoptosis and indicated that these results have significant applications as potential chemopreventive and chemotherapeutic agents.