Induction of G1 cell cycle arrest in human umbilical vein endothelial cells by flavone's inhibition of the extracellular signal regulated kinase cascade.

Dietary flavonoids have demonstrated anti-carcinogenic activity in several animal models, but their mechanisms of action have not yet been clearly established. Here, we show that flavone, a parent compound of flavonoids, inhibits the proliferation, migration, and capillary tube formation of human umbilical vein endothelial cells (HUVECs). Flow cytometric analysis showed that flavone arrests the cell cycle progression at G(1) phase in HUVECs. We observed the down-regulation of the hyperphosphorylated form of retinoblastoma gene product and cyclin-dependent kinases 2 and 4 in flavone-treated cells, but it had no affect on the expression of p53 and cyclin-dependent kinase inhibitors p21(CIP/Waf1) and p27(Kip). Flavone almost completely inhibited the activation of extracellular signal regulated kinase 1. The present results suggest that the flavone moiety of flavonoids is required for anti-proliferative activity of flavonoids and that anti-carcinogenic action of flavonoids in vivo was mediated, at least in part, by inhibiting angiogenesis.

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells.

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.