Inhibitory activity of flavonoids from Ephedrae Herba on hypoxia signaling in PANC-1 cells and the evaluation of their mechanisms.

Pancreatic cancer is a lethal disease with a very poor prognosis. Recent reports indicate that hypoxia signaling mediated by hypoxia-inducible factor (HIF) contributes to the progression of pancreatic cancer. Therefore, elucidating the inhibitor of hypoxia signaling may lead to the development of a candidate for new anticancer agents. During our screening program for HIF inhibitor from crude drug extracts, new acylated kaempferol glycosides, kaempferol 3-O-[4″-(E)-p-coumaroyl-3″-O-dihydroxypalmityl] rhamnoside (1) and kaempferol 3-O-[4″-(E)-p-coumaroyl-2″-O-dihydroxypalmityl] rhamnoside (2), were isolated from an acetone extract of Ephedrae Herba, together with eight known flavonol glycosides (3-10). The structures of novel compounds 1 and 2 were elucidated based on spectroscopic and chemical analyses. Using a cell-based HRE-driven luciferase reporter assay in a PANC-1 pancreatic cancer cell line, we found that these compounds demonstrated potent inhibitory activity on hypoxia signaling with IC50 values of 18.0 ± 0.6 and 13.3 ± 2.2 µM, respectively. Mechanistically, 2 reduced the amount of HIF-1α protein in the nuclear at 30 µM via the ubiquitin-proteasome pathway with no effect on the nuclear translocation of HIF proteins from cytosol and subsequently decreased Glut1 mRNA. These results indicate that 2 inhibits hypoxia signaling through a mechanism involving the reduction of HIF-1α protein levels and Glut1 mRNA and may have anti-pancreatic cancer effects.