Mitogen activated protein kinase signaling through MEK-ERK is an important growth signaling pathway in hepatocellular carcinoma (HCC). We have identified and characterized a novel MEK inhibitor, PD181461, through an in vitro cascade assay using bacterially purified glutathione-S-transferase (GST) fusion proteins of MEK and MAPK. PD181461 directly inhibits MEK (IC50= 50nM) without effects on activity of MAPK or 26 related kinases (<10μM) and is non-competitive with ATP or the ERK site on MEK consistent with an allosteric mechanism of inhibition. Five HCC cell lines (HepG2, Hep3B, SKHep, PLC and SUHC-1) were employed for in vitro and in vivo studies. MEK activity and ERK expression were determined by phospho-specific and total ERK immunoblot. Proliferation was determined by MTS assay as well as trypan blue excluded cell counts. Athymic nude mice with xenografted tumors were given PD181461 via orogastric lavage and bioavailability was confirmed with ex-vivo assays of MEK activity. MEK activity was inhibited by PD181461 in a time and concentration dependent manner in all cell lines with no change in total ERK expression. Inhibition of proliferation was strongly associated with inhibition of MEK activity. In in vivo studies, MEK activity was significantly inhibited in tumors of PD181461 treated animals compared to controls (p< 0.05). PD181461 prevented formation of tumor xenografts at 3 weeks whereas 100% of placebo controls formed tumors (p< 0.0001). PD181461 also inhibited growth of established tumors at 6 weeks (p< 0.05). PD181461 is a novel MEK inhibitor which has significant effects on HCC proliferation in vitro and in vivo. Treatments which inhibit MEK-ERK signaling may hold promise for treatment of HCC.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]