Hepatocyte growth factor enhances protein phosphatase Cdc25A inhibitor compound 5‐induced hepatoma cell growth inhibition via Akt‐mediated MAPK pathway

We have previously shown that Compound 5 (Cpd 5), an inhibitor of protein phosphatase Cdc25A, inhibits Hep3B human hepatoma cell growth. We now show that hepatocyte growth factor (HGF), a hepatocyte growth stimulant, can strongly enhance Cpd 5‐induced growth inhibition in Hep3B cells, and this enhancement in cell growth inhibition is correlated with a much stronger ERK phosphorylation when compared to cells treated with Cpd 5 or HGF separately. We found that HGF/Cpd 5‐induced ERK phosphorylation and cell growth inhibition were mediated by Akt (protein kinase B) pathway, since combination HGF/Cpd 5 treatment of Hep3B cells inhibited Akt phosphorylation at Ser‐473 and its kinase activity, which led to the suppression of Raf‐1 phosphorylation at Ser‐259. The suppression of Raf‐1 Ser‐259 phosphorylation caused the induction of Raf‐1 kinase activity, as well as hyper‐ERK phosphorylation. Transient transfection of Hep3B cells with dominant negative Akt c‐DNA further enhanced both Cpd 5‐ and HGF/Cpd 5‐induced ERK phosphorylation, while over‐expression of wild‐type Akt c‐DNA diminished their effects. In contrast, HGF antagonized the growth inhibitory actions of Cpd 5 on normal rat hepatocytes, thus showing a selective effect on tumor cells compared to normal cells. Our data suggest that Akt kinase negatively regulates MAPK activity at the Akt‐Raf level. Suppression of Akt activity by either combination HGF/Cpd 5 treatment or by dominant negative Akt c‐DNA transfection antagonizes the Akt inhibitory effect on Raf‐1, resulting in an enhancement of Cpd 5‐induced MAPK activation and cell growth inhibition. © 2004 Wiley‐Liss, Inc.