Abstract
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.
Copyright 2004 Wiley-Liss, Inc.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Animals
-
Antineoplastic Combined Chemotherapy Protocols / pharmacology
-
Cell Proliferation / drug effects
-
Cells, Cultured
-
DNA, Complementary / genetics
-
DNA, Complementary / pharmacology
-
Drug Screening Assays, Antitumor
-
Drug Synergism
-
Enzyme Inhibitors / pharmacology
-
Enzyme Inhibitors / therapeutic use
-
Extracellular Signal-Regulated MAP Kinases / drug effects
-
Extracellular Signal-Regulated MAP Kinases / metabolism
-
Growth Inhibitors / pharmacology*
-
Hepatocyte Growth Factor / pharmacology*
-
Hepatocyte Growth Factor / therapeutic use
-
Hepatocytes / drug effects*
-
Hepatocytes / enzymology
-
Humans
-
Liver Neoplasms, Experimental / drug therapy*
-
Liver Neoplasms, Experimental / enzymology
-
MAP Kinase Signaling System / drug effects*
-
MAP Kinase Signaling System / physiology
-
Mutation / genetics
-
Phosphorylation / drug effects
-
Protein Serine-Threonine Kinases / drug effects
-
Protein Serine-Threonine Kinases / genetics
-
Protein Serine-Threonine Kinases / metabolism*
-
Proto-Oncogene Proteins / drug effects
-
Proto-Oncogene Proteins / genetics
-
Proto-Oncogene Proteins / metabolism*
-
Proto-Oncogene Proteins c-akt
-
Proto-Oncogene Proteins c-raf / drug effects
-
Proto-Oncogene Proteins c-raf / metabolism
-
Rats
-
Rats, Inbred F344
-
Tumor Cells, Cultured
-
Vitamin K / analogs & derivatives*
-
Vitamin K / pharmacology*
-
cdc25 Phosphatases / antagonists & inhibitors
-
cdc25 Phosphatases / metabolism
Substances
-
2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone
-
DNA, Complementary
-
Enzyme Inhibitors
-
Growth Inhibitors
-
Proto-Oncogene Proteins
-
Vitamin K
-
Hepatocyte Growth Factor
-
AKT1 protein, human
-
Akt1 protein, rat
-
Protein Serine-Threonine Kinases
-
Proto-Oncogene Proteins c-akt
-
Proto-Oncogene Proteins c-raf
-
Extracellular Signal-Regulated MAP Kinases
-
CDC25A protein, human
-
Cdc25a protein, rat
-
cdc25 Phosphatases