Over-expression of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK in hepatocellular carcinoma: its role in tumor progression and apoptosis.
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the most common malignancies in South East Asia. Although activation of the MEK-MAPK is often associated with cellular growth, the role of MEK-MAPK in growth and survival of hepatocarcinoma cells has not been established.


METHODS
Immuno-histochemistry was used to localize phosphorylated MAPK and MEK1/2 in the tissues. 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay and ELISA were used to determine cell viability and cell proliferation. Deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to detect apoptotic cells. Western blots analysis was performed to determine the levels of proteins involved in the MEK-MAPK and apoptotic pathways. Transfection study was performed to assess the role of MEK-MAPK pathway in growth and survival of liver cancer cells.


RESULTS
We report that phosphorylation of MEK1/2 at Ser217/221 was detected by immuno-histochemistry in 100% (46 of 46) of HCCs examined. A positive signal was localized in the nuclei of hepatocarcinoma cells but not in dysplastic hepatocytes or stromal cells. Over-expression and phosphorylation of MAPK was also detected in 91% (42 of 46) and 69% (32 of 46) of HCCs examined, respectively. The percentage of cells showing positively for phosphorylated MEK1/2 increased with advancing tumor stage. In vitro, treatment of human HepG2 and Hep3B cells with MEK1/2 specific inhibitors U0126 and PD98059 led to growth inhibition and apoptosis. U0126 induced the release of cytochrome c and increased the cleavage of caspase-3, caspase-7, and poly ADP-ribose polymerase (PARP). Inhibition of phosphatidylinositol 3-kinase (PI-3K), c-Jun N-terminal kinase (JNK) and p38 kinase activities caused only a mild apoptosis in HepG2 and Hep3B cells. Activated MEK1-transfected cells were more resistant to UO126-induced apoptosis in vitro and formed larger tumors in SCID mice than mock-transfected cells.


CONCLUSIONS
In conclusion, our results demonstrate that MEK-MAPK plays an important role in the growth and survival of liver cancer cells and suggest that blocking MEK-MAPK activity may represent an alternative approach for the treatment of liver cancer.
Huynh
Hung
H
Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, Singapore General Hospital, Singapore 169610. cmrhth@nccs.com.sg
Nguyen
Thi Thanh Tuyen
TT
Chow
Kah-Hoe Pierce
KH
Tan
Puay Hoon
PH
Soo
Khee Chee
KC
Tran
Evelyne
E
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000473:pathology; D045744:Cell Line, Tumor; D018450:Disease Progression; D018572:Disease-Free Survival; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D016513:Mice, SCID; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation
D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases