Over-expression of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK in hepatocellular carcinoma: Its role in tumor progression and apoptosis

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Abstract
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. 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. 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. 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.