The role of protein kinases in hypoxia-inducible factor 1-alpha induced erythropoietin gene expression in hepatocellular carcinoma (Hep3B) cells.

摘要

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, signal transduction pathways as well as molecular mechanisms of HCC pathogenesis remain largely unexplored. Hypoxia and inflammation have been linked to an increased risk of liver cancer and resistance to chemotherapeutic agents. Earlier studies in our laboratory have identified specific protein kinase pathways that may contribute to the pathogenesis of HCC. The phosphatidylinositol-3-phosphate (PI3K) and the mitogen activated protein kinase (MAPK) pathways, although implicated in HCC proliferation and cell survival had not been studied in relationship to transcriptional activation of hypoxia-regulated genes. In our model of HCC, Hep3B cells, we explored the relationships between the transcription factors, hypoxia inducible factor-1 (HIF-1) and nuclear factor-kappa B (NF-kappaB), to upstream PI3K and MAPK pathways. AKT, a major effector of PI3K, was activated in a time dependent manner after hypoxia treatment for 0, 1 and 6 hrs. Pharmacological as well as genetic inhibition of the PI3K signaling pathway significantly decreased NF-kappaB and HIF-1-dependent transcriptional activation. Pharmacological or genetic inhibition of MEK1, an important member of the MAPK pathway, did not cause any significant change in HIF-1 trans-activation. In addition, the specific interaction between NF-kappaB and HIF-1 was explored in detail. Transient transfection in Hep3B cells of a genetic construct containing the HIF-1 gene promoter in which the NF-kappaB binding site was deleted completely abrogated HIF-1 trans-activation during hypoxia. Transient transfection of the whole HIF-1 gene promoter region and treatment with LY294002 (30 mumol/L), a selective pharmacological inhibitor of PI3K, significantly decreased HIF-1 transcriptional activation. Pharmacological inhibition of NF-kappaB with pyrrolidine dithiocarbamate (PDTC, 25 mumol/L) significantly decreased HIF-1 mRNA and protein levels. Finally, the importance of the PI3K/NF-kappaB/HIF-1 pathway in the survival of Hep3B cells was studied by TUNEL assay and DAPI staining. Inhibition of this pathway decreased proliferation and increase apoptosis of Hep3B cells. The data support the hypothesis that activation of the PI3K/NF-kappaB/HIF-1 signaling pathway in Hep3B cells plays an important role in cell survival and oncogenesis of HCC, and therefore is a potential target for therapeutic intervention.