PGE₂ Increase Adhesion, Migration And Invasion In Hepatocellular Carcinoma Cells Via The FAK/Paxillin/Erk Signaling Pathway

Background:Prostaglandin E₂ (PGE₂) is found to play a major role in promoting growth and inhibiting apoptosis in the cancer cells. Recently, PGE₂ is also found to correlate with malignant cell invasion. However, the effects of PGE₂ on migration and invasion and its mechanism have not been clarified yet in HCC. Focal adhesion kinase is a non-receptor cytoplasmic tyrosine kinase that plays a key role in the regulation of cell proliferation. FAK also appears to be essential for the regulation of cell adhesive and migratory properties. Recent studies show that integrins control the cell movement, morphology, cell growth and gene expression by associated with FAK and the other downstream signaling proteins. But the mechanism of FAK in HCC cell migration is not well known. Therefore, in this article, we studied the effects of PGE₂ on HCC cell adhesion, migration, invasion in vitro and its relationship with FAK/Paxillin/Erk2 signaling pathway. Objective:1. To identity whether PGE₂ can promote adhesion, migration and invasion of hepatocellular carcinoma cell line Huh7 in vitro.2. To investigate the expression of FAK, Paxillin and Erk2 protein and their phosphorylation level in Huh7 and Hep3B cell line after treated with PGE₂.3. To identity whether EGFR specific inhibitor AG1478 can promote adhesion, migration and invasion and to investigate the expression of Paxillin and Erk2 protein of Huh7 cell line in vitro.4. To identity whether EGFR specific inhibitor AG1478 can effect PGE₂-mediated adhesion, migration and invasion in Huh7 cells5. To investigate the role of FAK/Paxillin/Erk signal transduction pathway in any effect exerted by PGE₂ on the adhesion, migration and invasion abilities in hepatocellular carcinoma cells.Methods:1. Cell culture: human hepatoma cells (Hep3B and Huh7) were cultured in vitro as routine.2. After the treatment of PGE₂, the cell adhesion ability was tested by WST-1 assay, cell migration ability was identified by Transwell plate and invasion ability was tested by a Matrix gel coated Transwell plate.3. Western Blot analysis was used to detect the expression of FAK, Paxillin Erk2 and their phosphorylation level after the treatment of PGE₂.4. EGFR specific inhibitor AG1478 were added to culture media and the change of cell adhesion, migration and invasion and the expression of Paxillin and Erk2 were then tested.5. AG1478 were added to culture media and the change of cell adhesion, migration and invasion and the expression of Paxillin and Erk2 were then tested to determine whether EGFR signaling pathway was involved in the mechanism exerted by PGE₂.Results:1. PGE₂ treatment significantly increased the Huh7 cells adhesion, migration, and invasion. Compared with the control group, after treated with extrinsic source 10,20,50μmol/L PGE₂, Huh7 cells related adhesion ratio was increased 18.5%,57.9% and 54%, the increased migration ratio was 6.1%,20% and 23% and the incrased invasion ratio was 5%,30% and 25%, respectively.2. After treatedment with PGE₂ for 24h in Huh7 and Hep3B cells, Western Blot analysis showed that PGE₂ treatment induced the phosphorylation of FAK(Tyr³⁹⁷), Paxillin(Tyr³¹) and Erk, and it also showed remarkable increased synthesis of FAK and Paxillin, but not Erk2. 3. After the treatment with EGFR specific inhibitor--AG1478, the Huh7 cell related adhesion, migration and invasion ratio were inhibited 53%, 56% and 70%, respectively (P<0.01). And Western Blot analysis showed AG1478 decreased the synthesis of Paxillin and Erk2 29% and 18%, respectively.4. In Huh7 cell line, after the treatment with EGFR specific inhibitor--AG1478, the PGE₂-mediated adhesion, migration and invasion related ratio were inhibited by 69%, 74% and 66%, respectively. And the synthesis of Paxillin which was mediated by PGE₂ was also decreased by 40%.Conclusions:1. PGE₂ can significantly promote the adhesion, migration and invasion abilities of Huh7 hepatocellular carcinoma cells in a dose dependent manner in vitro.2. PGE₂ can induce the phosphorylation of FAK, Paxillin and Erk2 in a PGE₂-concentration dependent manner and also increase the synthesis of FAK and Paxillin, but not Erk2 so as to activate the FAK/Paxillin/Erk signaling pathway.3. EGFR signaling pathway may involve in the PGE₂-mediated adhesion, migration and invasion in hepatocellular carcinoma cells.4. PGE₂ greatly induced HCC cell adhesion, migration, and invasion by activating FAK/paxillin/Erk signaling pathway.