The Effects Of PGE₂ On Cell Growth And Infiltration In Hepatocellular Carcinoma Cells And The Related Mechanisms

Background:Prostaglandin E₂ (PGE₂) one of predominant metabolic products of arachidonic acid. It is widely known that PGE₂ synthesis is associated with cell growth and spread in many cancer cells. Cyclooxygenase (COX) is known as prostaglandin endoperoxide synthase. Cyclooxygenase has three isoforms: COX-1, COX-2 and COX-3. Among them, COX-2 is induced by a lot of stimuli such as cytokines, hormones, mitogens, and growth factors, which explains its up-regulation in various inflammatory diseases and human cancers. PGE₂ mediates its effects by binding to and activating 4 different G-protein-coupled receptors, EP1, EP2, EP3 and EP4. In our previous studies, all of the 4 EP receptors were expressed on the surface of hepatocellular carcinoma cells. Both COX-2 overexpression and exogenous PGE₂ improved cell growth, while the selective COX-2 inhibitor, celecoxib, suppressed cell survival and induced cell apoptosis in hepatocellular carcinoma cells.In current studies, we went on to explore the effects of PGE₂ on cell growth, adhesion, migration and invasion in hepatocellular carcinoma cells.Aims:1. To clarify the effects of PGE₂ on cell growth in hepatocellular carcinoma cells.2. To explore the effects of PGE₂ and related EP receptor on survivin expression in hepatocellular carcinoma cells.3. To investigate the effects of PGE₂ on cell adhesion, migration and invasion in hepatocellular carcinoma cells. 4. To define a role of focal adhesion kinase (FAK) in PGE₂-induced cell adhesion and migration in hepatocellular carcinoma cells.Materials and methods:1. The hepatocellular carcinoma cell lines HUH-7, Hep3B and HepG2 cells, and human embryonic kidney HEK293 cells, were all cultured in conventional conditions.2. The WST reagents were used to detect the cell viability rate and relative adhesion rate in hepatocellular carcinoma cells with PGE₂ or selective EP1 agonist (17-P-T-PGE₂) treatments.3. 12-well transwell units were used to detect relative migration rate and invasion rate in hepatocellular carcinoma cells with PGE₂ treatments.4. The pcDNA3 plasmid encoding human COX-2 (COX-2-pcDNA3) and EP1 receptor (EP1R-pcDNA3) were transfected in HUH-7 cells and HEK293 cells, respectively, to gain COX-2-overexpressed cells and EP1 receptor-stably expressed cells.5. RNA interference was used to suppress EP1 receptor or FAK protein expression in HUH-7 cells, in order to study the effects of these protein on cell biologic activities.6. Realtime PCR was used to detect survivin mRNA level in HUH-7 cells. Western Blot was used to detect the protein level of survivin, FAK and paxillin, and the phosphorylation of EGFR, Akt, FAK, paxillin and ERK.Results:1. In WST assays, the cell viabilities were increased to 123%, 112% and 109% in HUH-7, Hep3B and HepG2 cells when treated with 5μM PGE₂ for 24 h. However, 50μM celecoxib suppressed the cell viabilities to 5.4%, 1.2% and 25.6% of control, respectively.2. 5μM PGE₂ treatment increased survivin expression 2.3-fold in HUH-7 cell. COX-2 overexpression caused similar survivin upregulation, which was suppressed by 50μM celecoxib.3. 5μM selective EP1 agonist (17-P-T-PGE₂) or EP1 receptor transfection increased survivin expression more than 2 fold. Conversely, the PGE₂-induced survivin upregulation was blocked by selective EP1 antagonist or EP1 RNA interference.4. The phosphorylation of EGFR and Akt were elevated in 17-P-T-PGE₂-treated cells, while both EGFR and PI3K inhibitors suppressed survivin upregulation induced by 17-P-T-PGE₂.5. PGE₂ treatment significantly increased the relative adhesion rate, migration rate, and invasive rate by 158%, 122% and 128% in HUH-7 cells.6. PGE₂ treatment increased the synthesis and phosphorylation of FAK by 170% and 263%. RNA interference targeting FAK suppressed PGE₂-mediated cell adhesion and migration.7. PGE₂ treatment increased the phosphorylation of paxillin and Erk2, the down stream protein of FAK. PD98059, the specific inhibitor of MEK, suppressed PGE₂-mediated cell adhesion and migration.Conclusion:PGE₂ improved cell growth, adhesion, migration and invasion in hepatocellular carcinoma cells. The PGE₂/EP1/EGFR/PI3K pathway promoted cell growth by upregulating survivin expression. And the FAK/paxillin/ERK pathway was associated with PGE₂-induced cell adhesion, migration, and invasion.