| Objective:Recent studies have shown that cyclooxygenase-2(COX-2) play multiple roles in hepatocarcinogenesis including inhibition of apoptosis, immune suppression and promotion of angiogenesis and tumor invasion. Prostaglandin E2 (PGE2) has been found to primarily mediated the roles of COX-2 via their respective E series of prostaglandin (EP) receptors, which are G protein-coupled receptors (GPCR) superfamily of seven-transmembrane spanning proteins on the cell surface membrane. However, the precise signal pathway activated by PGE2 in the hepatocellular carcinoma (HCC) cells remains elusive.Wnt/β-catenin plays an important role in regulation of cell proliferation, movement and differentiation. Its activation has also been implicated in various stages of embryo development and tumorigenesis. Activation of the Wnt/β-catenin pathway occurs in approximately 30% to 40% of HCCs. In this study, we aimed to investigate whether the Wnt/(3-catenin pathway is involved in the mitogenic action of PGE2 in human hepatocellular carcinoma cells.Methods:Reverse transcription and polymerase chain reaction (RT-PCR) were used to determine the COX-2 mRNA and EP receptor mRNA levels in human HCC cell line Hep3B and human normal hepatocyte lines QSG7701. Cell counting kit-8 (CCK-8) assay was employed to investigate the effect of PGE2, selective EP2 receptor agonist butaprost and EP3/EP4 receptor agonist PGE1 alcohol, selective COX-2 inhibitor nimesulide, on cell proliferation. A small interfering RNA (siRNA) down regulating COX-2 mRNA was transfected into Hep3B cells with lipofectamine 2000 and cell viability was assayed by using CCK-8 kit. The inserting sequences coding hairpin RNA (shRNA) homologous to COX-2 gene were designed and COX-2 shRNA expression vectors were constructed and transfected into Hep3B cells with lipofectamine 20000. G418 was used to select a population of cells that stably expressed the shRNA. The vector that efficiently suppressed COX-2 mRNA expression was determined by detection of COX-2 mRNA levels in transfected cells applying RT-PCR. Cell proliferation was assayed to evaluate whether COX-2 gene knock-down in Hep3B cells resulted in growth inhibition.Hep3B cells were transiently transfected with Renilla constructs and either TOPflash or FOPflash TCF reporter constructs using lipofectamine 20000, and luciferase activities present in cellular lysates were assayed using the Dual-Luciferase Reporter System. Immunoblottings were used to detect changes of protein levels of COX-2,β-catenin and phospho-p-catenin (Ser33/37/Thr41), GSK-3βand phospho-GSK-3p(Ser9), Akt and phospho-Akt(Thr308) in PGE2 treated or COX-2 gene knock-down Hep3B cells. The effects of wortmannin, a specific phosphatidylinositol-3-kinase (PI3K) inhibitor, on PGE2-induced phosphorylation of the above-mentioned proteins andβ-catenin/TCF/LEF transcription activity were further investigated.Results and Discussions:Results from RT-PCR showed that COX-2 mRNA was expressed in human HCC cell line Hep3B and hardly in human normal hepatocyte line QSG7701. Though the Hep3B cells expressed mRNAs for all EP receptor subtypes, EP2 and EP4 receptors mRNAs were much more strongly expressed than EP1 and EP3 receptors mRNAs. CCK-8 assays provided the evidence that PGE2 significantly improved Hep3B cell viability in a time- and dose-dependent manner.24 h of PGE2 (0.1-100μmol/L) exposure showed no significant mitogenic effect in Hep3B cells, however, 10μmol/L of PGE2 increased cell viability by 22.57% (P<0.001) after 48 h of incubation with Hep3B cells. Treatment with PGE2 (0.1-10μmol/L) for 72 h resulted in 12.13%(P＜0.01),17.58%(P<0.01) and 33.07% (P＜0.001) of elevated cell viability in Hep3B cells respectively, whereas 100μmol/L of PGE2 suppressed cell proliferation. In the next step, we determined which EP receptor mediated the mitogenic effect of PGE2 using selective EP receptor agonists. Results showed that EP2 receptor agonist butaprost at the 20μmol/L concentration substantially increased Hep3B cell proliferation to an extent similar to that of 10μmol/L PGE2, however, the EP3/EP4 receptor agonist PGE1 alcohol at concentrations of 2-20μmol/L exhibited no significant mitogenic effect in Hep3B cells, and 200μmol/L of PGE1 alcohol decreased cell viability. These data indicated that the EP2 receptor played a predominant role in the mediation of the stimulatory effect of PGE2.To further confirm the mitogenic action of COX-2 and PGE2 in Hep3B cells, effect of selective COX-2 inhibitor nimesulide cell proliferation was investigated by CCK-8 assay. It was shown that 72 h of nimesulide (10-200μmol/L) exposure significantly suppresses Hep3B cells growth in a concentration-dependent manner (P＜0.01), with the 50 percent inhibitory concentration (IC50) being 77.33μmol/L. Nimesulide caused significant growth inhibition (P<0.01) on Hep3B cells when the concentration was up to 25μmol/L, and the inhibitory rate was 17.58% (P<0.001). Cell viability of Hep3B was decreased by 43.28%(P<0.001),54.98%(P<0.001) and 72.85%(P<0.001) respectively when incubated with 50,100 and 200μmol/L of nimesulide. It was also showed that PGE2 significantly impaired the effect of nimesulide on cell growth. 100μmol/L of nimesulide decreased the cell viability of Hep3B to 44.65% of the control, and exposure of both 10μmol/L of PGE2 and 100μmol/L of nimesulide caused the cell viability of Hep3B to 80.99% of the control. The evidence which PGE2 attenuated the inhibitory effect of nimesulide demonstrated that nimesulide suppresses cell proliferation of hepatoma cells through inhibition of COX-2 activity and PGE2 production.Results from RT-PCR indicated that COX-2 mRNA expression in Hep3B cells transfected with COX-2 siRNA obviously decreased. Viability of Hep3B cells transfected with COX-2 siRNA showed time-dependent decreases after 24-72 h of transfection, which decreased to 87.34%±4.07%,75.30%±7.55% and 63.58%±3.41% of the control of the cells tansfected with negative control siRNA. It was indicated that COX-2 gene knock-down in Hep3B cells resulted in a significant growth inhibition.PCR and plasmid sequenced results indicated the successful construction of shRNA expression vectors. The Hep3B cells which stably expressed COX-2 shRNA were obtained by being transfected with COX-2 shRNA expression vector and selected by G418. It was shown that levels of COX-2 mRNA in these cells decreased significantly when compared with either the cells that stably expressed scrambled shRNA or the cells not being teransfected. A 45.98% of growth reduction was observed in cells stably expressing COX-2 shRNA as compared with cells not being teransfected (P＜0.01). The fact that COX-2 gene silencing in Hep3B cells resulted in growth inhibition proved the role of COX-2 in cell proliferation of HCC.Protein levels ofβ-catenin and phospho-β-catenin (Ser33/37/Thr41) in PGE2 treated Hep3B cells were assayed by western blotting. As shown in our paper, treatment of PGE2 from 30 min to 8 h significantly inhibited the phosphorylation ofβ-catenin, whereas it increased theβ-catenin protein level in Hep3B cells. The results demonstrated that activation ofβ-catenin pathway was involved in the stimulatory effect of PGE2.To further confirm the connection of COX-2 andβ-catenin in Hep3B cells, we examined the effects of COX-2 gene knock-down on the phosphorylation ofβ-catenin. It was shown that protein levels of COX-2 andβ-catenin both obviously decreased in Hep3B cells stably expressing COX-2 shRNA. Phosphorylation ofβ-catenin was stimulated by COX-2 shRNA. It was indicated that COX-2/PGE2 activatedβ-catenin in HCC cells.Because P-catenin regulates gene expression via binding as a transcription factor in complex with the TCF/LEF transcription factor family to the promoter region of target genes, we further examined the effect of PGE2 and EP2 agonist butaprost on TCF/LEF reporter activity in Hep3B cells. Theβ-catenin/TCF/LEF transcription activity was assayed after transient transfection of a luciferase reporter construct under the control ofβ-catenin/TCF/LEF response element. The results showed that PGE2 (0.1-10μmol/L) significantly enhanced theβ-catenin/TCF/LEF reporter activity in a concentration-dependent manner. The activity were 122.05%±13.34%, 167.79%±27.70%(P＜0.05) and 201.08%±25.47%(P＜0.01) of the control respectively in Hep3B cells treated with 0.1,1 and 10μmol/L of PGE2. It was also showed that 20μmol/L of butaprost increased theβ-catenin/TCF/LEF reporter activity to 193.05% of the control. These results further conformed activation ofβ-catenin by PGE2. Results from Dual-Luciferase Reporter System assays showed that the P-catenin/TCF/LEF transcription activity in Hep3B cells stably expressing COX-2 shRNA was decreased by 37.07%(P＜0.05), which indicated that COX-2 gene knock-down inactivated P-catenin signal in HCC cells.Effects of the COX-2 inhibitor nimesulide onβ-catenin-dependent transcriptional activity in cultured Hep3B cells were also investigated. 100μmol/L of nimesulide significantly decreased theβ-catenin/TCF/LEF activity to 54.47% of the control, however, treatment with both 100μmol/L of nimesulide and 10μmol/L of PGE2 only decreased theβ-catenin/TCF/LEF activity to 86.49% of the control. It was demonstrated that nimesulide suppressed P-catenin-dependent transcription and subsequently proliferation of hepatoma cells through inhibition of COX-2 activity and PGE2 production.In order to explore whether B-catenin was required for the mitogenic activity of PGE2, effect of reduction of cellular B-catenin concentration by siRNA on PGE2-induced cell proliferation was determined. Results showed that B-catenin siRNA inhibited the growth-promoting effect of this metabolic product of COX-2. lOμmol/L of PGE2 enhanced the cell viability to 29.52% of the control in the cells transfected with negative control siRNA, however, PGE2 exerted no significant influence on cell growth in the cells transfected with B-catenin siRNA. The key role ofβ-catenin in the growth-promoting effect of PGE2 was apparently confirmed.To further explore the pathway leading to B-catenin activation, GSK-3B, a kinase that phosphorylated B-catenin, thereby promoting its ubiquitin-dependent proteolytic degradation, was investigated. Treatment of 10μmol/L of PGE2 from 15min to 1h significantly stimulated the phosphorylation of GSK-3B, whereas it exerted no influence on the protein levels of GSK-3B. Because Akt was the main upstream kinase that phosphorylated GSK-3B, we examined the direct effects with PGE2 on the phosphorylation of this protein. Results showed that levels of phospho-Akt remarkably increased after 15min to 2h of PGE2 stimulation. Wortmannin(WM), a specific PI3K inhibitor, was found to abolish both basal and PGE2-induced phosphorylation of Akt and GSK-3B. PGE2 increasedβ-catenin/TCF/LEF transcription activity by 111.49% in Hep3B cells not pretreated with WM, however, theβ-catenin/TCF/LEF activity was enhanced only by 41.71% in cells pretreated with 50nmol/L of WM for 15 min. These results demonstrated that PGE2 stimulate B-catenin/TCF/LEF-dependent gene transcription through activation of PI3K-Akt pathway and GSK-3B phosphorylation.Conclusions:PGE2 and EP2 receptor agonist butaprost significantly improved cell viability in Hep3B cells expressing COX-2mRNA and EP receptor mRNAs, whereas the selective COX-2 inhibitor and COX-2 gene knock-down remarkably suppressed cell growth. These results demonstrated the mitogenic activity of COX-2 and PGE2 in HCC cells, and the predominant role of EP2 receptor in the mediation of the stimulatory effect of PGE2. Treatment of PGE2 significantly inhibited the phosphorylation of P-catenin, whereas it increased the P-catenin protein level andβ-catenin/TCF/LEF reporter activity in Hep3B cells. Phosphorylation of P-catenin was stimulated, and protein levels ofβ-catenin andβ-catenin/TCF/LEF transcription activity was decreased in Hep3B cells stably expressing COX-2 shRNA. PGE2 attenuated the inhibitory effect of nimesulide on theβ-catenin/TCF/LEF activity.β-catenin gene knock-down significantly inhibited the growth-promoting effect of PGE2. The above-mentioned results indicated that COX-2 and its metabolic product PGE2 exerted mitogenic effects through activation ofβ-catenin pathway. PGE2 significantly stimulated the phosphorylation of Akt and GSK-3B in Hep3B cells, and PI3K inhibitor WM abolished PGE2-induced phosphorylation of these proteins. WM was also found to reduce the stimulatory effect onβ-catenin/TCF/LEF activity. These results demonstrated that PGE2 stimulate B-catenin/TCF/LEF-dependent gene transcription through activation of PI3K-Akt pathway and GSK-3B phosphorylation.Taken together, our study confirmed that COX-2 and its metabolic product PGE2 promoted human hepatocellular carcinoma cells Hep3B growth through the Wnt/β-catenin pathway.
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