Xiaoming Bai

Prostaglandin E2 upregulates survivin expression via the EP1 receptor in hepatocellular carcinoma cells

AIMS Cyclooxygenase-2 (COX-2)-controlled production of prostaglandin E(2) (PGE(2)) has been implicated in cell growth and metastasis in many cancers. Recent studies have found that COX-2 is co-expressed with survivin in many cancers. Survivin is a member of the inhibitor-of-apoptosis protein family. Some COX-2 inhibitors (e.g., celecoxib) can reduce the expression of survivin. However, little is known about the mechanism of PGE(2)-mediated expression of survivin. This study was designed to uncover the effect of PGE(2) on survivin expression in hepatocellular carcinoma cells. MAIN METHODS The effects of PGE(2) and EP1 agonist on survivin expression were examined in HUH-7 and HepG2 cells. Plasmid transfection and EP1 siRNA were used to regulate the expression of COX-2 and the EP1 receptor protein. KEY FINDINGS PGE(2) treatment increased survivin expression 2.3-fold. COX-2 overexpression resulted in a similar level of survivin upregulation. However, this effect was suppressed by treatment with celecoxib. EP1 receptor transfection or treatment with a selective EP1 agonist mimicked the effect of PGE(2) treatment. Conversely, the PGE(2)-induced upregulation of survivin was blocked by treatment with a selective EP1 antagonist or siRNA against the EP1 receptor. The phosphorylation of EGFR and Akt were elevated in EP1 agonist-treated cells, and both EGFR and PI3K inhibitors suppressed the upregulation of survivin induced by PGE(2) or EP1 agonist. SIGNIFICANCE PGE(2) regulates survivin expression in hepatocellular carcinoma cells through the EP1 receptor by activating the EGFR/PI3K pathway. Targeting the PGE(2)/EP1/survivin signaling pathway may aid the development of new therapeutic strategies for both the prevention and treatment of this cancer.

Prostaglandin E2 stimulates β1-integrin expression in hepatocellular carcinoma through the EP1 receptor/PKC/NF-κB pathway.

Prostaglandin E2 (PGE2) has been implicated in cell invasion in hepatocellular carcinoma (HCC), via increased β1-integrin expression and cell migration; however, the mechanism remains unclear. PGE2 exerts its effects via four subtypes of the E prostanoid receptor (EP receptor 1–4). The present study investigated the effect of EP1 receptor activation on β1-integrin expression and cell migration in HCC. Cell migration increased by 60% in cells treated with 17-PT-PGE2 (EP1 agonist), which was suppressed by pretreatment with a β1-integrin polyclonal antibody. PGE2 increased β1-integrin expression by approximately 2-fold. EP1 receptor transfection or treatment with 17-PT-PGE2 mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. 17-PT-PGE2 treatment induced PKC and NF-κB activation; PKC and NF-κB inhibitors suppressed 17-PT-PGE2-mediated β1-integrin expression. FoxC2, a β1-integrin transcription factor, was also upregulated by 17-PT-PGE2. NF-κB inhibitor suppressed 17-PT-PGE2-mediated FoxC2 upregulation. Immunohistochemistry showed p65, FoxC2, EP1 receptor and β1-integrin were all highly expressed in the HCC cases. This study suggested that PGE2 upregulates β1-integrin expression and cell migration in HCC cells by activating the PKC/NF-κB signaling pathway. Targeting PGE2/EP1/PKC/NF-κB/FoxC2/β1-integrin pathway may represent a new therapeutic strategy for the prevention and treatment of this cancer.

Prostaglandin E2 promotes liver cancer cell growth by the upregulation of FUSE-binding protein 1 expression

Liver cancer is a common human cancer with a high mortality rate and currently there is no effective chemoprevention or systematic treatment. Recent evidence suggests that prostaglandin E(2) (PGE(2)) plays an important role in the occurrence and development of liver cancer. However, the mechanisms through which PGE(2) promotes liver cancer cell growth are not yet fully understood. It has been reported that the increased expression of FUSE-binding protein 1 (FBP1) significantly induces the proliferation of liver cancer cells. In this study, we report that PGE(2) promotes liver cancer cell growth by the upregulation of FBP1 protein expression. Treatment with PGE2 and the E prostanoid 3 (EP3) receptor agonist, sulprostone, resulted in the time-dependent increase in FBP1 protein expression; sulprostone increased the viability of the liver cancer cells. The protein kinase A (PKA) inhibitor, H89, and the adenylate cyclase (AC) inhibitor, SQ22536, inhibited the cell viability accelerated by sulprostone. By contrast, the Gi subunit inhibitor, pertussis toxin (PTX), exhibited no significant effect. Treatment with PGE(2) and sulprostone caused a decrease in JTV1 protein expression, blocked the binding of JTV1 with FBP1, which served as a mechanism for FBP1 degradation, leading to the decreased ubiquitination of FBP1 and the increase in FBP1 protein expression. Furthermore, H89 and SQ22536 prevented the above effects of JTV1 and FBP1 induced by PGE(2) and sulprostone. These findings indicate that the EP3 receptor activated by PGE(2) may couple to Gs protein and activate cyclic AMP (cAMP)-PKA, downregulating the levels of JTV1 protein, consequently inhibiting the ubiquitination of FBP1 and increasing FBP1 protein expression, thus promoting liver cancer cell growth. These observations provide new insights into the mechanisms through which PGE(2) promotes cancer cell growth.

Prostaglandin E2 receptor EP1 phosphorylate CREB and mediates MMP2 expression in human cholangiocarcinoma cells

Cyclooxygenase-2 (COX-2) and COX-2-induced prostaglandin E2 (PGE2) have been implicated in all stages of malignant tumorigenesis. Although many aspects of matrix metalloproteinase (MMP2) on tumor invasion have been studied, the exact mechanism of PGE2-induced MMP2 overproduction has not been clearly defined. We have previously demonstrated that PGE2-enhanced extracellular signal-regulated kinase (Erk) phosphorylation via EP1 signaling pathway involved in PGE2-induced cell proliferation. Based on the identification of the transcription factor cyclic AMP response element-binding protein (CREB) as an important regulator of MMP2 and Erk phosphorylate CREB at ser133, we hypothesize that CREB may be implicated in the signaling of PGE2 stimulation to MMP2 overproduction via EP1 receptor. In the study, we investigated the role of EP1 receptor on PGE2-induced MMP2 expression and delineated the signaling pathway that contributes to EP1 receptor modulation of MMP2 in human cholangiocarcinoma cells. We found PGE2 or selective EP1 receptor agonist 17-P-T-PGE2-stimulated MMP2 expression and selective EP1 receptor antagonist SC-51322 or EP1 receptor siRNA abrogated PGE2-induced MMP2 expression. Intracellular calcium chelator BAPTA-AM, the selective inhibitor of EGFR AG1478 and the selective inhibitor of Erk PD98059 blocked EP1 receptor activation-induced CREB phosphorylation and MMP2 expression. A novel dominant-negative (D-N) inhibitor protein of the CREB, termed A-CREB, attenuated EP1 receptor activation-induced MMP2 expression. Our findings suggest that PGE2-enhanced MMP2 expression is, at least in part, mediated through EP1 receptors and calcium signaling pathway-induced CREB phosphorylation in human cholangiocarcinoma cells.

Prostaglandin E2 receptor EP1-mediated phosphorylation of focal adhesion kinase enhances cell adhesion and migration in hepatocellular carcinoma cells

The prostaglandin E₂ (PGE₂) EP1 receptor has been implicated in hepatocellular carcinoma (HCC) cell invasion. However, little is known about the mechanisms of EP1 receptor-mediated cell adhesion and migration. We previously showed that PGE₂ promotes cell adhesion and migration by activating focal adhesion kinase (FAK). The present study was designed to elucidate the association between the EP1 receptor and FAK activation in HCC cells and to investigate the related signaling pathways. The effects of PGE₂, EP1 agonist 17-phenyl trinor-PGE₂ (17-PT-PGE₂), PKC and EGFR inhibitors on FAK activation were investigated by treatment of Huh-7 cells. Phosphorylation of FAK Y397 and c-Src Y416 was investigated by western blotting. Cell adhesion and migration were analyzed by WST and transwell assays, respectively. Protein kinase C (PKC) activity was measured with a PKC assay kit. The results showed that 17-PT-PGE₂ (3 µM) increased FAK Y397 phosphorylation by more than 2-fold and promoted cell adhesion and migration in Huh-7 cells. In transfected 293 cells, expression of the EP1 receptor was confirmed to upregulate FAK phosphorylation, while the EP1 receptor antagonist sc-19220 decreased PGE₂-mediated FAK activation. PKC activity and c-Src Y416 phosphorylation were enhanced after 17-PT-PGE₂ treatment. Both PKC and c-Src inhibitor suppressed the 17-PT-PGE₂-upregulated FAK phosphorylation, as well as 17-PT-PGE₂-induced cell adhesion and migration. In addition, exogenous epidermal growth factor (EGF) treatment increased FAK phosphorylation. The EGF receptor (EGFR) inhibitor also suppressed 17-PT-PGE₂-upregulated FAK phosphorylation. Our study suggests that the PGE₂ EP1 receptor regulates FAK phosphorylation by activating the PKC/c-Src and EGFR signal pathways, which may coordinately regulate adhesion and migration in HCC.

Prostaglandin E2 promotes human cholangiocarcinoma cell proliferation, migration and invasion through the upregulation of β-catenin expression via EP3-4 receptor

Prostaglandin E2 (PGE2) is involved in cholangiocarcinoma cell proliferation, migration and invasion through E prostanoid receptors, including EP1, EP2 and EP4. However, the functions and the mechanisms of those splice variants of EP3 receptors in promoting liver cancer cell growth and invasion remain to be elucidated. In our previous studies, four isoforms of EP3 receptors, EP3-4, EP3-5, EP3-6 and EP3-7 receptors, were detected in CCLP1 and HuCCT1 cells. However, the functions of these receptors in these cells have yet to be determined. It was reported that β-catenin is closely correlated with malignancy, including cholangiocarcinoma. The present study was designed to examine the effects of 4-7 isoforms of EP3 in promoting cholangiocarcinoma progression and the mechanisms by which PGE2 increases β-catenin protein via EP3 receptors. The results showed that PGE2 promotes cholangiocarcinoma progression via the upregulation of β-catenin protein, and the EP3-4 receptor pathway is mainly responsible for this regulation. These findings reveal that PGE2 upregulated the cholangiocarcinoma cell β-catenin protein through the EP3-4R/Src/EGFR/PI3K/AKT/GSK-3β pathway. The present study identified the functions of EP3 and the mechanisms by which PGE2 regulates β-catenin expression and promoted cholangiocarcinoma cell growth and invasion.

Cyclooxygenase-2 induced β1-integrin expression in NSCLC and promoted cell invasion via the EP1/MAPK/E2F-1/FoxC2 signal pathway

Cyclooxygenase-2 (COX-2) has been implicated in cell invasion in non-small-cell lung cancer (NSCLC). However, the mechanism is unclear. The present study investigated the effect of COX-2 on β1-integrin expression and cell invasion in NSCLC. COX-2 and β1-integrin were co-expressed in NSCLC tissues. COX-2 overexpression or Prostaglandin E2 (PGE2) treatment increased β1-integrin expression in NSCLC cell lines. β1-integrin silencing suppressed COX-2-mediated tumour growth and cancer cell invasion in vivo and in vitro. Prostaglandin E Receptor EP1 transfection or treatment with EP1 agonist mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. EP1 agonist treatment promoted Erk1/2, p38 phosphorylation and E2F-1 expression. MEK1/2 and p38 inhibitors suppressed EP1-mediated β1-integrin expression. E2F-1 silencing suppressed EP1-mediated FoxC2 and β1-integrin upregulation. ChIP and Luciferase Reporter assays identified that EP1 agonist treatment induced E2F-1 binding to FoxC2 promotor directly and improved FoxC2 transcription. FoxC2 siRNA suppressed β1-integrin expression and EP1-mediated cell invasion. Immunohistochemistry showed E2F-1, FoxC2, and EP1R were all highly expressed in the NSCLC cases. This study suggested that COX-2 upregulates β1-integrin expression and cell invasion in NSCLC by activating the MAPK/E2F-1 signalling pathway. Targeting the COX-2/EP1/PKC/MAPK/E2F-1/FoxC2/β1-integrin pathway might represent a new therapeutic strategy for the prevention and treatment of this cancer.

Prostaglandin E2 upregulates β1 integrin expression via the E prostanoid 1 receptor/nuclear factor κ-light-chain-enhancer of activated B cells pathway in non-small-cell lung cancer cells.

The prostaglandin E2 (PGE2) E prostanoid (EP)1 receptor shown to be associated with lung cancer cell invasion. However, the mechanism of EP1 receptor-mediated cell migration remains to be elucidated. β1 integrin is an essential regulator of the tumorigenic properties of non-small-cell lung carcinoma (NSCLC) cells. To date, little is known regarding the association between the EP1 receptor and β1 integrin expression. The present study investigated the effect of EP1 receptor activation on β1 integrin expression and cell migration in NSCLC cells. A total of 34 patients with clinical diagnosis of NSCLC and 10 patients with benign disease were recruited for the present study. The expression levels of the EP1 receptor and β1 integrin expression were studied in resected lung tissue using immunohistochemistry. A statistical analysis was performed using Stata se12.0 software. The effects of PGE2, EP1 agonist 17-phenyl trinor-PGE2 (17-PT-PGE2) and the nuclear factor κ-B (NF-κB) inhibitor on β1 integrin expression were investigated on A549 cells. The expression of β1 integrin and the phosphorylation of NF-κB‑p65 Ser536 was investigated by western blot analysis. Cell migration was assessed by a transwell assay. The results demonstrated that β1 integrin and EP1 receptor expression exhibited a positive correlation of evident significance in the 44 samples. The in vitro migration assay revealed that cell migration was increased by 30% when the cells were treated with 5 µM 17-PT-PGE2 and that the pre-treatment of β1 integrin monoclonal antibody inhibited 17-PT-PGE2‑mediated cell migration completely. PGE2 and 17-PT-PGE2 treatment increased β1 integrin expression. RNA interference against the EP1 receptor blocked the PGE2-mediated β1 integrin expression in A549 cells. Treatment with 17-PT-PGE2 induced NF-κB activation, and the selective NF-κB inhibitor pyrrolidinedithiocarbamate inhibited 17-PT-PGE2-mediated β1 integrin expression. In conclusion, the present study indicated that the PGE2 EP1 receptor regulates β1 integrin expression and cell migration in NSCLC cells by activating the NF-κB signaling pathway. Targeting the PGE2/EP1/β1 integrin signaling pathway may aid in the development of new therapeutic strategies for the prevention and treatment of this type of cancer.