Intense Innate Immune Responses and Severe Metabolic Disorders in Chicken Embryonic Visceral Tissues Caused by Infection with Highly Virulent Newcastle Disease Virus Compared to the Avirulent Virus: A Bioinformatics Analysis.

The highly virulent Newcastle disease virus (NDV) isolates typically result in severe systemic pathological changes and high mortality in Newcastle disease (ND) illness, whereas avirulent or low-virulence NDV strains can cause subclinical disease with no morbidity and even asymptomatic infections in birds. However, understanding the host's innate immune responses to infection with either a highly virulent strain or an avirulent strain, and how this response may contribute to severe pathological damages and even mortality upon infection with the highly virulent strain, remain limited. Therefore, the differences in epigenetic and pathogenesis mechanisms between the highly virulent and avirulent strains were explored, by transcriptional profiling of chicken embryonic visceral tissues (CEVT), infected with either the highly virulent NA-1 strain or the avirulent vaccine LaSota strain using RNA-seq. In our current paper, severe systemic pathological changes and high mortality were only observed in chicken embryos infected with the highly virulent NA-1 strains, although the propagation of viruses exhibited no differences between NA-1 and LaSota. Furthermore, virulent NA-1 infection caused intense innate immune responses and severe metabolic disorders in chicken EVT at 36 h post-infection (hpi), instead of 24 hpi, based on the bioinformatics analysis results for the differentially expressed genes (DEGs) between NA-1 and LaSota groups. Notably, an acute hyperinflammatory response, characterized by upregulated inflammatory cytokines, an uncontrolled host immune defense with dysregulated innate immune response-related signaling pathways, as well as severe metabolic disorders with the reorganization of host-cell metabolism were involved in the host defense response to the CEVT infected with the highly virulent NA-1 strain compared to the avirulent vaccine LaSota strain. Taken together, these results indicate that not only the host's uncontrolled immune response itself, but also the metabolic disorders with viruses hijacking host cell metabolism, may contribute to the pathogenesis of the highly virulent strain in ovo.

Prostaglandin E2 promotes human cholangiocarcinoma cell proliferation, migration and invasion through the upregulation of ß-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 ß-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 ß-catenin protein via EP3 receptors. The results showed that PGE2 promotes cholangiocarcinoma progression via the upregulation of ß-catenin protein, and the EP3-4 receptor pathway is mainly responsible for this regulation. These findings reveal that PGE2 upregulated the cholangiocarcinoma cell ß-catenin protein through the EP3-4R/Src/EGFR/PI3K/AKT/GSK-3ß pathway. The present study identified the functions of EP3 and the mechanisms by which PGE2 regulates ß-catenin expression and promoted cholangiocarcinoma cell growth and invasion.

Prostaglandin E2 promotes the cell growth and invasive ability of hepatocellular carcinoma cells by upregulating c-Myc expression via EP4 receptor and the PKA signaling pathway.

Hepatocellular carcinoma (HCC) represents a major health problem worldwide. Prostaglandin E2 (PGE2), the predominant product of cyclooxygenase-2, has been implicated in hepatocarcinogenesis. However, the underlying molecular mechanisms remain to be further elucidated. c-myc, a cellular proto-oncogene, is activated or overexpressed in many types of human cancer, including HCC. The present study was designed to investigate the internal relationship and molecular mechanisms between PGE2 and c-Myc in HCC, and to define its role in HCC cell growth and invasion. Our results showed that PGE2 significantly upregulated c-Myc expression at both the mRNA and protein levels, and knockdown of c-Myc blocked PGE2-induced HCC cell growth and invasive ability in human HCC Huh-7 cells. The effect of PGE2 on c-Myc expression was mainly through the EP4 receptor, and EP4 receptor-mediated c-Myc protein upregulation largely depended on de novo biosynthesis of c-Myc mRNA and its protein. EP4 receptor signaling activated GS/AC and increased the intracellular cAMP level in Huh-7 cells. The adenylate cyclase (AC) activator forskolin mimicked the effects of the EP4 receptor agonist on c-Myc expression, while the AC inhibitor SQ22536 reduced EP4 receptor-mediated c-Myc upregulation. These data confirm the involvement of the GS/AC/cAMP pathway in EP4 receptor-mediated c-Myc upregulation. Moreover, the phosphorylation levels of CREB protein were markedly elevated by EP4 receptor signaling, and by using specific inhibitor and siRNA interference, we demonstrated that PKA/CREB was also involved in the EP4 receptor-mediated c-Myc upregulation. In summary, the present study revealed that PGE2 significantly upregulates c-Myc expression at both mRNA and protein levels through the EP4R/GS/AC/cAMP/PKA/CREB signaling pathway, thus promoting cell growth and invasion in HCC cells. Targeting of the PGE2/EP4R/c-Myc pathway may be a new therapeutic strategy to prevent and cure human HCC.

Prostaglandin E2 accelerates invasion by upregulating Snail in hepatocellular carcinoma cells.

Our previous studies showed that prostaglandin E2 (PGE2) promotes hepatoma cell growth and migration, as well as invasion; however, the precise mechanism remains elusive. Snail and p65 protein levels were detected in human samples with hepatocellular carcinoma (HCC) by immunohistochemistry (IHC) staining. HCC cell lines (Huh-7 and Hep3B) were used for in vitro experiments. PGE2/Akt/NF-κB pathway was investigated in Huh-7 and Hep3B cells after treatment with PGE2, EP4 receptor (EP4R) agonist, Akt inhibitor, and NF-κB inhibitor, respectively, by real-time reverse transcription (RT)-PCR, Western blotting, and immunofluorescence (IF) staining. In vitro cell invasion assay was performed to evaluate the effect of PGE2 on tumor invasiveness. Knockdown of EP4R was carried out in Huh-7 cells through plasmid-based small interfering RNA (siRNA) approach to confirm the regulation of PGE2 on Snail by EP4R. Dual luciferase reporter assay was performed to assess Snail promoter activity in Huh-7 cell after treatment with EP4R agonist. We found that the protein levels of Snail were higher in HCC tissues than those in control and that PGE2 and EP4R agonist treatment significantly increased Snail expression in Huh-7 and Hep3B cells. EP4R agonist also profoundly promoted invasiveness of Huh-7 cells. Knockdown of the EP4R by siRNA completely blocked the PGE2-induced upregulation of Snail expression and reduced invasiveness of Huh-7 cells. We failed to find that EP4R-induced upregulation of Snail was reversed by inhibition of cAMP response element-binding protein (CREB), a canonical downstream target of EP4R. Alternatively, EP4R agonist treatment significantly increased the levels of phosphorylated EGFR and Akt both in Huh-7 and Hep3B cells. AG1478, an EGFR inhibitor, blocked the phosphorylation of Akt. The levels of phosphorylated IκB increased in Huh-7 cells after treatment with EP4R agonist for 30 min. The levels of phosphorylated p65 started to increase in Huh-7 cells treated with EP4R agonist for 4 h, and p65 translocated into the nucleus. In EP4R-agonist-treated Hep3B, the levels of phosphorylated p65 were also increased compared to the control group. The phosphorylation levels of p65 were significantly decreased in Huh-7 and Hep3B cells after treatment with the Akt signaling inhibitor LY294002 and EP4R agonist for 24 h. Treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) at 10 µM for 24 h blocked EP4R-agonist-induced Snail upregulation in Huh-7 and Hep3B cells. Furthermore, we obtained human Snail promoter sequence from TRED-Promoter Database and identified a putative binding site of NF-κB in the sequence through TFSEARCH analysis. Subsequently, we treated Huh-7 cells with EP4R agonist or EP4R agonist and PDTC (NF-κB antagonist) and found significantly increased Snail promoter activity after EP4R agonist treatment for 12 h. The increased Snail promoter activity could be partially abolished by additional PDTC treatment. In addition, p65 protein levels were found increased together with Snail in HCC tissues compared to normal liver tissues. In conclusion, PGE2 activates Akt/NF-κB signaling and then upregulates Snail via the EP4R/EGFR to promote migration and invasion in hepatoma cells. These findings may help future evaluation of novel chemo-preventive strategies for HCC.

Prostaglandin E2 upregulates ß1 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. ß1 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 ß1 integrin expression. The present study investigated the effect of EP1 receptor activation on ß1 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 ß1 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 ß1 integrin expression were investigated on A549 cells. The expression of ß1 integrin and the phosphorylation of NF-κB­p65 Ser536 was investigated by western blot analysis. Cell migration was assessed by a transwell assay. The results demonstrated that ß1 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 ß1 integrin monoclonal antibody inhibited 17-PT-PGE2­mediated cell migration completely. PGE2 and 17-PT-PGE2 treatment increased ß1 integrin expression. RNA interference against the EP1 receptor blocked the PGE2-mediated ß1 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 ß1 integrin expression. In conclusion, the present study indicated that the PGE2 EP1 receptor regulates ß1 integrin expression and cell migration in NSCLC cells by activating the NF-κB signaling pathway. Targeting the PGE2/EP1/ß1 integrin signaling pathway may aid in the development of new therapeutic strategies for the prevention and treatment of this type of cancer.

Prostaglandin E2 receptor EP2 mediates Snail expression in hepatocellular carcinoma cells.

Prostaglandin E2 (PGE2) has been shown to influence cell invasion and metastasis in several types of cancer, including hepatocellular carcinoma (HCC). however, the molecular mechanisms underlying it remain to be further elucidated. Snail, as one of key inducers of epithelial-mesenchymal transition (EMT), plays pivotal roles in HCC invasion and metastasis. The present study was designed to evaluate the possible signaling pathways through which PGE2 regulates Snail protein expression in HCC cell lines. PGE2 markedly enhanced Huh-7 cell invasion and migration ability by upregulating the expression level of Snail protein, and EP2 receptor played an important role in this process. Src, EGFR, Akt and mTOR were all activated and involved in the regulation of snail protein expression. Our findings suggest that PGE2 could upregulate the expression level of Snail protein through the EP2/Src/EGFR/Akt/mTOR pathway in Huh-7 cells, which promotes HCC cell invasion and migration.

Prostaglandin E2 promotes hepatocellular carcinoma cell invasion through upregulation of YB-1 protein expression.

Prostaglandin E2 (PGE2) has been implicated in hepatocellular carcinoma cell invasion. Recently, it was reported that Y box-binding protein 1 (YB-1) is closely correlated with malignancy. This study was designed to examine the mechanisms by which PGE2 increases YB-1 expression and promotes HCC cell invasion. PGE2 greatly enhanced HCC cell invasion through upregulation of the YB-1 protein, and the EP1 receptor is mainly responsible for this regulation. Src and EGFR were both activated by PGE2, which in turn increased the phosphorylation levels of p44/42 MAPK. Src, EGFR and p44/42 MAPK were all involved in PGE2-induced YB-1 expression. Chemical inhibitors and RNAi analysis all confirmed the role of mTOR complex 1 in YB-1 expression induced by PGE2. Furthermore, YB-1 was able to regulate the expression of a series of EMT-associated genes, which indicated that YB-1 could have the potential to control the epithelial-mesenchymal transition process in HCC cells. These findings reveal that PGE2 upregulated YB-1 expression through the EP1/Src/EGFR/p44/42 MAPK/mTOR pathway, which greatly enhanced HCC cell invasion. This study for the first time describes the mechanisms through which PGE2 regulates YB-1 expression and promotes HCC cell invasion.

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.