EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

PGE2-induced colon cancer growth is mediated by mTORC1

The inflammatory prostaglandin E₂ (PGE₂) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE₂ directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE₂-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE₂ increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE₂ EP₄ receptor was responsible for transducing the signal to mTORC1. Moreover, PGE₂ increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE₂-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE₂ increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE₂ mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.     

Prostaglandin E2/EP1 Signaling Pathway Enhances Intercellular Adhesion Molecule 1 (ICAM-1) Expression and Cell Motility in Oral Cancer Cells

Oral squamous cell carcinoma has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. However, the effects of COX-2 on human oral cancer cells are largely unknown. We found that overexpression of COX-2 or exogenous PGE₂ increased migration and intercellular adhesion molecule 1 (ICAM)-1 expression in human oral cancer cells. Using pharmacological inhibitors, activators, and genetic inhibition of EP receptors, we discovered that the EP1 receptor, but not other PGE receptors, is involved in PGE₂-mediated cell migration and ICAM-1 expression. PGE₂-mediated migration and ICAM-1 up-regulation were attenuated by inhibitors of protein kinase C (PKC)δ, and c-Src. Activation of the PKCδ, c-Src, and AP-1 signaling pathway occurred after PGE₂ treatment. PGE₂-induced expression of ICAM-1 and migration activity were inhibited by a specific inhibitor, siRNA, and mutants of PKCδ, c-Src, and AP-1. In addition, migration-prone sublines demonstrated that cells with increased migration ability had higher expression of COX-2 and ICAM-1. Taken together, these results indicate that the PGE₂ and EP1 interaction enhanced migration of oral cancer cells through an increase in ICAM-1 production.     

Evidence for a Pro-Proliferative Feedback Loop in Prostate Cancer: The Role of Epac1 and COX-2-Dependent Pathways

Objective

In human prostate cancer cells, a selective Epac agonist, 8-CPT-2Me-cAMP, upregulates cell proliferation and survival via activation of Ras-MAPK and PI- 3-kinase-Akt-mTOR signaling cascades. Here we examine the role of inflammatory mediators in Epac1-induced cellular proliferation by determining the expression of the pro-inflammatory markers p-cPLA2, COX-2, and PGE₂ in prostate cancer cells treated with 8-CPT-2Me-cAMP.

Methods

We employed inhibitors of COX-2, mTORC1, and mTORC2 to probe cyclic AMP-dependent pathways in human prostate cancer cells. RNAi targeting Epac1, Raptor, and Rictor was also employed in these studies.

Results

8-CPT-2Me-cAMP treatment caused a 2–2.5-fold increase of p-cPLA2^S505, COX-2, and PGE₂ levels in human prostate cancer cell lines. Pretreatment of cells with the COX-2 inhibitor SC-58125 or the EP4 antagonist AH-23848, or with an inhibitor of mTORC1 and mTORC2, Torin1, significantly reduced the Epac1-dependent increase of p-cPLA2 and COX-2, p-S6-kinase^T389, and p-AKT^S473. In addition, Epac1-induced protein and DNA synthesis were greatly reduced upon pretreatment of cells with either COX-2, EP4, or mTOR inhibitors. Transfection of prostate cancer cells with Epac1 dsRNA, Raptor dsRNA, or Rictor dsRNA profoundly reduced Epac1-dependent increases in p-cPLA2 and COX-2.

Conclusion

We show that Epac1, a downstream effector of cAMP, functions as a pro-inflammatory modulator in prostate cancer cells and promotes cell proliferation and survival by upregulating Ras-MAPK, and PI 3-kinase-Akt-mTOR signaling.     

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E₂ (PGE₂) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE₂ formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β).     

mTORC2 regulates PGE2-mediated endothelial cell survival and migration

Prostaglandin E₂ (PGE₂) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE₂-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE₂-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE₂-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE₂-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE₂-mediated endothelial cell responses.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

Prostaglandin E2 modulates components of the Wnt signaling system in bone and prostate cancer cells

Both Wnt signaling and prostaglandin E₂ (PGE₂) play pivotal roles in bone development, remodeling, osteoporosis and prostate cancer (PCa) bone metastases. We investigated the effects of PGE₂ on Wnt signaling in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells. We demonstrate that low dose PGE₂ (0.1 μM) promotes Wnt signaling while higher doses of PGE₂ (1.0-10 μM) inhibit these same parameters in osteoblast-lineage cells. The differential effects of low vs high-dose PGE₂ on pre-osteoblasts may be attributed to dose-dependent modulation of prostaglandin receptor (EP) subtype expression; with lower doses increasing the expression the cAMP-stimulatory EP4 receptor subtype and higher doses increasing the expression of the cAMP-inhibitory EP3 receptor subtype. Moreover, we demonstrate that high expression levels of COX-2 and PGE₂ promote the secretion of Wnt inhibitors from prostate cancer cells. These data demonstrate that there are dose-dependent effects of PGE₂ on Wnt activation in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells which may have clinical implications in the management.     

Adenosine and Prostaglandin E2 Cooperate in the Suppression of Immune Responses Mediated by Adaptive Regulatory T Cells

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4⁺CD25⁻ T cells and incubated with autologous immature dendritic cells, irradiated COX-2⁺ or COX-2⁻ tumor cells, and IL-2, IL-10, and IL-15 (each at 10–15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429–1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E₂ (PGE₂) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2⁺ tumor induced a greater number of Tr1 than COX-2⁻ tumor (p < 0.05). Tr1 induced by COX-2⁺ tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE₂ (p < 0.05) than Tr1 induced by COX-2⁻ tumor. Inhibitors of ectonucleotidase activity, A_2A and EP₂ receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE₄ inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE₂ and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.     

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

Objective Naturally occurring regulatory T (T_R) cells suppress autoreactive T cells whereas adaptive T_R cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive T_R cells express COX-2 and produce PGE₂ that suppress effector T cells in a manner that is reversed by COX-inhibitors. Methods and results Here we demonstrate that CRC patients have elevated levels of PGE₂ in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ T_R cells. Depletion of T_R cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity. Conclusion We suggest that adaptive T_R cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE₂-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting T_R cells and the PGE₂–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.     

Prostaglandin E2 produced by inducible COX-2 and mPGES-1 promoting cancer cell proliferation in vitro and in vivo

Aim

Many cancers originate and flourish in a prolonged inflammatory environment. Our aim is to understand the mechanisms of how the pathway of prostaglandin E₂ (PGE₂) biosynthesis and signaling can promote cancer growth in inflammatory environment at cellular and animal model levels.

Main methods

In this study, a chronic inflammation pathway was mimicked with a stable cell line that over-expressed a novel human enzyme consisting of cyclooxygenase isoform-2 (COX-2) linked to microsomal (PGE₂ synthase-1 (mPGES-1)) for the overproduction of pathogenic PGE₂. This PGE₂-producing cell line was co-cultured and co-implanted with three human cancer cell lines including prostate, lung, and colon cancers in vitro and in vivo, respectively.

Key findings

Increases in cell doubling rates for the three cancer cell types in the presence of the PGE₂-producing cell line were clearly observed. In addition, one of the four human PGE₂ subtype receptors, EP₁, was used as a model to identify PGE₂-signaling involved in promoting the cancer cell growth. This finding was further proven in vivo by co-implanting the PGE₂-producing cells line and the EP₁-positive cancer cells into the immune deficient mice, after that, it was observed that the PGE₂-producing cells promoted all three types of cancer formation in the mice.

Significance

This study clearly demonstrated that the human COX-2 linked to mPGES-1 is a pathway that, when mediated by the EP, is linked to promoting cancer growth in a chronic inflammatory environment. The identified pathway could be used as a novel target for developing and advancing anti-inflammation and anti-cancer interventions.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Role of leukotriene B4 in celecoxib-mediated anticancer effect

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has anticancer effect on many cancers associated with chronic inflammation by both COX-2-dependent and COX-2-independent mechanisms. The non-COX-2 targets of celecoxib, however, are still a matter of research. Leukotriene B₄ (LTB₄) has been implicated in prostate and colon carcinogenesis, but little is known about the potential role of LTB₄ in celecoxib-mediated anticancer effect. In this study, we evaluated whether LTB₄ was involved in celecoxib-mediated inhibitory effect on human colon cancer HT-29 cells and human prostate cancer PC-3 cells. Our data showed that survival of both cell lines was obviously suppressed after celecoxib treatment for 72 h in a concentration-dependent manner. However, only in HT-29 cells, this inhibitory effect could be reversed by LTB₄, which promoted survival of HT-29 cells rather than PC-3 cells. Consistent with these results, lioxygenase (LOX) potent inhibitor nordihydroguaiaretic acid (NDGA) had a higher inhibitory effect on HT-29 cells than PC-3 cells. Additionally, ELISA results showed that celecoxib could suppress expression of LTB₄ in both cell lines, whereas, inhibition of PGE₂ was only detected in HT-29 cells. These results indicate that the anticancer effect of celecoxib is COX-2-independent in HT-29 and PC-3 cells and in HT-29 cells primarily via down-regulating LTB₄ production.