EP4

HCA-7 cell proliferation enhanced by PGE2 is inhibited by L-161982 (10 μM; 2 hours) [1]. In HCA-7 cells, PGE2-stimulated ERK phosphorylation is blocked by L-161982 (10 μM; 1 hour) [1]. In oral squamous cell carcinoma Tca8113 cells, L-161982 causes apoptosis, cell cycle arrest, and inhibits prostaglandin E2-induced proliferation [3].

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It was found that the EP4 receptor agonist, PGE1-OH, could mimick PGE2 rescued the inhibitory effect of celecoxib and induced cell growth via ERK phosphorylation, and the EP4 receptor antagonist, L-161,982, completely blocked PGE2-stimulated ERK phosphorylation and proliferation of Tca8113 cells. Furthermore, L-161,982 may induce apoptosis and block cell cycle progression at s phase by upregulating Bax and p21 protein levels and by downregulating Bcl-2, CDK2, and cyclin A2 protein levels.[1]
L-161,982 blocks PGE2-stimulated cell proliferation of HCA-7 cells.[2]
L-161,982 blocks PGE2-stimulated ERK phosphorylation.[2]
CREB phosphorylation by ERK is attenuated by L-161,982.[2]
L161982 suppress Th 17 differentiation of Naïve T cells in vitro. [3]

In mice with CIA, L-161982 (5 mg/kg; intraperitoneal injection; once daily for 2 weeks) decreases the development of arthritic lesions and their progression [2].

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L161982 treatment reduced arthritis lesions and lesion progression in CIA mice. L161982 reduced plasma and tissue IL-17 and MCP-1 expression in CIA mice. L161982 increased the ratio of Treg cells in CIA mice but could not promote the growth of Treg cells directly. [3]

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CIA mice treated with L161982 showed reduced arthritis scores, joint swellings, cracked cartilage surface, and less hyperplasia in the connective tissue of the articular cavity. Plasma and tissue IL-17 and MCP-1 decreased, while the proportion of Treg cells is increased both in the spleen and lymph nodes of CIA mice. Otherwise, L161982 have no direct effect on Tregs proliferation. Conclusion: Although less effective than Celecoxib, L161982 also resulted in a reduction of ankle joint inflammation in CIA mice. L161982 reduces the RA severity in CIA mice through inhibition of IL-17 and MCP-1, increasing Treg cells, and reducing inflammation[3].

Cell proliferation assay [1]
Cell Types: HCA-7 Cell
Tested Concentrations: 10 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Blocks PGE2-induced cell proliferation.

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Cell proliferation assay [2]
Cell proliferation in response to different treatments was measured using sulforhodamine B (SRB) assay as previously described. Briefly, ~8×104 HCA-7 cells were plated per well in a 6-well plate and allowed to grow for 24 h. Later the cells were serum starved for 24 h and pre-incubated with L-161,982 (10 μM) for 2 h before stimulating the cells with PGE2 for 72 h. Later the viable cells were fixed with cold 50% trichloroacetic acid (final concentration 10%) for 1 h at 4°C. Cells were washed with deionized water and stained by incubating with 0.4% SRB dye for 10 min at room temperature. Then the cells were washed with 1% acetic acid and the bound SRB dye was solubilized with 1M unbuffered Tris, mixed well, and the optical density (O.D.) was measured using a plate reader (Biomek @ 540 nm).

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In vitro proliferation assay [3]
CD4+CD25+ cells were purified from mice splenocytes by using the CD4+CD25+ Regulatory T Cell Isolation Kit by a negative selection procedure. Cells were cultured at a density of 2 x 103cells per well, and were stimulated with 0.5 μg/ml soluble mouse anti-CD3, 1 μg/ml anti-CD28 and 700 pg/ml PEG2 with or without 150 pg/ml L161982. After five days incubation at 37 ° C and 5% CO2, the cells were pulsed with BrdU (100 μM) and were assessed for BrdU incorporation 4 h later. Results are expressed as optical density (OD) at 405 nm.

Animal/Disease Models: 6 to 8 weeks old female DBA/1 mice (collagen-induced arthritis (CIA) mouse model) [1]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection; one time/day for 2 weeks
Experimental Results: 35 days after immunization, joint swelling diminished and arthritis scores diminished.

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Establishment of CIA mouse model and dosing regimen [3]
Forty mice were randomly divided into five groups, 8 mice per group: the control group and four CIA groups. For CIA groups, 200 μg of chicken type II collagen dissolved in DMSO was mixed with an equal volume of Freund’s Complete Adjuvant and emulsified in ice bath. Of this emulsion, 100 μl was administrated through intradermal injection at the base of the tail and this immunization was boosted 3 weeks later. For the model control group, the emulsion with Freund’s Complete Adjuvant but without chicken type II collagen was injected according to the same protocol. For the remaining CIA treatment groups, mice were treated firstly as the CIA group and then administered with 5 mg/kg of L161982 by intraperitoneal injections (IP), 200 U celecoxib by intragastrical injections (IA) or 100 μl PBS (IP) respectively as the previous studies. All injections were administered once per day for 2 weeks.

[1]. The EP4 antagonist, L-161,982, induces apoptosis, cell cycle arrest, and inhibits prostaglandin E2-induced proliferation in oral squamous carcinoma Tca8113 cells.J Oral Pathol Med. 2017 Nov;46(10):991-997.

[2]. The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells.Exp Cell Res. 2007 Aug 15;313(14):2969-79.

[3]. L161982 alleviates collagen-induced arthritis in mice by increasing Treg cells and down-regulating Interleukin-17 and monocyte-chemoattractant protein-1 levels.BMC Musculoskelet Disord. 2017 Nov 16;18(1):462.

N-[2-[4-[[3-butyl-5-oxo-1-[2-(trifluoromethyl)phenyl]-1,2,4-triazol-4-yl]methyl]phenyl]phenyl]sulfonyl-3-methyl-2-thiophene carboxamide is a member of the biphenyl class of compounds.

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Background: Recent studies have shown that cyclooxygenase 2 (COX-2) inhibitors may enhance the toxicity of anticancer drugs to tumor cells (including oral squamous cell carcinoma (OSCC)), but long-term use can lead to side effects such as gastric ulcers and myocardial infarction. This study aimed to investigate the effect of prostaglandin E2 (PGE2), a downstream product of COX-2, on the proliferation of the human oral squamous cell carcinoma cell line Tca8113, and to explore the effects of PGE2 receptors, particularly the EP4 receptor, on the growth of Tca8113 cells. Methods: To assess the effects of PGE2 and EP receptors on Tca8113 cells, we performed CCK8 assays, Western blotting, cell cycle analysis, and apoptosis assays. Results: We found that the EP4 receptor agonist PGE1-OH mimics PGE2, reverses the inhibitory effect of celecoxib, and induces cell proliferation through ERK phosphorylation; while the EP4 receptor antagonist L-161,982 completely blocks PGE2-stimulated ERK phosphorylation and Tca8113 cell proliferation. In addition, L-161,982 may induce apoptosis and arrest the S phase of the cell cycle by upregulating the levels of Bax and p21 proteins and downregulating the levels of Bcl-2, CDK2 and cyclin A2 proteins. Conclusion: Our results indicate that the EP4 receptor mediates PGE2-induced cell proliferation through the ERK signaling pathway, and inhibiting the EP4 receptor may represent an alternative therapeutic strategy for the prevention and treatment of oral squamous cell carcinoma (OSCC). [1]

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Increasing evidence suggests that elevated prostaglandin E2 (PGE2) levels can increase intestinal epithelial cell proliferation, thereby playing a role in colorectal tumorigenesis. PGE2 exerts its effects through four G protein-coupled PGE receptor (EP) isoforms, named EP1, EP2, EP3, and EP4. Prostaglandin E2 (PGE2) stimulation of human colon cancer cell proliferation requires elevated phosphorylation levels of extracellular signal-regulated kinases (ERK1/2). However, the specific EP receptors involved in this process remain unclear. Our evidence suggests that the selective EP4 receptor antagonist L-161,982 completely blocks PGE2-induced ERK phosphorylation and proliferation in HCA-7 cells. To identify downstream target genes in the ERK1/2 signaling pathway, we found that PGE2 induces expression of the ERK1/2 downstream early growth response gene 1 (EGR-1) and regulates its expression at the transcriptional level. PGE2 treatment induces phosphorylation of the cyclic adenosine monophosphate response element-binding protein (CREB) Ser133 site in HCA-7 cells and enhances CRE-mediated luciferase activity. Studies using dominant-negative CREB mutants (ACREB) have provided clear evidence that CREB is involved in PGE(2)-driven egr-1 transcription in HCA-7 cells. In summary, this study reveals that egr-1 is a target gene of PGE(2) in HCA-7 cells and is regulated through the newly discovered EP4/ERK/CREB pathway. Finally, our results support the possibility that antagonizing the EP4 receptor may provide a new therapeutic approach for colorectal cancer. [2]

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Background: To investigate the effects of L161982 (an EP4 antagonist) on a mouse model of collagen-induced arthritis (CIA) and its potential mechanisms. Methods: A CIA mouse model was first established by immunizing DBA/1 mice with chicken type II collagen. The CIA group was administered L161982 once daily for 2 weeks via intraperitoneal injection (IP) of 5 mg/kg, intragastric instillation of 200 U celecoxib, or intraperitoneal injection of 100 μl PBS. At the end of the study, the total arthritis score and histopathological examination results were assessed to determine the severity of CIA. The expression of IL-17 and monocyte chemoattractant protein-1 (MCP-1) in plasma and tissues was detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining (IHC), respectively. The proportion of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in lymph nodes and spleen was measured. We also used BrdU assay and flow cytometry to detect the proliferation of Treg cells isolated after L161982 treatment and the Th17 polarization ratio of naive T cells. Results: L161982 treatment reduced arthritis scores, joint swelling, and cartilage surface cracks in CIA mice, and also decreased connective tissue proliferation in the joint cavity. The levels of IL-17 and MCP-1 in plasma and tissues of CIA mice were decreased, while the proportion of Treg cells in spleen and lymph nodes increased. Furthermore, L161982 had no direct effect on Treg cell proliferation; in vitro, a reduced trend of Th17 polarization was observed in naive T cells treated with L161982. Conclusion: Although L161982 was less effective than celecoxib, it could also alleviate ankle joint inflammation in CIA mice. L161982 reduced the severity of RA in CIA mice by inhibiting IL-17 and MCP-1, increasing Treg cells, and reducing inflammation. The mechanism by which plasma or tissue IL-17 levels decreased after L161982 administration may be due to the inhibition of CD4+ T cell differentiation into Th-17 cells. [3]

C₃₂H₂₉F₃N₄O₄S₂

654.72

654.158

C, 58.70; H, 4.46; F, 8.71; N, 8.56; O, 9.77; S, 9.79

147776-06-5

9961192

White to off-white solid powder

1.37g/cm3

> 145 °C

1.633

8.071

1

9

10

45

1190

0

MMDNKTXNUZFVKD-UHFFFAOYSA-N

InChI=1S/C32H29F3N4O4S2/c1-3-4-13-28-36-39(26-11-7-6-10-25(26)32(33,34)35)31(41)38(28)20-22-14-16-23(17-15-22)24-9-5-8-12-27(24)45(42,43)37-30(40)29-21(2)18-19-44-29/h5-12,14-19H,3-4,13,20H2,1-2H3,(H,37,40)

N-[2-[4-[[3-butyl-5-oxo-1-[2-(trifluoromethyl)phenyl]-1,2,4-triazol-4-yl]methyl]phenyl]phenyl]sulfonyl-3-methylthiophene-2-carboxamide

L161982; 147776-06-5; L-161,982; L-161982; L 161,982; N-[2-[4-[[3-butyl-5-oxo-1-[2-(trifluoromethyl)phenyl]-1,2,4-triazol-4-yl]methyl]phenyl]phenyl]sulfonyl-3-methylthiophene-2-carboxamide; L161982; N-[2-[4-[[3-butyl-5-oxo-1-[2-(trifluoromethyl)phenyl]-1,2,4-triazol-4-yl]methyl]phenyl]phenyl]sulfonyl-3-methyl-2-thiophenecarboxamide; 2-Thiophenecarboxamide, N-[[4'-[[3-butyl-1,5-dihydro-5-oxo-1-[2-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-4-yl]methyl][1,1'-biphenyl]-2-yl]sulfonyl]-3-methyl-; L 161982

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~152.74 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)