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].

---

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].

---

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]

---

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.

---

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).

---

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.

---

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-thiophenecarboxamide is a member of biphenyls.

---

Background: Recent studies suggest that cyclooxygenase 2 (COX-2) inhibitors may enhance the toxic effects of anticancer drugs on tumor cells, including oral squamous cell carcinoma (OSCC), but its long-term use can cause side effects such as stomach ulcers and myocardial infarction. Our aim was to investigate proliferative effects of a downstream product of COX-2, prostaglandin E2 (PGE2), in human oral squamous carcinoma cell line Tca8113 and explore the effects of PGE2 receptors, especially EP4 receptor, on the growth of Tca8113 cells. Methods: To evaluate the effects of PGE2 and EP receptors on Tca8113 cells, CCK8 assay, Western blotting, cell cycle analysis, and apoptosis assay were performed. Results: We 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. Conclusions: Our results indicate that EP4 receptor mediates PGE2-induced cell proliferation through ERK signaling, and inhibition of EP4 receptor may represent an alternative therapeutic strategy for the prevention and treatment of OSCC.[1]

---

Accumulating evidence indicates that elevated levels of prostaglandin E(2) (PGE(2)) can increase intestinal epithelial cell proliferation, and thus play a role in colorectal tumorigenesis. PGE(2) exerts its effects through four G-protein-coupled PGE receptor (EP) subtypes, named the EP1, EP2, EP3, and EP4. Increased phosphorylation of extracellular regulated kinases (ERK1/2) is required for PGE(2) to stimulate cell proliferation of human colon cancer cells. However, the EP receptor(s) that are involved in this process remain unknown. We provide evidence that L-161,982, a selective EP4 receptor antagonist, completely blocks PGE(2)-induced ERK phosphorylation and cell proliferation of HCA-7 cells. In order to identify downstream target genes of ERK1/2 signaling, we found that PGE(2) induces expression of early growth response gene-1 (EGR-1) downstream of ERK1/2 and regulates its expression at the level of transcription. PGE(2) treatment induces phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 residue and CRE-mediated luciferase activity in HCA-7 cells. Studies with dominant-negative CREB mutant (ACREB) provide clear evidence for the involvement of CREB in PGE(2) driven egr-1 transcription in HCA-7 cells. In conclusion, this study reveals that egr-1 is a target gene of PGE(2) in HCA-7 cells and is regulated via the newly identified EP4/ERK/CREB pathway. Finally our results support the notion that antagonizing EP4 receptors may provide a novel therapeutic approach to the treatment of colon cancer.[2]

---

Background: To investigate the effects and potential mechanism of L161982 (a kind of EP4 antagonist) on the collagen-induced arthritis (CIA) mice model. Methods: The CIA mice model were first established by immunizing with Chicken Type II Collagen on DBA/1 mice. The CIA groups were administered once a day for 2 weeks with either 5 mg/kg L161982 by intraperitoneal injections (IP), 200 U celecoxib by intragastrical injections, or 100 μl PBS (IP). At the end of the study, total arthritis score and histopathologic examination were assessed to determine CIA severity. The plasma and tissue expressions of IL-17 and monocyte chemoattractant protein-1 (MCP-1) were detected by enzyme-linked immunosorbent assay (ELISA) and Immunohistochemical staining (IHC) respectively; The number of CD4+CD25+Foxp3+ regulatory T cells (Treg) determined as a proportion of total CD4+ cells in the lymph nodes and spleen. We also tested the proliferation of isolated Tregs and the ratio of Th17 polarization of Naïve T cells under the treatment of L161982 by BrdU assay and flow cytometry respectively. Results: 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; a decreased tendency of Th17 polarization in vitro were observed in L161982-treated naïve T cells. 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. The mechanism of the reduction of IL-17 in plasma or tissue after administration of L161982 might be potentially derived from the suppression of CD4+ T cells 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.

---

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