The Anti-tumor Activity Of Novel EP4 Receptor Antagonist And Detection Of EP4/β-arrestin Interaction

The tumor microenvironment plays an important role in tumor initiation,progression and metastasis.PGE_2,a pro-inflammatory factor,and its actions lead to immunosuppressive condition.EP4 receptor has emerged as a major contributor to PGE₂-mediated involvement of development of tumors by affecting the function of immune cells.Consequently,the new EP4 receptor antagonists have significant potential for tumor immunotherapy.In this study,we screened a series of compounds by calcium flux assay.A representative compound YJ-4-8 was selected for further experiments.Results showed that YJ-4-8 could inhibit downstream pathway of EP4,such as c AMP accumulation(IC₅₀=18.7±0.6 n M),CRE reporter gene(IC₅₀=5.2±0.4 n M)and ERK1/2 activation.At the same time,YJ-4-8 showed high selectivity for the EP4 receptor over other EP receptors.In competitive experiments,YJ-4-8 was shown to be a PGE₂ competitive antagonist.Meanwhile YJ-4-8 could significantly down-regulate the expression of genes associated with immune suppression（IL-6,IL-1β,ARG1,etc.）caused by PGE₂in Raw264.7 cells.In order to verify the anti-tumor activity of YJ-4-8 in vivo,we established a CT-26 allograft model for abnormal activation of PGE₂-EP4 in colon cancer and found YJ-4-8 could remarkably suppress the growth of tumor without causing systematical toxicity in mice.Cytotoxicity assay suggested that YJ-4-8 could not kill tumor cells directly(IC₅₀>100μM).We then analyzed the cytotoxic T lymphocyte in the tumor tissue,and found that the infiltration of CD8⁺T（CD45⁺CD8⁺）cells was significantly increased in the YJ-4-8 administration group.We also observed the increasing of CD8⁺T cells in the immunofluorescence experiment,indicating the improved anti-tumor immunity.In summary,YJ-4-8 was a novel,potent and specific EP4 receptor antagonist,and it could suppress the growth of tumor through the enhaced anti-tumor immunity.The study showed EP4 receptor antagonists may provide a novel therapeutic approach to tumor immunotherapy.β-arrestin is a critical transducer and regulator protein for GPCRs.β-arrestin recruitment can be used to the detection of GPCRs activation.In this study,we constructed two experimental systems for detection of EP4/β-arrestin interaction:the Tango arrestin assay and the Nano Bit protein-protein interaction（PPI）assay.YJ-4-8could inhibit EP4/β-arrestin interation induced by PGE₂ in these two assays.These assays can be futher used to evaluate the acticvity of GPCRs ligands.