Journal: British Journal of Pharmacology

Article Title: Small‐molecule inhibition of prostaglandin E receptor 2 impairs cyclooxygenase‐associated malignant glioma growth

doi: 10.1111/bph.14622

Figure Lengend Snippet: PGE2 mediates cAMP signalling in human malignant glioma cells via EP2 receptor. (a) Stimulation with PGE2 (10 μM) or forskolin (100 μM) induced the biosynthesis of cAMP in human GBM cells LN229 and SF767, measured by a cell‐based assay using the time‐resolved FRET (TR‐FRET; n = 5, *P < 0.05 compared with control group, one‐way ANOVA and post hoc Dunnett's test). Data are shown as mean + SEM. (b) PGE2 increased cAMP in GBM cells in a concentration‐dependent manner. The PGE2 EC50s: 150 nM for LN229 and 3 nM for SF767 cells. Note that the cell response was approximately maximized with 0.1 and 1 μM PGE2 for SF767 and LN229 cells respectively. Exploratory data are shown as mean ± SEM (n = 4). (c) Chemical structures of EP2‐selective antagonist TG4‐155 and the current lead compound TG6‐10‐1. (d) Radioligand binding assay was performed to evaluate the affinity of TG6‐10‐1 to the human EP2 receptor by measuring its inhibition of binding of [3H]‐PGE2 (3 nM) to the cell membrane homogenates (Jiang et al., 2012). The average of two independent measurements of radio‐labelled PGE2 binding was plotted against the increasing concentrations of TG6‐10‐1, and the error bars were not displayed. The compound showed an IC50 of 350 nM and a K i of 175 nM on the human EP2 receptor. (e) With systemic administration to mice (10 mg·kg−1, p.o.), compound TG6‐10‐1 showed a plasma terminal t 1/2 of 1.8 hr and brain‐to‐plasma concentration ratio of 1.2 (Ganesh, Jiang, & Dingledine, 2014; Ganesh, Jiang, Yang, & Dingledine, 2014). Exploratory data are shown as mean ± SEM (n = 3 mice per time point). The compound Schild K B values for eight canonical prostanoid receptors are also indicated: 74.6, 762, 7.98, 2,380, 7,740, 202, 3,640, and 193 ng·ml−1 for DP1, EP1, EP2, EP3, EP4, FP, IP, and TP receptors respectively. (f) The PANCAN‐normalized expression of PGD2 receptor DP1 in human LGG and GBM tissues (n = 530 for LGG and 172 for GBM, N.S.: not significant, Student's t‐test). Data are shown as mean + SEM. (g) The relationship between survival probability of glioma patients (n = 690) and the expression of the DP1 receptor gene in their tumours was shown by Kaplan–Meier estimator with post hoc log‐rank test (P = 0.4469). (h) Inhibition of cAMP production in human GBM cells LN229 and SF767 by TG4‐155 and TG6‐10‐1 in response to stimulation of PGE2 (1 μM for LN229 cells; 0.1 μM for SF767 cells). Data were normalized and presented as a percentage of maximum response; points represent mean ± SEM (n = 6)

Article Snippet: The cells were lysed in 10 μl lysis buffer containing the FRET acceptor cAMP‐d2, and 1 min later, another 10 μl lysis buffer with anti‐cAMP‐cryptate was added (Cisbio Bioassays, Cat # 62AM4PEC).

Techniques: Cell Based Assay, Concentration Assay, Radio Ligand Binding Assay, Inhibition, Binding Assay, Expressing