Neuroblastoma cells produce PGE₂ and dmPGE₂ increases cell viability.

<p>(A) Neuroblastoma cells produce PGE₂. SK-N-BE(2) and SK-N-SH cells were cultured with or without 40 µM of arachidonic acid (AA) for 48 h and 10 ng/mL IL-1β for 12 h. Cell homogenates were incubated with 80 µM of arachidonic acid and the concentration of produced PGE₂ was measured using LC-MS/MS. (B) PGE₂ increases neuroblastoma cell viability. SK-N-BE(2) and SK-N-SH cells were incubated in a serum-free medium for 24 h before adding different concentrations of dmPGE₂. Cell viability was measured using MTT-assay after 24, 48, 72 or 96 h. Values are representative of two independent experiments and data are expressed as mean (±SD) in percentage of control at 24 h. A statistical analysis was performed using 2-way ANOVA p<0.0001 for both concentration and incubation time. (C) PGE₂ rescues neuroblastoma cells from celecoxib induced apoptosis. SK-N-BE(2) cells were incubated in 35 µM celecoxib alone or in combination with 5 µM dmPGE₂. After 48 h cell viability was assessed using MTT-assay. Mean (±SD) of six replicate wells is shown; values are representative of three independent experiments. Statistical analysis was performed using 2-sided t test P<0.0001.</p

dmPGE₂ increases intracellular Ca²⁺ and cAMP concentrations followed by phosphorylation of Akt.

<p>(A) Intracellular calcium mobilization in response to dmPGE₂. SK-N-SH cells were loaded with the calcium fluorescent dye Fluo-4/AM before the addition of 1 µM dmPGE₂ or (B) pre-treatment with 2 mM EGTA before exposure to 1 µM dmPGE₂. The fluorescence intensity was followed using a confocal laser scanning microscope and representative single-cell recordings are shown. The arrows indicate when dmPGE₂ is added. (C) Intracellular accumulation of cAMP in response to dmPGE₂. SK-N-SH cells were incubated overnight in a medium without serum before the addition of 1 µM of dmPGE₂. Pretreatment with 10 µM NF 449, which is a Gαs inhibitor, before the incubation in dmPGE₂ for 10 min inhibited the production of cAMP. Forskolin, 10 µM for 10 min, was used as a positive control. The graph shows mean (±SD) in % of untreated control of three independent experiments. A statistical analysis was performed using 2-sided t-test, P<0.05. (D) PGE₂ induces phosphorlyation of Akt. SK-N-BE(2) and SK-N-SH cells were grown in the presence of serum (Ctr) before 24 h of culturing in the absence of serum (0 h) prior to the addition of 1 µM of dmPGE₂. Cells were further incubated in dmPGE₂ for 1, 2, 4, 6, 12 or 24 h and protein extracts were subjected to western blotting to detect phosphorylated Akt(ser473). An antibody detecting unphosphorylated Akt was used to exclude differences in total protein expression. β-actin was used to control for equal protein loading. The western blots are representative of three independent experiments.</p

EC₅₀ of EP1-4 receptor antagonists on neuroblastoma cell viability <i>in vitro</i>.

<p>Abbreviations: EC₅₀; effective concentration decreasing neuroblastoma cell viability with 50%,</p>a<p>MYCN amplification;</p>b<p>Multidrug-resistant phenotype.</p