Figure 1.
Neuroblastoma expresses all four PGE2 receptors.
(A) Immunohistochemistry showing specific expression of EP1-4 in tumor cells of a primary human neuroblastoma. (B) Antibody specific blocking peptides were used to control for non-specific binding. Magnification ×400. (C) RT-PCR detecting EP1-4 mRNA in human neuroblastoma cell lines. The myelocytic cell line, U-937, was used as positive control. β-actin was used to ensure equal cDNA load. (D) Western blot detecting bands at 42, 52, 52 and 65 kDa corresponding to EP1, EP2, EP3 and EP4, respectively, in protein extracts from human neuroblastoma cell lines. U-937 cells were used as a positive control. β-actin was used to ensure equal protein loading. The western blots are representative of two independent experiments. (E) Immunofluorescence staining identifying EP1 and EP4 receptor expression in the cellular membrane, in the cytoplasm, and in the nuclear membrane of neuroblastoma cells. EP2 and EP3 were detected in the cellular membrane and in the cytoplasm. Cells were double stained with DAPI to visualize cell nucleus. Magnification ×400.
Figure 2.
Neuroblastoma cells produce PGE2 and dmPGE2 increases cell viability.
(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.
Figure 3.
dmPGE2 increases intracellular Ca2+ and cAMP concentrations followed by phosphorylation of Akt.
(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.
Table 1.
EC50 of EP1-4 receptor antagonists on neuroblastoma cell viability in vitro.