Human articular chondrocytes express ChemR23 and chemerin; ChemR23 promotes inflammatory signalling upon binding the ligand chemerin21-157

Chemerin is a chemotactic peptide which directs leukocytes expressing the chemokine-like receptor ChemR23 towards sites of inflammation. ChemR23 is a G protein-coupled receptor which binds several different ligands, and it is also expressed by other cell types such as adipocytes. In addition to chemotaxis, recent reports suggest that ChemR23 is capable of mediating either inflammatory or anti-inflammatory effects, depending on the type of ligand it binds. In the present study, we aimed to clarify whether human chondrocytes express ChemR23 and chemerin, and whether chemerin/ChemR23 signalling could affect secretion of inflammatory mediators. Tissue sections were taken from human knee joints and labelled with antibodies towards chemerin and ChemR23. Chondrocytes from cartilage tissue were isolated, cultured and assessed for chemerin and ChemR23 expression by PCR and immunolabelling. Receptor activation and intracellular signalling were studied by assessment of phosphorylated mitogen activated protein kinases (MAPKs) and phosphorylated Akt after stimulating cells with recombinant chemerin21-157. Biological effects of chemerin21-157 were investigated by measuring secretion of pro-inflammatory cytokines and metalloproteases in cell supernatants. Both serially cultured human articular chondrocytes and resident cells in native cartilage expressed chemerin and ChemR23. Stimulating cells with chemerin21-157 resulted in phosphorylation of p44/p42 MAPKs (ERK 1/2) and Akt (Ser 473). Also, significantly enhanced levels of the pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and the matrix metalloproteases MMP-1, MMP-2, MMP-3, MMP-8 and MMP-13 were detected. These results demonstrate that human chondrocytes express both the receptor ChemR23 and the ligand chemerin. Chemerin21-157 stimulation engaged signal-transduction pathways that further promoted inflammatory signalling in chondrocytes, as judged by an enhanced secretion of cytokines and metalloproteases. Taken together, the previously reported chemotaxis and the present findings suggest that the receptor and its ligand may play pivotal roles in joint inflammation

Autocrine Prostaglandin E2 Signaling Promotes Tumor Cell Survival and Proliferation in Childhood Neuroblastoma

Background: Prostaglandin E2 (PGE2) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE2 production, the expression pattern and localization of PGE2 receptors and intracellular signal transduction pathways activated by PGE2. Principal Findings: A high expression of the PGE2 receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE2 and stimulation of serum-starved neuroblastoma cells with PGE2 increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE 2 (dmPGE2) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE2. Similarly, PGE 2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner

Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment

PPM1D Is a Therapeutic Target in Childhood Neural Tumors

Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers

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

Rho-associated kinase is a therapeutic target in neuroblastoma

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma

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