Original Article
Induction of chemokine (C-C motif) ligand 2 by sphingosine-1-phosphate signaling in neuroblastoma

https://doi.org/10.1016/j.jpedsurg.2014.04.001Get rights and content

Abstract

Background/purpose

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Preliminary data derived from a human angiogenesis array in NB showed that the bioactive lipid sphingosine-1-phosphate (S1P) induced the secretion of several angiogenesis-related proteins including the important inflammatory factor chemokine (C-C motif) ligand 2 (CCL2). In the present study, we investigated the mechanism of S1P-induced CCL2 expression in NB.

Methods

Quantitative real-time PCR and CCL2 ELISA were conducted to detect the mRNA expression and protein secretion of CCL2 in NB cells. Gain and loss of function studies were performed by using specific S1PR antagonists, adenoviral transduction and siRNA transfection. Macrophage F4/80 receptor in NB xenografts was detected by quantitative real-time PCR and immunohistochemistry staining.

Results

S1P induced CCL2 mRNA expression and protein secretion in a time- and concentration-dependent manner in NB cells. Blockade of S1P2 signaling using the selective S1P2 antagonist JTE-013 inhibited S1P-induced CCL2 expression. Overexpression of S1P2 by adenoviral transduction increased CCL2 secretion while knockdown of S1P2 by siRNA transfection decreased S1P-induced CCL2 secretion in NB cells. Macrophage infiltration, as detected by F4/80 staining, was significantly decreased in JTE-013-treated NB xenografts.

Conclusions

Taken together, our data for the first time demonstrate that S1P induced the macrophage-recruiting factor CCL2 expression in NB cells via S1P2, providing new insights into the complicated functions of S1P2 in cancer.

Section snippets

Materials

S1P was purchase from Biomol (Plymouth Meeting, PA) and JTE-013 was from Tocris Bioscience (Ellisville, MO). Fatty-acid free BSA was purchased from Sigma (Saint Louis, MO).

Cell culture, adenoviral transduction and siRNA transfection

SK-N-AS cell line was obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Sigma, Saint Louis, MO) supplemented with 10% FBS (Hyclone, Logan, UT), 1 × MEM NEAA (Gibco, Grand Island, NY) and penicillin-streptomycin (Gibco) at 37 °C in a humidified chamber of 5% CO2. SK-N-BE(2) cell line, originally from

S1P induced CCL2 expression in NB cells

To determine the effect of S1P on CCL2 expression in NB, the SK-N-AS cell line was utilized since it has an S1P receptor (S1PR) expression profile consistent with that of human NB specimens, as demonstrated in our previous findings [8]. Quantitative real-time PCR and CCL2 ELISA were performed to detect the mRNA expression and protein secretion of CCL2 induced by S1P. We found that S1P at a concentration as low as 10 nM, was able to significantly induce CCL2 mRNA expression and protein secretion

Discussion

Our preliminary data obtained from human angiogenesis array assay showed that the bioactive lipid S1P, which we have shown to be an important regulator of VEGF expression [8], also induces the secretion of the chemokine CCL2. Quantitative real-time PCR and CCL2 ELISA confirmed that S1P induced CCL2 mRNA expression and protein secretion in both MYCN amplified and non-amplified NB cells (Fig. 1). The induction is an early phase and continued event. Five S1PRs have been reported to bind

Acknowledgments

This work was supported by NIH grant R01CA168903 (F.F.), the Seraph Foundation and the Burr Curtis Surgical Endowment.

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