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