Sphingosine-1-phosphate inhibits motility of human breast cancer cells independently of cell surface receptors

Exogenous sphingosine-1-phosphate (SPP) inhibits chemotactic motility of se veral transformed cell lines. We have found that SPP at high micromolar con centrations decreased chemotaxis of estrogen-independent (MDA-MB-231 and BT 549) and estrogen-dependent (MCP-7 and ZR-75-1) human breast cancer cells. Because SPP has been implicated as a Lipid-signaling molecule with novel d ual intra- and intercellular actions, it was of interest to determine wheth er the effect of SPP on chemotactic motility of human breast cancer cells i s mediated intracellularly or through the recently identified endothelial d ifferentiation gene (EDG) family of G protein-coupled SPP receptors. There was no detectable specific binding of [P-32]Spp to MDA-MB-231 Or MCP-7 cell s; however, reverse transcription-PCR analysis revealed that both MDA-MB-23 1 and MCF-7 cells expressed moderate levels of EDG-3, neither expressed EDG -1, and EDG-5 mRNA was expressed in MCF-7 but not in MDA-MB-231 cells. In c ontrast to SPP, sphinganine-1-phosphate, which binds to and signals through SPP receptors EDG-1, EDG-3, and EDG-5, had no effect on chemotactic motili ty of MDA-MB-231 or MCF-7 cells. To further discriminate between intracellu lar and receptor-mediated actions of SPP, we used caged SPP, a photolyzable derivative of SPP that elevates intracellular levels of SPP after illumina tion. Caged SPP inhibited chemotactic motility of MDA-MB-231 cells only upo n UV irradiation. In addition, in MCF-7 cells, overexpression of sphingosin e kinase, the enzyme that produces SPP, inhibited chemotactic motility comp ared with vector-transfected cells and markedly increased cellular SPP leve ls in the absence of detectable secretion. Our results suggest that the inh ibitory effect of SPP on chemotactic motility of human breast cancer cells is likely mediated through intracellular actions of SPP rather than through cell surface receptors.