Data Availability StatementAll relevant data are contained within the manuscript. Rho-ROCK pathway, by decreasing calpain activity, and by decreasing the expression, secretion and activity of MMP2 and, to a lesser extent, MMP9. Our results thus unveil a novel function for the S1P2 receptor in attenuating thyroid cancer cell invasion. Introduction Sphingosine 1-phosphate (S1P), a bioactive lipid, is usually a regulator of many cellular processes, including cancer cell invasion and migration . S1P can bind to five G-protein coupled receptors (S1P1-5), which activate downstream signaling pathways . In many cancer cells, including follicular thyroid cancer ML-1 cells, S1P promotes migration and invasion by activating S1P1, 3 and downstream PI3K-Akt and Rac signaling pathways [3C8]. However, S1P inhibits migration and invasion by activating S1P receptor 2 and the downstream Rho-ROCK signaling pathway and by inhibiting Rac activity in many cell types , including human anaplastic thyroid cancer C643 cells . In some cell types, however, S1P2 can also enhance migration . The small GTP-ase Rac promotes invasion  and has been shown to regulate S1P-evoked matrix metalloproteinase 2 and -9 (MMP2 and -9) secretion in cancer cells . The MMPs are zinc-dependent proteolytic enzymes, which are expressed and secreted into the extracellular matrix by cancer cells . The inactive zymogen forms of MMP2 and MMP9 are activated by calpains (calcium-dependent proteolytic enzymes), which cleave the pro-peptide domains. MMP2 and MMP9 use mainly collagen IV as substrate and digest the basement membrane to promote cell invasion in cancer cells [13,15C18]. Previous Prasugrel (Maleic acid) studies show that increased expression and activity of MMP2 and MMP9 in thyroid cancer cells promotes invasion . Recently, we have reported that S1P induces secretion and activity of MMP2 and MMP9 through S1PR1,3, and that these MMPs are important for the S1P-evoked invasion of thyroid cancer ML-1 cells . However, the role of MMP2 and MMP9 in S1P-evoked inhibition of invasion of thyroid cancer cells remained Prasugrel (Maleic acid) unknown. In the present study, we have investigated the expression, secretion and activity of MMP2 and MMP9 in thyroid cancer C643 cells where S1P inhibits invasion. Our results show for the first time that S1P can attenuate the expression, secretion and activity of MMP2 and MMP9. This occurs via a S1P2-evoked activation of the Rho-ROCK pathway, and an inhibition of calpain activity. We propose that S1P-evoked inhibition of invasion is Prasugrel (Maleic acid) usually mediated, at least in part, by effects on MMP2 and MMP9. Materials and methods DMEM, BSA, fatty acid-free BSA (FAF-BSA), Mitomycin C, ethidium bromide, SB-3CT and JumpStart Taq DNA polymerase were purchased from Sigma Aldrich Corporation (St. Louis, MO, USA). RPMI 1640 medium was from Lonza (Basel, Switzerland). S1P was purchased from Prasugrel (Maleic acid) Biomol (Plymouth, PA, USA). FBS, penicillin/streptomycin (P/S), L-Glutamine, trypsin, F-12 Hams nutrient medium and OptiMEM were from Gibco Life Technologies (Grand Island, NY, USA). Primary antibodies against S1P2 and S1P3 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit IgG was purchased from Bio-Rad Laboratories (Hercules, CA, USA). MMP2 and anti-mouse IgG antibodies were from Cell Signaling Technology (Denver, MA, USA). MMP9, S1P1, S1P4, S1P5 antibodies and the calpain activity assay kit were purchased from Abcam (Cambridge, MA, USA). Human collagen type IV and cell culture plastic-ware were from Becton Dickinson (Bedford, MA, USA). Transwell migration inserts were from Corning, Prasugrel (Maleic acid) Inc. (Corning, NY, USA). The bicinchoninic acid protein assay reagent kit was from Pierce. All the chemicals and reagents used were of molecular biology and reagent grades. JTE-013 was from Tocris Biosciences (Ellisville, MO, USA). “type”:”entrez-protein”,”attrs”:”text”:”VPC23019″,”term_id”:”1643589982″,”term_text”:”VPC23019″VPC23019 was purchased from Avanti Polar Lipids (Alabuster, AL, USA). Y27632 was obtained from Calbiochem (San Diego, CA, USA). C3 Transferase was from Cytoskeleton, Inc. (Denver, CO, USA). MMP2 siRNA test was used when three or more means were compared. The GraphPad Prism 5 software (GraphPad Software Inc.; San Diego, CA) was used for the statistical analyses. P-values 0.05 were considered statistically significant. Results S1P inhibits expression, secretion and activity of MMP2 and MMP9 in C643 cells Previously, we have shown that S1P (100 nM, 6 h) inhibits migration and invasion of human anaplastic thyroid cancer C643 and follicular thyroid cancer FTC-133 cells towards lipid-stripped serum. This attenuated migration is usually mediated by S1P2 and the Rho-ROCK pathway . Interestingly, S1P promotes migration and invasion in human follicular thyroid Rabbit polyclonal to ZNF146 cancer ML-1 cells [6C8, 21] and increases the secretion and activity of MMP2 and MMP9 through S1P1,3 . This effect on MMP2/9, at least in.