S1P induced changes in epithelial ovarian cancer proteolysis, invasion, and attachment are mediated by Gi and Rac.
OBJECTIVE
We previously demonstrated that sphingosine 1-phosphate (S1P) bimodally regulates epithelial ovarian cancer (EOC) cell invasiveness: low-concentration S1P stimulates invasion similar to lysophophatidic acid (LPA), while high-concentration S1P inhibits invasion. In this study, we investigated the mechanisms through which S1P affects EOC cell proteolysis, invasion, and adhesion in two cultured epithelial ovarian cancer cell lines.


METHODS
G-protein Gi was inhibited by pertussis toxin (PTX) and GTP binding protein Rac by NSC23766. S1P conditioned media of DOV13 and OVCA429 cells were evaluated via gel zymography, fluorometric gelatinase assay, urokinase plasminogen activator (uPA) activity assay, and Western Blot for MT1-MMP. Cell invasion was analyzed in Matrigel chambers. Membrane-N-cadherin was localized via fluorescence microscopy.


RESULTS
Zymography revealed pro-MMP2 in conditioned media of EOC cells regardless of treatment. Gelatinase activity was increased by low-concentration S1P. In DOV13 cells this effect was Gi and Rac dependent. In all OVCA429 and control DOV13 cells, PTX enhanced gelatinolysis, suggesting an MMP2-inhibitory pathway via Gi. MT1-MMP was decreased Gi-dependently by high-concentration S1P. Rac inhibition significantly counteracted low-S1P enhancement and high-S1P reduction of DOV13 invasiveness; and uPA activity in conditioned media of invading cells correlated significantly. Immunohistochemistry revealed Gi-dependent clustering of membrane-N-cadherin in DOV13 cells treated with 0.5 microM S1P or 10 microM LPA.


CONCLUSIONS
S1P influences EOC invasion by regulating ECM-proteolysis and cell-cell attachment via MMP2, uPA, and membrane-N-cadherin. Furthermore, this study illustrates that the net effect of S1P on each of these processes reflects a complex interplay of multiple GPCR pathways involving Gi and downstream Rac.
Department of Obstetrics and Gynecology, New York University School of Medicine, New York, NY 10016, USA.
Kathleen M
KM
Devine
Yoel
Y
Smicun
Joanie Mayer
JM
Hope
David A
DA
Fishman
D015703:Antigens, CD / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology*; D053511:Matrix Metalloproteinase 14 / Q000096:biosynthesis; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D037342:Pertussis Toxin / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D015703:Antigens, CD; C050361:CDH2 protein, human; D015820:Cadherins; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D037342:Pertussis Toxin; D014568:Urokinase-Type Plasminogen Activator; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020744:rac GTP-Binding Proteins; D013110:Sphingosine; C032881:lysophosphatidic acid