S1P induced changes in epithelial ovarian cancer proteolysis, invasion, and attachment are mediated by Gi and Rac

Abstract

OBJECTIVES-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-proteinGi 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 Gidependently 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 0.5µM S1P or 10µM 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.

Survival benefit of sphingosin‐1‐phosphate and receptors expressions in breast cancer patients

“…Previous study has been shown that S1P stimulates OVCA invasion at low concentration (0.5 μmol/L), but inhibits its invasion at high concentration (20 μmol/L) 37. In our study, the minimum dosage required to inhibit colony formation of BCA cells was 4 μmol/L (Figure 4A).…”

Extracellular and intracellular sphingosine-1-phosphate in cancer

“…This is achieved by the activation or secretion of proteases, including metalloproteinases and components of the plasminogen activator system. S1P has been shown to upregulate the expression of urokinase plasminogen activator [96,216], uPAR [95], and plasminogen activator inhibitor 1 [95], as well as metalloproteinase 2 [216]. Surprisingly, while S1PR2 generally inhibits motility, it increases glioma invasiveness [95,201].…”

Sphingolipids in cancer

“…SphK1-specific inhibitor decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of anti-apoptotic Bcl-2 protein [105]. In ovarian carcinoma cells, lack of ceramide generation and altered sphingolipid metabolism may be correlated with drug resistance [106,107], and S1P-induced epithelial ovarian cancer cells invasion by regulating proteolysis cell-cell attachment [108,109]. Recently, it has been reported that resveratrol, a phytoalexin that exerts cytotoxic effects on cancer cells, induces apoptosis in HL-60 cells through increasing accumulation of ceramides by induction of LASS genes and repression of SphK1 and GCS genes.…”