We previously found that lysophophatidic acid (LPA) and sphingosine-1-phosphate (S1P) affect invasion and metastasis of epithelial ovarian cancer (EOC) cells. LPA and low dose S1P (0.5 μM) stimulate, while 20 μM S1P inhibits invasion in attached cells. However, high concentration S1P has an opposite, enhancing effect when applied to invading cells. Therefore, we compared attachment molecules induced by these bioactive phospholipids in long-term attached cells vs. reattached invading cells. Finally, we evaluated LPA and S1P effects on cell-cell adhesion.
Attached Dov13 cells were treated for 4 hours with 40 μM LPA or 0.5 μM or 20 μM S1P. Some of the cells were then detached and reattached to the same plates, as a model for invading cells. At 3, 6 and 24 hours, cells were lysed and cell extracts were fractionated into membrane (hydrophobic) and cytoplasmic (hydrophylic) fractions. N-cadherin, γ- and β-catenins and focal adhesion kinase (FAK) in both fractions were analyzed by western blot analysis using specific antibodies. For cell-cell adhesion assay, treated cells were detached, labeled and applied to wells coated with a monolayer of untreated Dov13 cells, incubated, washed and counted.
In attached cells, membrane N-cadherin, γ- and β-catenins and FAK were all increased by 20 μM S1P and reduced by LPA. In reattaching cells, N-cad was initially depleted from the membrane but recovered by 24 hours. LPA and 0.5 μM S1P prolonged N-cad recovery, while 20 μM S1P accelerated it to 6 hours. FAK was depleted from the membrane of all reattaching cells up to 24 hours. Both γ-and β-catenins were upregulated by 20 μM S1P at 6-24 hours and by LPA only at 6 hours. Cytoplasmic proteins were only slightly affected by treatments. Treatment with 40 μM LPA inhibited cell-cell adhesion by half (p=0.002) and 0.5 μM S1P inhibited by 26% (p=0.017). A highly reproducible 20% increase in adhesion was induced by 20 μM S1P.
Our results indicate major differences in the membrane attachment molecules between attached and reattaching cells. FAK is completely depleted from the membrane of reattaching cells. Cell adhesion and membrane recovery of N-cad inversely correlated with EOC cell invasion, indicating a negative role of membrane N-cad in cell invasion. Membrane recovery of γ- and β-catenins in reattaching cells paralleled the recovery of N-cad in S1P treated cells, yet not in LPA treated cells. In LPA treated cells the membrane γ- and β-catenins were highly elevated by 6 hours when N-cad was depleted, suggesting that the catenins were linked in these cells to other membrane protein(s) than N-cadherin.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA