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. 2008 Jul;7(7):1993-2002.
doi: 10.1158/1535-7163.MCT-08-0088.

S1P Differentially Regulates Migration of Human Ovarian Cancer and Human Ovarian Surface Epithelial Cells

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Free PMC article

S1P Differentially Regulates Migration of Human Ovarian Cancer and Human Ovarian Surface Epithelial Cells

Dongmei Wang et al. Mol Cancer Ther. .
Free PMC article

Abstract

Epithelial ovarian cancer (EOC) arises from the epithelial layer covering the surface of ovaries and i.p. metastasis is commonly observed at diagnosis. Sphingosine-1-phosphate (S1P), a bioactive lipid signaling molecule, is potentially involved in EOC tumorigenesis. We have found that S1P is elevated in human EOC ascites. We show that physiologically relevant concentrations of S1P stimulate migration and invasion of EOC cells but inhibit migration of human ovarian surface epithelial (HOSE) cells. In addition, S1P inhibits lysophosphatidic acid (LPA)-induced cell migration in HOSE but not in EOC cells. We have provided the first line of evidence that the expression levels of S1P receptor subtypes are not the only determinants for how cells respond to S1P. Although S1P(1) is expressed and functional in HOSE cells, the inhibitory effect mediated by S1P(2) is dominant in those cells. The cellular preexisting stress fibers are also important determinants for the migratory response to S1P. Differential S1P-induced morphology changes are noted in EOC and HOSE cells. Preexisting stress fibers in HOSE cells are further enhanced by S1P treatment, resulting in the negative migratory response to S1P. By contrast, EOC cells lost stress fibers and S1P treatment induces filopodium-like structures at cell edges, which correlates with increased cell motility. In addition, inhibition of the protein kinase C pathway is likely to be involved in the inhibitory effect of S1P on LPA-induced cell migration in HOSE cells. These findings are important for the development of new therapeutics targeting S1P and LPA in EOC.

Figures

Figure 1
Figure 1
S1P- and LPA-induced cell migration and invasion in HEY EOC cells. A. Dose curve of S1P-induced migration. B. Cell migration in response to LPA (5 μM) and/or S1P (0.1 μM). C. Cell invasion in response to LPA (5 μM) or S1P (0.1 μM). D. S1P was added to bottom, top or both chambers to modulate cell migration. Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical analyses were conducted by comparing each case with the control (Ctrl) unless specified.
Figure 2
Figure 2
The effect of S1P in cell migration in HOSE cells. A. Dose curve of S1P-induced migration in T103p53i cells. B and C. Primary HOSE n605 and immortalized HOSE 642 cell migration in response to LPA (1 μM) and/or S1P (0.5 μM). D. T103p53i cell migration in the presence of LPA (1 μM) and/or S1P (0.5 μM) with S1P was added to the bottom or the top chamber. Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical analyses were conducted by comparing each case with the control (Ctrl) unless specified.
Figure 3
Figure 3
The role of S1P receptors in migration of EOC and HOSE cells. A. S1P1-3 expression in EOC and HOSE cells detected by RT-PCR. B. Effect of S1P with or without the S1P2 antagonist JTE013 (1 μM), the S1P1/3 antagonist VPC23019 (1 μM) on HEY cell migration and S1P2 stably expressing HEY cells' migration in response to S1P. C. Migration response of T103p53i cells to SEW-2871 (a) and to S1P (0.1 μM) with or without the S1P2 antagonist JTE013 (1 μM), the S1P1/3 antagonist VPC23019 (1 μM) (b). D. Selective depletion of S1P2 expression by target siRNA but not control siRNA was identified by RT-PCR. Migration response to S1P was test 72 hrs after siRNA transfection. Student's t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical analyses were conducted by comparing each case with the control (Ctrl) unless specified.
Figure 4
Figure 4
Cell morphology of EOC and HOSE cells and response to S1P treatment. Two EOC cells (HEY and OCC1) (A) and two HOSE cells (T103p53i and n615 p3) (B) were plated onto glass slides, treated with or without S1P (0.5 μM) or LPA (5 μM) for 10 min as indicated, and then F-actin was stained with TRITC-phalloidin. The pictures are representative images of two independent experiments.
Figure 5
Figure 5
MEK-regulated stress fibers were correlated with cell migration in response to S1P. A. HEY cells were treated with U0126 (10 μM) for 24 hrs or transfected with MEK 2A and then stained F-actin with TRITC-phalloidin. B. 24 hrs after treatment with U0126 (10 μM or 48 hrs after MEK 2A transfection, cell migration in response to S1P (0.1 μM) was tested. C. T103p53i cells were transfected with MEK 2E and then stained F-actin with TRITC-phalloidin. D. Forty-eight hrs after transfection, cell migration in response to S1P (0.5 μM) was tested. Student's t test, *, P < 0.05; ***, P < 0.001.
Figure 6
Figure 6
Activation of the PKC pathway by PMA pretreatment reversed the inhibitory effect of S1P. T103p53i cells were untreated or pretreated with PMA (0.1 μM) for 1 hr, Gö6976 (1 μM) for 30 min, or Gö6976 (1 μM) for 30 min followed by PMA (0.1 μM) for 1 hr. Cell migration in response to LPA or/and S1P was then tested. Student's t test, P < 0.05; **, P < 0.01; ***, P < 0.001; compared with the correspondent control (Ctrl, the first bar in the same group). Student's t test, +, P < 0.05; ++, P < 0.01; +++, P < 0.001; compared with the correspondent PMA results (0.1 μM PMA, the second bar in the same group).

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