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. 2017 Aug 24;8(43):74947-74961.
doi: 10.18632/oncotarget.20471. eCollection 2017 Sep 26.

Sphingosine Kinase 1/sphingosine-1-phosphate (S1P)/S1P Receptor Axis Is Involved in Ovarian Cancer Angiogenesis

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Free PMC article

Sphingosine Kinase 1/sphingosine-1-phosphate (S1P)/S1P Receptor Axis Is Involved in Ovarian Cancer Angiogenesis

Lan Dai et al. Oncotarget. .
Free PMC article

Abstract

Sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) signaling pathway has been implicated in a variety of pathological processes of ovarian cancer. However, the function of this axis in ovarian cancer angiogenesis remains incompletely defined. Here we provided the first evidence that SphK1/S1P/S1PR1/3 pathway played key roles in ovarian cancer angiogenesis. The expression level of SphK1, but not SphK2, was closely correlated with the microvascular density (MVD) of ovarian cancer tissue. In vitro, the angiogenic potential and angiogenic factor secretion of ovarian cancer cells could be attenuated by SphK1, but not SphK2, blockage and were restored by the addition of S1P. Moreover, in these cells, we found S1P stimulation induced the angiogenic factor secretion via S1PR1 and S1PR3, but not S1PR2. Furthermore, inhibition of S1PR1/3, but not S1PR2, attenuated the angiogenic potential and angiogenic factor secretion of the cells. in vivo, blockage of SphK or S1PR1/3 could attenuate ovarian cancer angiogenesis and inhibit angiogenic factor expression in mouse models. Collectively, the current study showed a novel role of SphK1/S1P/S1PR1/3 axis within the ovarian cancer, suggesting a new target to block ovarian cancer angiogenesis.

Keywords: S1P receptor (S1PR); angiogenesis; ovarian cancer; sphingosine kinase 1 (SphK1); sphingosine-1-phosphate (S1P).

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. Immunohistochemical staining of SphK1, SphK2, CD34 and CD105 in epithelial ovarian cancer tissues
(A) Low SphK1 expression; (B) high SphK1 expression; (C) low SphK2 expression; (D) high SphK2 expression; (E) low MVDCD34 area; (F) high MVDCD34 area; (G) low MVDCD105 area; (H) high MVDCD105 area. Magnification, 200× (A–H).
Figure 2
Figure 2. Effect of SphK inhibition by SKI-II on angiogenesis in vitro
(A) Cell migration assay. Endothelial cells were stimulated with the culture media (CM) collected from the ovarian cancer cells precultured with or without SKI-II (2.5μM). The migrated cells were stained, photographed and counted. (B) Cell invasion assay. Endothelial cells were stimulated with CM, the invaded cells were photographed and counted. (C) Tube formation assay. Endothelial cells suspended in CM were placed on the matrigel to form tube like structures, which were then photographed and quantificated. (D) Effect of SKI-II on the S1P secretion of ovarian cancer cells. (E) Effect of SKI-II on the VEGF, IL-8 and IL-6 secretion of ovarian cancer cells. All experiments were repeated three times, with three replicates in each group (* p<0.05 vs. Control group).
Figure 3
Figure 3. Effect of SphK1 or SphK2 blockage on angiogenesis in vitro
(A) SKOV3 cells were transfected with control-siRNA, SphK1-siRNA or SphK2-siRNA. Expression of SphK1 or SphK2 mRNA levels were determined by PCR and normalized to GAPDH mRNA. (B) Protein levels of SphK1 and SphK2 were determined by western blot. (C) S1P levels in the culture media (CM) from siRNA transfected cells were determined by ELISA. (D) Representative images of the migration, invasion and tube formation assays. Endothelial cells were stimulated with the CM collected from siRNA transfected cells with or without S1P (1μM) addition. Migrated cells, invaded cells and tube like structures were photographed. (E) Statistical analysis of the migrated cells, invaded cells and tube like structures. (F) Effect of siRNA transfection with or without S1P addition on the VEGF, IL-8 and IL-6 secretion of ovarian cancer cells. All experiments were repeated three times, with three replicates in each group (* p<0.05 vs. Control group).
Figure 4
Figure 4. S1PR1 and S1PR3 mediate S1P-induced VEGF, IL-8 and IL-6 expression
(A) Immunohistochemical staining of S1PR1, S1PR2 and S1PR3 in human normal ovarian tissue and ovarian cancer tissue. (B) SKOV3 cells were transfected with control-siRNA, S1PR1-siRNA, S1PR2-siRNA or S1PR3-siRNA. Expression levels of S1PR1, S1PR2 and S1PR3 were determined by PCR and normalized to GAPDH mRNA. (C) S1PR1, S1PR2 and S1PR3 proteins were determined by western blot. (D) Effects of S1PR1-siRNA on VEGF, IL-8 and IL-6 secretion in response to S1P treatment. (E) Effects of S1PR2-siRNA on VEGF, IL-8 and IL-6 secretion in response to S1P treatment. (F) Effects of S1PR3-siRNA on VEGF, IL-8 and IL-6 secretion in response to S1P treatment. All experiments were repeated three times, with three replicates in each group (* p<0.05 vs. Control group).
Figure 5
Figure 5. Effect of S1PR1-3 inhibition on angiogenesis in vitro
(A) Representative images of the migration, invasion and tube formation assays. Endothelial cells were stimulated with CM collected from the ovarian cancer cells precultured with VPC23019 (300nM) or JTE-013 (1μM). Migrated cells, invaded cells and tube like structures were photographed. (B) Statistical analysis of the migrated cells, invaded cells and tube like structures. (C) Effect of VPC23019 and JTE-013 on the VEGF, IL-8 and IL-6 secretion of ovarian cancer cells. All experiments were repeated three times, with three replicates in each group (* p<0.05 vs. Control group).
Figure 6
Figure 6. Effect of SphK blockage on angiogenesis in vivo
(A) Representative images of disseminated tumors in intraperitoneal ovarian cancer xenograft model treated with PBS (n=8) or SKI-II (n=8). Tumor number (B) and tumor weight (C) were quantificated. (D) Immunohistochemical staining for CD31, CD34, CD105, VEGF, IL-8 and IL-6 was performed. (E) The number of CD31-positive, CD34-positive or CD105-positive vessels was quantificated. (F) Statistical analysis of integrated optical density (IOD)/area of VEGF, IL-8 and IL-6. (G) S1P levels in the tumor tissue were significantly deceased in the SKI-II treated group (* p<0.05 vs. PBS group).
Figure 7
Figure 7. Effect of S1PR1/3 blockage on angiogenesis in vivo
(A) Representative images of disseminated tumors in intraperitoneal ovarian cancer xenograft model treated with PBS (n=8) or VPC23019 (n=8). Tumor number (B) and tumor weight (C) were quantificated. (D) Immunohistochemical staining for CD31, CD34, CD105, VEGF, IL-8 and IL-6 was performed. (E) The number of CD31-positive, CD34-positive or CD105-positive vessels was quantificated. (F) Statistical analysis of IOD/area of VEGF, IL-8 and IL-6 (* p<0.05 vs. PBS group).

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