Effect of S1P5 on proliferation and migration of human esophageal cancer cells

Abstract

Aim: To investigate the sphingosine 1-phosphate (S1P) receptor expression profile in human esophageal cancer cells and the effects of S1P5 on proliferation and migration of human esophageal cancer cells.

Methods: S1P receptor expression profile in human esophageal squamous cell carcinoma cell line Eca109 was detected by semi-quantitative reverse transcription polymerase chain reaction. Eca109 cells were stably transfected with S1P5-EGFP or control-EGFP constructs. The relation between the responses of cell proliferation and migration to S1P and S1P5 expression was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and migration assay, respectively.

Results: Both normal human esophageal mucosal epithelium and Eca109 cells expressed S1P1, S1P2, S1P3 and S1P5, respectively. Esophageal mucosal epithelium expressed S1P5 at a higher level than Eca109 cell line. S1P5 over-expressing Eca109 cells displayed spindle cell morphology with elongated and extended filopodia-like projections. The proliferation response of S1P5-transfected Eca109 cells was lower than that of control vector-transfected cells with or without S1P stimulation (P < 0.05 or 0.01). S1P significantly inhibited the migration of S1P5-transfected Eca109 cells (P < 0.001). However, without S1P in transwell lower chamber, the number of migrated S1P5-transfected Eca109 cells was greater than that of control vector-transfected Eca109 cells (P < 0.001).

Conclusion: S1P binding to S1P5 inhibits the proliferation and migration of S1P5-transfected Eca109 cells. Esophageal cancer cells may down-regulate the expression of S1P5 to escape the inhibitory effect.

Keywords: Esophageal cancer; Migration; Proliferation; Sphingosine 1-phosphate; Sphingosine 1-phosphate 5.

Figure 1

Expression of sphingosine 1-phosphate (S1P) receptors in normal human esophageal mucosal epithelium and Eca109 cell line. N: Normal human esophageal mucosal epithelium; E: Eca109 cell line.

Figure 2

S1P5 receptor overexpression in Eca109 cells causes cell spindle change with elongated and extended filopodia-like projections in a medium containing 10% FBS or 0.1% fatty acid-free BSA (× 200). A, B: Eca109/control-EGFP; C, D: Eca109/S1P5-EGFP.

Figure 3

MTT assay showing proliferation of control-EGFP or S1P5-EGFP-transfected Eca109 cells. ^aP < 0.05, ^bP < 0.01 vs control group.

Figure 4

Effect of S1P on migration of S1P5-EGFP-transfected Eca109 cells and control-EGFP Eca109 cells. A: Eca109 cells are allowed to migrate for 8 h, and stained with crystal violet (× 200); B: Results are expressed as percentage in cell migration of S1P-treated cells compared to vehicle (DMSO)-treated cells; C: Results are expressed as migration cell number. ^aP < 0.05, ^bP < 0.001 indicate a statistically significant effect between S1P5-EGFP and control-EGFP transfected Eca109 cells treated by indicated concentrations of S1P or vehicle. NS: Not significant.

Figure 5

Putative signaling pathway of S1P5.