Figure 3
P2Y 2 R activation by ATP or uridine 5′-triphosphate (UTP) regulated the expression of adhesion molecules in endothelial cells (ECs) and increased the adhesion and invasion of MDA-MB-231 cells in ECs. (A) Conditioned media (CM) was obtained from MDA-MB-231 cultured in serum-free media for 16 h with or without apyrase (10 U/ml). Then, ECs were treated with CM from MDA-MB-231 for 6 h, and ICAM-1 and VCAM-1 expression was determined by western blotting. Significance compared to the control, *P <0.05; significance compared to CM, #P <0.05. (B) ECs were treated with the indicated doses of ATP or UTP for 6 h, and protein expression levels were determined. (C, D) Control- or P2Y2R-siRNA-transfected ECs were treated with ATP or UTP (10 μM) for 6 h, and ICAM-1 and VCAM-1 protein expression levels were determined. Significance compared to the control, *P <0.05, **P <0.01; significance compared to ATP or UTP, #P <0.05, ##P <0.01. (E, F) Control- or P2Y2R-siRNA-transfected MDA-MB-231 and ECs were treated with ATP or UTP (10 μM) for 6 h. Then, MDA-MB-231 were seeded onto ECs (E) or ECs in Matrigel-coated cell culture inserts (F). After 30 minutes the remaining cell suspension (MDA-MB-231) was withdrawn, and the number of adherent cells was counted under a light microscope and quantified (E). After 24 h, the numbers of cancer cells that had invaded through the EC-Matrigel-coated insert membranes were evaluated by staining with DAPI and quantified (F). Significance compared to the control, **P <0.01; significance compared to ATP or UTP, ##P <0.01.