COX-2 inhibition by celecoxib or specific siRNA inhibits vascular channel formation. (a) Phase contrast images show vascular channel formation in growth factor reduced matrigel of MDA-MB-231, treated with vehicle or 40 mmol/l celecoxib. Images were captured 48 hours after plating using a phase contrast microscope. (part i) With vehicle treatment, MDA-MB-231 cells form well differentiated tubular structures. (part ii) With celecoxib treatment, differentiation into channels was significantly reduced in MDA-MB-231 cells. (part iii) Addition of 50 ng/ml PGE2 to MDA-MB-231 cells treated with 40 μM celecoxib could reverse the inhibitory effect of celecoxib. (b) COX-2 expression decreases in MDA-MB-231 cells with siRNA treatment. COX-2 protein expression was measured by Western blot. Treatment with a COX-2 siRNA for 48 hours significantly inhibited COX-2 expression at siRNA concentrations of 10, 50, and 100 nmol/l. Data shown are representative of three independent experiments. (c) Inhibition of vascular channel formation in MDA-MB-231 cells with celecoxib and COX-2-specific siRNA treatment. Quantitative analysis of vascular channel formation: the number of vascular channels was determined by counting the number of connected cells in five randomly selected fields, using 200 × magnification, and dividing that number by the total number of cells in the same field. Raw data from five standardized fields for each treatment from three separate experiments are shown. Treatment with 40 and 60 mmol/l celecoxib and treatment with a 50 nmol/l concentration of COX-2 siRNA for 48 hours caused significant decrease in the number of channels formed by MDA-MB-231 cells, and addition of 50 ng/ml of PGE2 was able to reverse the effect observed with treatment with 40 mmol/l celecoxib. P values represent significant difference between vehicle control and celecoxib treatment. COX, cyclo-oxygenase; PG, prostaglandin; siRNA, small interfering RNA.