Article Figures & Data
Figures
- Figure 1.
Dose dependency of the growth inhibitory effect of celecoxib, DMC, and rofecoxib in HUVECs. Cells were seeded into 96-well plates (1,500 cells per well) in six replicates in Medium 200 containing 2% fetal bovine serum, 1 μg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL basic fibroblast growth factor, and 10 μg/mL heparin. After 24 hours of incubation, test agents were added at the indicated concentrations. After 72 hours of drug exposure, cells were stained with crystal violet, and cell viability was determined as described in Materials and Methods. Cell growth over the treatment period was expressed as a percentage of that in the vehicle (DMSO)-treated group. Points, mean; bars, SD. Experiments were done at least thrice.
- Figure 2.
Dose-dependent effects of celecoxib and DMC on HUVEC apoptosis. HUVECs were exposed to varying concentrations of celecoxib or DMC for 24 hours, respectively, under the conditions described in the legend of Fig. 1. A, quantitative measurement of the formation of nucleosomes by the Cell Death Detection ELISA. Columns, mean (n = 3); bars, SD. B, induction of PARP cleavage by celecoxib. PARP proteolysis to the apoptosis-specific 85-kDa fragment was monitored by Western blotting.
- Figure 3.
Dose-dependent effects of celecoxib and DMC on the kinase activity of Akt in drug-treated HUVECs. Cells were treated with the test reagent at the indicated concentrations for 2 hours in Medium 200 containing 2% fetal bovine serum, 1 μg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL basic fibroblast growth factor, and 10 μg/mL heparin. Akt kinase activity in the cell lysates was analyzed as described in Materials and Methods. Points, mean (n = 3); bars, SD.
- Figure 4.
Effects of celecoxib and DMC on retinoblastoma (Rb) phosphorylation and CDK2 kinase activity of immunoprecipitated cyclin E complexes in HUVECs. A, time-dependent inhibition of RbThr821 phosphorylation. HUVECs were treated with 20 μmol/L celecoxib (Cele) or DMC for 24 and 72 hours and were harvested for pRbThr821 and total retinoblastoma determinations by ELISA, respectively, as described in Materials and Methods. pRbThr821/total retinoblastoma ratio was determined and expressed as a percentage of the ratio in the vehicle (DMSO)–treated group. B, dose-dependent inhibition of the CDK2 kinase activity of immunoprecipitated cyclin E complexes. Cell lysates of HUVECs were treated with cyclin E antibodies, and the CDK2 kinase activity of the immune complex was analyzed as described in Materials and Methods. Points, mean (n = 3); bars, SD. C, celecoxib at 10–30 μmol/L does not affect Thr160 phosphorylation levels of CDK2, excluding the involvement of the CDK-activating kinase in inhibiting CDK2 kinase activity.
- Figure 5.
A, celecoxib at 10–30 μmol/L does not affect the expression levels of cyclins, cyclin-dependent inhibitors, and CDKs. HUVECs were treated with 10–30 μmol/L celecoxib for 72 hours followed by Western blot determination for indicated cell cycle regulatory proteins. β-Actin served as a loading control for each treatment. B, celecoxib and DMC at 10–30 μmol/L have no major impact on the phosphorylation status of ERKs. HUVECs were treated with both agents at the indicated concentrations for 24 hours. Western blot analyses for the respective phosphorylated and total proteins.
- Figure 6.
A, effect of celecoxib, DMC, rofecoxib, indomethacin, and roscovitine on neovascularization in the CAM assay. CAMs of fertile 8-day-old White Leghorn chicken eggs were treated the test agent at the indicated doses. After 72 hours, vascular densities in the CAMs were determined as described in Materials and Methods. Vascular densities in the CAMs were expressed as a percentage of that determined in the vehicle-treated control group. Columns, mean (n = 8); bars, SD. B, images of representative CAMs after treatment with 15 nmol celecoxib or DMC for 72 hours. C, DMC-mediated inhibition of Akt phosphorylation in CAMs at different time intervals.
Tables
- Table 1.
Cell cycle phase distribution of HUVECs treated with celecoxib or DMC at the indicated concentrations in 2% fetal bovine serum–supplemented medium for 24 hours
Cells in Cell Cycle Phases (%) G0-G1 S G2-M DMSO vehicle 53.3 ± 4.4 11.9 ± 0.8 33.7 ± 5.7 Celecoxib (μmol/L) 10 62.55 ± 5.7 9.56 ± 1.9 24.2 ± 4.7 15 64.6 ± 4.0 9.4 ± 1.7 22.9 ± 3.0 20 65.9 ± 3.9 8.6 ± 2.0 22.5 ± 3.0 25 69.3 ± 3.8 6.3 ± 1.6 22.9 ± 3.2 DMC (μmol/L) 10 64.3 ± 4.1 8.5 ± 1.7 23.4 ± 3.4 15 65.2 ± 3.6 7.8 ± 1.6 22.9 ± 3.5 20 69.7 ± 7.1 6.1 ± 2.4 20.8 ± 3.1 25 73.2 ± 3.0 4.2 ± 2.9 22.1 ± 1.5 Rofecoxib (μmol/L) 10 53.4 ± 4.0 10.5 ± 2.8 32.8 ± 0.7 15 52.9 ± 3.2 11.7 ± 2.0 33.6 ± 2.7 20 54.2 ± 3.7 10.9 ± 1.7 34.0 ± 2.3 25 54.0 ± 3.2 11.2 ± 2.8 34.2 ± 1.8 NOTE: Control cells received DMSO vehicle. Each tabulated percentage represents the average of three independent experiments.
Volume 3, Issue 12
