Different cell cycle modulation by celecoxib at different concentrations

Abstract

Abstract Different cyclooxygenase (COX)-2 inhibitors were known to cause different cell cycle changes. We investigated whether this different effect on cell cycle change was due to concentration-dependent effect. We investigated the effects of celecoxib, a COX-2 selective inhibitor, on cell cycle regulation in irradiated cancer cells that express high or low levels of COX-2. Four stably COX-2 knocked-down or overexpressed cell lines were treated with various concentrations of celecoxib with or without radiation. Celecoxib differentially modulated the cell cycle according to the concentrations applied. G1 arrest was induced at lower concentrations, whereas G2/M arrest was induced at higher concentrations in each cell line tested. Radiation-induced G2/M arrest was enhanced at lower concentrations but reduced at higher concentrations. The cutoff values to divide lower and higher concentrations were cell-type specific. Celecoxib treatment activated Cdc25C and inhibited p21 expression in both unirradiated and irradiated cells, regardless of COX-2 expression. Apoptosis was induced in irradiated cells 48 hours after treatment with celecoxib dependent of COX-2. These results imply that celecoxib deactivates the G2 checkpoint via both Cdc25C- and p21-dependent pathways in irradiated cells, which subsequently die by secondary apoptosis. Cell cycle modulating effects in irradiated cells resulting from treatment with celecoxib may have clinical importance with regard to the potential application of celecoxib in cancer patients undergoing radiotherapy.

FIG. 1.

Cell cycle changes after treatment with celecoxib and/or radiation in AN (A), AS (B), HCT-116-mock (C), and HCT-116-COX-2 (D) cells. Cells were exposed to various celecoxib concentrations or vehicle (dimethyl sulfoxide [DMSO]) for 4 hours, and then exposed to 0, 9, or 6 Gy of γ-rays. After an additional 20-hour incubation in a medium containing either drug or vehicle, the cells were harvested, fixed, and the number of cells in different cell cycle phases was measured by flow cytometry. The white column represents G₂/M phase; black column, S phase; slanting lines, G₀/G₁ phase. All data are mean values from the results of three independent experiments. Error bars were omitted to more clearly show the data columns.

FIG. 2.

Western blot analysis for expression and phosphorylation of Cdk1 and cyclin B1 in irradiated or unirradiated cells expressing high or low levels of COX-2. AN/AS (A, B) or HCT-116-mock/HCT-116-COX-2 (C, D) cells were pretreated with various concentrations of celecoxib for 4 hours, exposed to 9 or 6 Gy radiation, and further incubated for 20 hours for 100 or 80 μM celecoxib treatment, or up to 68 hours for 50 or 40 μM celecoxib treatment, respectively. The cells were harvested and used for Western blot analyses.

FIG. 3.

Western blot analysis for expression and phosphorylation of Cdc25C and p21 in irradiated or unirradiated cells expressing high or low levels of COX-2. AN/AS (A, B) or HCT-116-mock/HCT-116-COX-2 (C, D) cells were pretreated with various concentrations of celecoxib for 4 hours, exposed to 9 or 6 Gy radiation, and further incubated for 20 hours for 100 or 80 μM celecoxib treatment, or up to 68 hours for 50 or 40 μM celecoxib treatment, respectively. The cells were harvested and Western blot analyses were performed to determine the expression and phosphorylation levels of Cdc25C and p27.

FIG. 4.

Apoptosis induction after celecoxib treatment either without (left panels) or with (right panels) radiation in AN and AS cells (A) or HCT-116-mock and HCT-116-COX-2 cells (B). Cells were exposed to various celecoxib concentrations or vehicle (DMSO) for 4 hours, and then exposed to 9 Gy (AN/AS cells) or 6 Gy (HCT-116-mock and HCT-116-COX-2 cells) of γ-rays. After an additional 20-hour incubation in a medium containing either drug or vehicle, the cells were harvested, fixed, and the number of cells that had undergone apoptosis (sub-G₁) was evaluated by flow cytometric analysis. Each column represents the mean value±SEM from three independent experiments. N.S., not statistically significant; *Cb, celecoxib; ^†RT, radiation treatment; ^‡p<0.05.

FIG. 5.

Detection of caspase-3 activation by Western blot analysis of cleaved caspase-3 in irradiated or unirradiated cells expressing high or low levels of COX-2. Cells were exposed to 100 μM or 80 μM celecoxib for 4 hours and then exposed to 9 or 6 Gy of radiation in AN/AS cells (A) or HCT-116-mock/HCT-116-COX-2 cells (B), respectively. After additional 20 or 44 hours of incubation in the medium containing either the drug or the vehicle, the cells were harvested and Western blot analyses were performed. ns; not statistically significant. † and ‡; p<0.05.