Figure 1.
Celecoxib induced a potent anti-proliferative effect on CGCCA cells.
(A) CGCCA cells with strong positive cytoplasmic immunoreactivity for COX-2 (400X). (B) Concentration-dependent anti-proliferative effect of celecoxib on CGCCA cells. CGCCA cells were incubated with various concentrations (12.5, 25, 50, and 100 µM) of celecoxib for 24 h. Cell proliferation was determined using the BrdU assay. (C) After 3 days of celecoxib treatment, cell viability was measured by the MTT assay. (D) Celecoxib (50 µM) induced growth repression in CGCCA cells in a time-dependent manner. Results are presented as % of the control. Each value is the mean ± SD of 3 to 5 determinations. *p<0.05, **p<0.001 (versus control).
Figure 2.
Celecoxib induced cell cycle arrest at the G2 phase in CGCCA cells.
CGCCA cells were treated with the indicated concentration of celecoxib for 1 day, and the cell cycle distribution was analysed. (A and B) After 1 day of celecoxib treatment, CGCCA cells were analysed by flow cytometry to determine cell cycle distribution. (C) Treated CGCCA cells were stained with PI to show condensed chromatin (white arrow) in M-phase cells. In the right lower panel, the (+) and (–) are examples of cells with and without condensed chromatin, respectively. (D) Ratio of CGCCA cells at M-phase. (E) Expression levels of Cdc25C and CDK1 in CGCCA cells after 1 day of treatment with the indicated concentrations of celecoxib as measured using western blot. Results are presented as % of the control. Each value is a mean ± SD of 3 to 5 determinations. *p<0.05, **p<0.001 (versus control).
Figure 3.
Celecoxib induced apoptosis in CGCCA cells.
CGCCA cells were treated with the indicated concentrations of celecoxib for 2 days. (A) TUNNEL assay was used to identify apoptotic cells under different concentrations of celecoxib. Cell morphology and the nuclei were revealed by Harris modified hematoxylin solution counter-staining. The arrows indicated cells positive (+) for TUNNEL assay. The examples of positive (+) and negative (–) cells for TUNNEL assay were shown in the right lower corner panel (B) The apoptotic index of CGCCA cells treated with different concentrations of celecoxib. Each value is a mean ± SD of 3 to 5 determinations. *p<0.05, **p<0.001 (versus control).
Figure 4.
Detection of rat CCA by animal PET and SUV of Tumor, Liver, and Tumor/Liver ratio.
(A). Transverse, sagittal, and coronal views of fused CT and PET scans of representing control rats revealed CCA expressing areas of the liver in which the 18F-FDG uptake was increased from baseline 1 to 5 weeks after the experiment (i.e., weeks 20, 21, and 25). (B) Transverse, sagittal, and coronal views of fused CT and PET scans of representing rats treated with celecoxib revealed CCA expressing areas of the liver in which the 18F-FDG uptake was slightly increased from baseline1 to 5 weeks after the experiment (i.e., weeks 20, 21, and 25). The arrows indicated the “hottest point” with highest 18F-FDG uptake. (C) The tumour SUVmean of control rats was initially elevated (week 21) and then decreased to a lower level at the last scan (week 25); however, the tumour SUVmean of rats treated with celecoxib was decreased initially (week 21) and remained at a constant level until the last scan (week 25). (D) The liver SUVmean decreased gradually in control and treated rats during this experiment. (E) The tumour-to-liver (T/L) ratio of SUV showed trend of elevation until the last scans in the control group. In the treatment group, the T/L ratio of SUV was significantly decreased 1 week after celecoxib treatment (control, 1.85±0.12; celecoxib, 1.52±0.04; p<0.05).
Figure 5.
COX-2 expression in rat CCA and apoptosis detection in treated rat CCA.
(A) Rat CCA tissue revealed positive cytoplasmic immunoreactivity for COX-2 (×400). (B) Apoptotic DNA fragmentation of rat CCA tissue after treatment with celecoxib (160 mg/kg body weight) for 5 weeks visualised by the DNA laddering assay. No apoptotic DNA fragmentation was observed in the control group treated with DMSO buffer for 5 weeks. (C, D, E) TUNNEL assay for determination of rat CCA apoptosis. The positive cells for TUNNEL assay with brown stains are shown in the positive control (TACS-Nuclease treated tissues ) figure (D). No apoptosis was detected in the control group (C). (E) Apoptosis cells were detected in rat CCA tissues treated with celecoxib (160 mg/kg body weight).
Table 1.
Cox’s Proportional Hazards Analysis.