Combination of celecoxib and PD184161 exerts synergistic inhibitory effects on gallbladder cancer cell proliferation

Abstract

Cyclooxygenase-2 (COX-2) and extracellular signal-regulated kinase 1/2 (ERK1/2) may serve as potential targets in various types of cancer; however, the roles of these proteins in gallbladder carcinoma (GBC) have not been reported previously. In the present study, the expression levels of COX-2 and phospho (p)-ERK1/2 in GBC were examined and the biological activities of celecoxib and PD184161 (specific inhibitors of COX-2 and p-ERK1/2, respectively) on the proliferation, cell cycle and apoptosis of the GBC-SD and NOZ human GBC cell lines were evaluated by a series of in vitro and in vivo studies. COX-2 and p-ERK1/2 protein expression levels were found to be significantly elevated in GBC tissues as well as in GBC-SD and NOZ cells. Treatments with celecoxib and PD184161 significantly inhibited GBC-SD and NOZ cell growth in a concentration-dependent manner, and their combination produced a synergistic inhibitory effect. In addition, celecoxib and PD184161 significantly inhibited tumor growth in xenograft nude mice. Celecoxib treatment led to G1 arrest via the upregulation of p21 and p27 expression in GBC-SD and NOZ cells, whereas PD184161 did not affect cell cycle distribution. The combination of celecoxib and PD184161 was able to promote cell apoptosis by triggering a collapse of mitochondrial membrane potential and activating caspase-3-mediated apoptosis. In conclusion, COX-2 and p-ERK1/2 protein may serve as potential targets for GBC chemotherapy, and the combination of celecoxib and PD184161 could significantly inhibit GBC cell growth, induce cell G1 arrest and trigger cell apoptosis of GBC cells.

Keywords: PD184161; apoptosis; celecoxib; gallbladder carcinoma.

Figure 1.

Expression of COX-2 and p-ERK1/2 protein in GBC cell lines and tissues. (A) COX-2 and (B) p-ERK1/2 were upregulated in GBC tissues as compared with normal tissues. (C) COX-2 and p-ERK1/2 protein expression levels in GBC-SD and NOZ human GBC cells were higher than that in HIBEpiC human biliary epithelial cells. (D) Expression of COX-2 was decreased following treatment with celecoxib compared with the control (no treatment), and was decreased markedly more following treatment with a combination of celecoxib and PD184161. (E) Similar alterations were observed in p-ERK1/2 levels following treatment with PD184161 alone or in combination with celecoxib. COX-2, cyclooxygenase-2; p-ERK1/2, phospho-extracellular signal-regulated kinase 1/2; GBC, gallbladder carcinoma.

Figure 2.

Celecoxib and PD184161 inhibited GBC-SD and NOZ cell growth. Celecoxib caused the concentration-dependent inhibition of (A) GBC-SD and (B) NOZ cell growth. Similarly, PD184161 caused the concentration-dependent inhibition of (C) GBC-SD and (D) NOZ cell growth. Combined treatment with 10 µM PD184161 and 2–4 µM celecoxib significantly inhibited (E) GBC-SD and (F) NOZ cell proliferation compared with 10 µM PD184161 single treatment; and combined treatment with 2 µM celecoxib and 10–20 µM PD184161 significantly inhibited (G) GBC-SD and (H) NOZ cell proliferation compared with 2 µM celecoxib single treatment. (I) Isobologram analysis indicated that celecoxib and PD184161 exert synergistic inhibitory effects on GBC cell growth, as data points ‘c’ and ‘d’ (indicating half-maximal inhibitory concentrations of the drugs used in combination) were positioned below the line that indicates an additive effect. *P<0.05 vs. blank control group.

Figure 3.

Celecoxib combined with PD184161 inhibited xenograft tumor growth. In (A) GBC-SD and (B) NOZ xenografts, tumor volumes in the co-treatment groups were significantly less than that of the single treatment and control groups. In (C) GBC-SD and (D) NOZ xenografts, tumor weights in the co-treatment groups were significantly less than that of the single treatment and control groups. Data are presented as the mean ± standard deviation. *P<0.05 vs. the blank control group; ^#P<0.05, combined treatment group vs. all other groups.

Figure 4.

Celecoxib induced G1 phase arrest GBC-SD and NOZ cells. (A) Flowjo software was used for the analysis of flow cytometry data. The images shown are representative of three independent experiments. (B and C) The proportion of the G1 population increased significantly following treatment with celecoxib or combination treatment, whereas PD184161 had no significant effect on cell cycle distribution. (D) The expression levels of p21 and p27 in GBC-SD cells were significantly increased following treatment with celecoxib or combination treatment, whereas they did not change significantly following treatment with PD184161. Data are presented as the mean ± standard deviation. *P<0.05 vs. control.

Figure 5.

Combination of celecoxib and PD184161 induced apoptosis in gallbladder carcinoma cells. (A) Representative flow cytometry results from three independent experiments are shown. Lower left quadrants represent the Annexin^–/PI^– viable cells, lower right quadrants represent the Annexin⁺/PI^– early apoptotic cells, and the right upper quadrants represent Annexin⁺/PI⁺ late apoptotic cells. In (B) GBC-SD and (C) NOZ cells, the apoptotic cell population did not increase following treatment with 4 µM celecoxib or 20 µM PD184161 alone; however, it was elevated following treatment with a combination of 4 µM celecoxib and 20 µM PD184161. Data are presented as the mean ± standard deviation. *P<0.05 vs. other groups. PI, propidium iodide.

Figure 6.

Celecoxib and PD184161 induced the collapse of the mitochondrial membrane potential. (A) Flow cytometric analysis was used to assess the ratio of red/green fluorescence (aggregate/monomeric JC-1) in GBC-SD and NOZ cells following treatment with celecoxib and PD184161. In (B) GBC-SD and (C) NOZ cells, following 2 h of treatment, the red fluorescence was significantly decreased while the green fluorescence was significantly increased, indicating a reduction in mitochondrial membrane potential. Data are presented as the relative ratio of the mean fluorescence intesnsity of aggregate (red) and monomeric (green) JC-1. (D) Western blot analysis indicated that treatment with celecoxib and PD184161 induced cytochrome c release from mitochondria to cytoplasm and the activation of caspase-3. *P<0.05 vs. 0h.