Antitumor and anti-metastatic effects of cyclooxygenase-2 inhibition by celecoxib on human colorectal carcinoma xenografts in nude mouse rectum

Abstract

We examined the effects of the preferential cyclooxygenase-2 (COX-2) inhibitor celecoxib on tumorigenesis, angiogenesis, apoptosis, vascular endothelial growth factor (VEGF) protein expression and metastasis in HT-29 human colorectal carcinoma cell xenografts in nude mouse rectum. COX-2 mRNA expression was examined in the xenograft and metastatic sites. The antitumor effect of celecoxib in the xenografts was evaluated by measuring the weight of the peri-ano-rectal tumor. The anti-metastatic effect of celecoxib was assessed by quantification of lymph node and lung metastases by amplification of a cancer-related human DNA by TaqMan PCR. The effects of celecoxib on angiogenesis, apoptosis, prostaglandin E2 (PGE2) production and VEGF protein expression in the xenografts were evaluated by means of microvessel density (MVD) counting, terminal deoxynucleotidyl transferase-mediated nick end labeling assay, quantitative enzyme-linked immunosorbent assay and western blotting, respectively. The rectal xenograft model showed lymph node and lung metastases with enhanced expression of COX-2 mRNA in each organ. Celecoxib inhibited rectal xenograft growth in a dose-dependent manner as follows: 150 ppm, 33.0% (p=0.000220); 750 ppm, 46.4% (p=0.0000292); 1500 ppm, 63.4% (p=0.0000109). Celecoxib inhibited lymph node metastasis in a dose-dependent manner as follows: 150 ppm, 86.7% (p=0.0263); 750 ppm, 90.3% (p=0.00638); 1500 ppm, 96.0% (p=0.000894). Celecoxib also inhibited lung metastasis as follows: 750 ppm, 53.3% (p=0.0107); 1500 ppm, 78.3% (p=0.00022). Celecoxib (1500 ppm) significantly inhibited PGE2 production by 68.4% (p=0.000157) and MVD counting by 48.2% (p=1.3x10-12) and induced apoptosis 2.5-fold (p=3.0x10-14) in the rectal xenograft. Celecoxib suppressed VEGF protein expression in the rectal xenograft. These studies demonstrate that celecoxib reduces the growth and metastatic potential of colorectal carcinoma in mice through COX-2 inhibition, anti-angiogenesis and apoptosis induction. The studies using HT-29 human colorectal carcinoma cell xenografts in nude mouse rectum also provide important information that supports that the COX-2 inhibitor celecoxib has a high potential for use as a clinical agent for inhibition of hematological and lymphatic metastases of colorectal cancer.

Figure 1

Microscopic findings of the lung of a mouse with the HT-29 cell rectal xenograft. Black arrows show lung metastasis. Scale bar, 100 μm.

Figure 2

Macroscopic findings of a rectal xenograft model 6 weeks after tumor inoculation in vehicle-treated mouse (A) and celecoxib-treated mouse (B). White ring shows peri-ano-rectal tumor and black ring shows para-aortic lymph node metastasis.

Figure 3

Effect of celecoxib on HT-29 rectal xenograft growth. The weight of the rectal xenograft of celecoxib-treated mice and vehicle-treated control mice bearing the HT-29 rectal xenograft was measured. Celecoxib repressed growth of rectal xenograft in a dose-dependent manner.

Figure 4

Quantification of metastasized tumor cell amounts in para-aortic lymph node (A) and lung (B) of mice with the HT-29 rectal xenografts treated with various concentrations of celecoxib. The amount of metastasized tumor cells was calculated by amplification of the human β-globin gene and shown as the number of tumor cells in the organ. Celecoxib inhibited lymph node and lung metastases in mice with rectal xenograft in a dose-dependent manner.

Figure 4

Quantification of metastasized tumor cell amounts in para-aortic lymph node (A) and lung (B) of mice with the HT-29 rectal xenografts treated with various concentrations of celecoxib. The amount of metastasized tumor cells was calculated by amplification of the human β-globin gene and shown as the number of tumor cells in the organ. Celecoxib inhibited lymph node and lung metastases in mice with rectal xenograft in a dose-dependent manner.

Figure 5

Effect of celecoxib on angiogenesis in rectal xenograft. (A) Representative micrograph of immunohistochemistry with an anti-mouse CD31 antibody of the rectal xenograft of a mouse treated with vehicle and celecoxib-treated mouse. Black arrows show CD31-positive microvessels. Scale bar, 100 μm. (B) Vascularity was measured by the average number of vessels per field counted in 8 random areas at ×20 magnification. MVD was calculated as the average number of vessels per mm². Celecoxib significantly inhibited angiogenesis in the rectal xenograft.

Figure 5

Effect of celecoxib on angiogenesis in rectal xenograft. (A) Representative micrograph of immunohistochemistry with an anti-mouse CD31 antibody of the rectal xenograft of a mouse treated with vehicle and celecoxib-treated mouse. Black arrows show CD31-positive microvessels. Scale bar, 100 μm. (B) Vascularity was measured by the average number of vessels per field counted in 8 random areas at ×20 magnification. MVD was calculated as the average number of vessels per mm². Celecoxib significantly inhibited angiogenesis in the rectal xenograft.

Figure 6

Effect of celecoxib on PGE₂ production in rectal xenograft as measured by quantitative ELISA. Celecoxib significantly suppressed PGE₂ production in the rectal xenograft.

Figure 7

Effect of celecoxib on VEGF expression in rectal xenograft as detected by western blotting. HT-29 human colon cancer cells and rectal xenograft of vehicle-treated control mice showed stronger VEGF expression than rectal xenograft of mice treated with 1500 ppm celecoxib.

Figure 8

Effect of celecoxib on tumor cell apoptosis in rectal xenograft as detected by TUNEL assay. (A) Representative micrograph of TUNEL assay in the rectal xenograft of a mouse treated with vehicle and celecoxib-treated mouse. Black arrow shows the apoptotic cells. Scale bar, 50 μm. (B) The apoptotic index was calculated as follows: apoptotic index (%) = (no. of apoptotic cells/no. of total tumor cells × 100). Celecoxib significantly induced tumor cell apoptosis.

Figure 8

Effect of celecoxib on tumor cell apoptosis in rectal xenograft as detected by TUNEL assay. (A) Representative micrograph of TUNEL assay in the rectal xenograft of a mouse treated with vehicle and celecoxib-treated mouse. Black arrow shows the apoptotic cells. Scale bar, 50 μm. (B) The apoptotic index was calculated as follows: apoptotic index (%) = (no. of apoptotic cells/no. of total tumor cells × 100). Celecoxib significantly induced tumor cell apoptosis.