Increase of cyclooxygenase-2 inhibition with celecoxib combined with 5-FU enhances tumor cell apoptosis and antitumor efficacy in a subcutaneous implantation tumor model of human colon cancer

Abstract

Background: The purpose of this study was to investigate the anti-tumor effect and explore the mechanisms of celecoxib (a selective cyclooxygenase-2 inhibitor) combined with 5-fluorouracil (5-FU) on the treatment of human colorectal cancer in a BALB/C nude mouse subcutaneous xenograft model.

Methods: Effects of celecoxib combined with 5-FU on the proliferation of xenograft carcinoma induced by HT-29 were investigated. The apoptotic cells were detected by electron microscope and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. Immunohistochemistry and Western blot were used to estimate the expression of cytochrome C, caspase-3 and caspase-9.

Results: Compared with the control group, treatment groups showed significant inhibition of tumor growth. More apoptotic cells existed after treatment with celecoxib combined with 5-FU. Cytochrome C, caspase-3 and caspase-9 were increased in treated groups, and more obviously in the drug combination group. Cyclooxygenase-2 (COX-2) were decreased after treatment with celecoxib only or combined with 5-FU. And the combined group showed a greater decrease.

Conclusions: Celecoxib combined with 5-FU could inhibit the growth of tumors in vivo by inducing apoptosis and activation of the cytochrome C dependency apoptosis signal pathway. A decrease of COX-2 and an increase of cytochrome C, caspase-3 and caspase-9 may be involved in this process.

Figure 1

Comparison of tumor inhibition rate in each group. The tumor inhibition rate i the control group, the 5-FU group, the celecoxib group and the 5-FU + celecoxib group was 0%, 27.81%, 53.02% and 78.37%, respectively. P <0.05 among all groups.

Figure 2

The influence of 5-FU and celecoxib on tumor growth. The anti-tumor effect on the 5-FU + celecoxib group was better than the other groups.

Figure 3

Comparison of TUNEL positive rate in each group. The rate of tumor apoptosis in the control group, 5-FU group, celecoxib group and 5-FU + celecoxib group was 9.5%, 47.5%, 68.5% and 80.1%, respectively, P <0.05 among all groups.

Figure 4

Detection of apoptotic tumor cells by TUNEL assay. TUNEL-positive cells (apoptotic cells) were stained black-brown (magnification: × 400). There were more apoptotic cells in the 5-FU + celecoxib group than in the other groups. (A, control group; B, 5-FU group; C, celecoxib group; D, 5-FU + celecoxib group).

Figure 5

Observation of apoptotic tumor cells by fluorescence microscope. Apoptotic cells were stained green fluorescence (magnification: ×400). There were more apoptotic cells in the 5-FU + celecoxib group than in the other groups. (A, control group; B, 5-FU group; C, celecoxib group; D, 5-FU + celecoxib group).

Figure 6

Observation of apoptotic tumor cells by transmission electron microscope (Magnification: × 6000). The morphology alteration of apoptotic tumor cells in the 5-FU + celecoxib group was more typical than that in other groups. (A, control group; B, 5-FU group; C, celecoxib group; D, 5-FU + celecoxib group).

Figure 7

Detection of protein expression of cytochrome C, caspase-3 and caspase-9 by Western blot. The protein expression of cytochrome C, caspase-9 and caspase-3 in the 5-FU + celecoxib group was significantly stronger than that in other groups.

Figure 8

Detection of protein expression of COX-2 by Western blot. In the 5-FU group and 5-FU + celecoxib group, COX-2 expression was suppressed more than in the control group and celecoxib group. COX-2 expression was inhibited more obviously after the combined drug treatment.