Journal of Cancer Prevention 2020; 25(1): 27-37
Published online March 30, 2020
© Korean Society of Cancer Prevention
1Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, 2Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, 3CHA Cancer Prevention Research Center, CHA Bio Complex, 4Digestive Disease Center, CHA University Bundang Medical Center, Seongnam, Korea
Correspondence to :
Young-Joon Surh, E-mail: email@example.com, https://orcid.org/0000-0001-8310-1795
Ki Baik Hahm, E-mail: firstname.lastname@example.org, https://orcid.org/0000-0002-2971-7166
*These authors contributed equally to this work as co-correspondence authors.
**Current affiliation: College of Medical Sciences, Department of Biotechnology and Functional Foods, Jeonju University, Jeonju, Korea.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
COX-2 has been inappropriately overexpressed in various human malignancies, and is considered as one of the representative targets for the chemoprevention of inflammation-associated cancer. In order to assess the role of COX-2 in colitis-induced carcinogenesis, the selective COX-2 inhibitor celecoxib and COX-2 null mice were exploited in an azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted murine colon carcinogenesis model. The administration of 2% DSS in drinking water for 1 week after a single intraperitoneal injection of AOM produced colorectal adenomas in 83% of mice, whereas only 27% of mice given AOM alone developed tumors. Oral administration of celecoxib significantly lowered the incidence as well as the multiplicity of colon tumors. The expression of COX-2 and inducible nitric oxide synthase (iNOS) was upregulated in the colon tissues of mice treated with AOM and DSS, and this was inhibited by celecoxib administration. Likewise, celecoxib treatment abrogated the DNA binding of NF-κB, a key transcription factor responsible for regulating expression of aforementioned pro-inflammatory enzymes, which was associated with suppression of IκBα degradation. In the COX-2 null (COX-2–/–) mice, there was about 30% reduction in the incidence of colon tumors, and the tumor multiplicity was also markedly reduced (7.7 ± 2.5 vs. 2.43 ± 1.4, P < 0.01). As both pharmacologic inhibition and genetic ablation of COX-2 gene could not completely suppress colon tumor formation following treatment with AOM and DSS, it is speculated that other pro-inflammatory mediators, including COX-1 and iNOS, should be additionally targeted to prevent inflammation-associated colon carcinogenesis.
Keywords: Chemoprevention, Celecoxib, Colitis, Colon cancer, COX-2
Patients with inflammatory bowel disease (IBD) face an increased lifetime risk of developing colorectal cancer (CRC). Although early detection and appropriate removal of polyps represent essential components for the prevention of sporadic CRC, intervention with an efficient anti-inflammatory strategy may provide the better opportunity in the management of the colitis-associated cancer [1-3].
While normal colonic mucosa does not express COX-2, this enzyme is abnormally upregulated during the colorectal carcinogenesis in an ‘adenoma-carcinoma’ or an ‘inflammation-dysplasia-carcinoma’ sequence . As aberrant COX-2 overexpression accelerates carcinogenesis by stimulating cell proliferation and rendering cancerous cells resistant to apoptosis, COX-2 inhibition has been considered to be a rational strategy for the chemoprevention of CRC. In line with this notion, offsprings from COX-2 null mice mated to
However, other studies have revealed that COX-2 may have a negligible or even an opposite effect on colon inflammation and possibly, carcinogenesis. Both myeloid cell- and endothelial cell-specific
In the current study, we attempted to evaluate the chemopreventive effects of pharmacological inhibition and genetic ablation of COX-2 in the azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted intestinal tumorigenesis in mice, an experimental model that mimics the human colitis-associated CRC.
Male Institute of Cancer Research (ICR) mice (Daehan Biolink Experimental Animal Center, Daejeon, Korea) five to six week of age and the
Male ICR mice were divided into 7 groups (Fig. 1) for use in a colitis-associated murine carcinogenesis experiment . Group 1 mice were given a vehicle only, Group 2 mice treated with 2% DSS alone in drinking water for 7 days, Group 3 mice treated with a single intraperitoneal (i.p.) dose (10 mg/kg) of AOM, and Group 4 mice given a single i.p. injection of AOM followed by 2% DSS in the drinking water for 7 days. The remaining three groups were assigned for evaluating the effect of celecoxib treatment; Group 5 same as Group 4 treated additionally with 0.5% CMC alone served as a control group, Group 6 given 0.1 mmole/kg celecoxib orally for 14 weeks, and Group 7 was given 0.25 mmole/kg celecoxib for 14 weeks. To further verify the role of COX-2 in the AOM-initiated and DSS-promoted carcinogenesis , we compared the incidence and the multiplicity of AOM plus DSS-induced colon carcinogenesis between
The extracted colon tissue was spread onto a plastic sheet, fixed in 10% formalin for 16 hours, and prepared for paraffin block. The paraffin sections were subjected to the hematoxylin and eosin (H&E) staining to assess the severity of histopathological colitis. Colon cancer cells associated with ulcerative colitis was verified microscopically by a pathologist. The tumor incidence and the multiplicity were calculated by the following formulae; tumor incidence (%) = (number of tumor-bearing mice / total number of mice) × 100 and tumor multiplicity = number of tumors / number of tumor-bearing mice.
The paraffin-embedded sections were deparaffinized with xylene and stained with 0.5% toluidine blue in acetate buffer (pH 4.0). Staining with acidic toluidine blue gave rise to a light blue background, which permitted mapping of the metachoronic mast cells with purplish blue-staining granules in their cytoplasm in relation to the other tissue components within the specimen. To count the mast cells, these sections were examined under a microscope with × 100 or × 400 magnification, and a grid eyepiece (0.0625 mm2) was placed over the cross-sectional area of tissues.
The colon tissue was collected, placed immediately in liquid nitrogen and pulverized in a mortar. The pulverized colon tissue was homogenized in ice-cold lysis buffer (150 mM NaCl, 0.5% Triton-X 100, 50 mM Tris-HCl [pH 7.4], 20 mM EGTA, 1 mM dithiothreitol [DTT], 1 mM Na3VO4 and protease inhibitor cocktail tablet [Roche Molecular Biochemicals, Mannheim, Germany]). Lysates were centrifuged at 13,000 ×
Total RNA was isolated from the harvested colon tissue by appropriate treatment using the TRIzol® reagent (Life Technologies, Milan, Italy) and 2 mg of total RNA was reverse transcribed by Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Promega, Madison, WI, USA). PCR was performed by using the Primix Ex Taq Kit (Takara, Chiba, Japan) with specific primers. The PCR reaction was preformed at respective thermal cycles of 1 minute at 95°C for denaturation and 1 minute at 72°C for annealing. Amplification products were resolved on 1.2% agarose gel, stained with ethidium bromide and photographed under UV light.
For the preparation of nuclear and cytosolic extracts from mouse colon, the tissue was collected, placed immediately in liquid nitrogen and pulverized in a mortar. The pulverized colon tissue was homogenized in ice-cold hypotonic buffer A (10 mM HEPES [pH7.8], 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride [PMSF]). After 20 minutes incubation on ice, the nuclear fraction was separated from the cytosolic fraction by centrifugation for 5 minutes at 12,000 ×
Electrophoretic mobility shift assay was performed using a DNA-protein binding detection kit (Gibco BRL, Grand Island, NY, USA) according to the manufacturer’s protocol. Briefly, the NF-κB oligonucleotide probe (5’-AGT TGA GGG GAC TTT CCC AGG C-3’) was labeled with [γ-32P] ATP by T4 polynucleotide kinase and purified on a Nick column (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The binding reaction was carried out in a total volume of 25 µL containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% (v/v) glycerol, 0.1 mg/mL sonicated salmon sperm DNA, 10 µg of nuclear extracts, and 100,000 cpm of the labeled probe. After 50 minutes incubation at room temperature, 2 µL of 0.1% bromophenol blue was added, and samples were electrophoresed through a 6% non-denaturating PAGE at 150 V in a cold room for 2 hours. Finally, the gel was dried and exposed to an X-ray film.
Gelatin gels (7.5%) were prepared according to the standard procedure. For preparing the running gel, gelatin stock solution (20 mg/mL in double-distilled H2O) was diluted to get the concentration of 0.2%. For this procedure, the Bio Rad Mini Protean II electrophoresis apparatus was used. During gel solidification, colon tissue protein samples (typically 10-25 µg) were mixed with Tris-glycine SDS sample buffer (2 × 0.5 M Tris-HCl [pH 6.8], glycerol, 10% SDS and 0.1% bromophenol blue in diluted with deionized water) and then stand for 10 minutes at room temperature. Colon tissue samples were added to wells and then electrophoresed in 1 × Tris-glycine SDS running buffer (125 mM Tris-HCl [pH 8.3], 1.23 M glycine and 0.5% SDS) according to the standard running conditions. After running, the zymogram renaturing buffer (10 × 25% Triton X-100 in double distilled water] was diluted with deionized water (1 : 9, v/v), and the gel was incubated in the buffer (100 mL for one or two mini-gels) with gentle agitation for 30 minutes at room temperature. After incubation, the zymogram renaturing buffer was decanted and replaced with 1 × zymogram developing buffer (Tris-base, Tris-acid, NaCl, and CaCl2). The gel was equilibrated for 30 minutes at room temperature with gentle agitation and replaced in the fresh 1 × zymogram developing buffer. After overnight incubation at 37°C, the product was stained for 30 minutes in the staining buffer (Coomassie Blue R-250, methanol, acetic acid and distilled water). Gels were destained with an appropriate Coomassie R-250 destaining solution (methanol : acetic acid : water [50 : 10 : 40, v/v]). Areas of protease activity appeared as clear bands against a dark blue background where the protease had digested the substrate.
Results are expressed as the means ± SD. Data were analyzed by one-way ANOVA and Student’s
In our previous studies, administration of a single i.p. dose of AOM (10 mg/kg) alone caused colon tumor formation in about 15% of treated mice with an average number of 1.7 tumors per mouse, while administration of 2% DSS alone in drinking water for 7 consecutive days failed to induce colon tumor formation. However, the administration of AOM followed by DSS in drinking water resulted in the 84% incidence of colon tumors (21 mice among 25 mice developed colon tumors) with the average number of 7.16 ± 2.7 tumors per mouse. Macroscopically, nodular, polypoid or caterpillar-like tumors were observed mostly in the middle and distal colon of animals treated with AOM plus DSS.
In order to determine whether DSS-induced colitis promoted colon carcinogenesis induced by AOM, we counted the number of mast cells. Mast cells are tissue-resident immune cells (granulocytes), which play a key role in inflammatory reactions . The infiltration of mast cells has been considered to be associated with IBD  and inflammation-associated colon tumors [15,18]. Proportion of mast cells increased significantly in colon tumor of the AOM plus DSS-treated mice compared with the normal colonic mucosa (Fig. 2A). Since mast cells represent an important source of TNF-α, which is implicated in the pathogenesis of IBD, we measured the levels of this cytokine in colonic mucosa. As illustrated in Figure 2B, there was a significant increase in the colonic TNF-α production in mice treated with AOM plus DSS.
Enhanced expression and secretion of matrix metalloproteinases (MMPs) are essential for tumor invasion and metastasis . Compared with the normal distal colon, the AOM plus DSS-treated colon exhibited markedly elevated expression of some MMPs, such as MMP2, MMP9, MMP13, and MT2-MMP (Fig. 2C) as well as their catalytic activities determined by zymography (Fig. 2D). In addition, the serum levels of TNF-α, interleukin-6, and nitric oxide were significantly increased in the AOM plus DSS-treated mice (Fig. 2E-2G).
Inappropriately elevated expression of COX-2 has been implicated in pathogenesis of inflammation-associated malignancies including colitic cancer [12,20]. COX-2 expression was significantly increased at both transcriptional (Fig. 3A) and translational (Fig. 3B) levels in the AOM plus DSS-treated group compared to the vehicle-treated control group. Repeated experiments showed a significant difference in the colonic COX-2 expression between the control and the AOM plus DSS-treated animals, with the ratio of
The findings that COX-2 was overexpressed in colitis-associated carcinogenesis prompted us to determine whether a pharmacologic inhibition of this pro-inflammatory enzyme could prevent DSS-promoted colon carcinogenesis. Oral administration of celecoxib (0.25 mmole/kg) for 14 consecutive weeks resulted in substantial reduction (83.3% vs. 44.4%) in the incidence (Fig. 4A) and the multiplicity (6.00 ± 2.75 vs. 1.75 ± 0.96 tumors/mouse,
NF-κB regulates the expression of COX-2 and iNOS, and promotes inflammation-associated tumorigenesis . Therefore, we determined whether celecoxib could suppress the activation of this transcription factor in the colon tissue of mice treated with AOM plus DSS. Under physiologic conditions, NF-κB is sequestered in the cytoplasm by forming an inactive complex with IκBα protein. Upon exposure of cells to pro-inflammatory stimuli, IκBα translocation rapid phosphorylation and degradation. This allows release of NF-κB and translocation to the nucleus, where it binds to the promoter regions of target genes including
Besides the pharmacologic inhibition of COX-2 by a selective inhibitor, we determined whether ablation of
In line with the findings that COX-2 expression is substantially increased in inflamed mucosa and further elevated in dysplastic and cancerous lesions , COX-2 inhibition by NSAIDs has been chemopreventive in familial or sporadic CRC [24,25]. In accord with results of our present study, celecoxib was proven to be effective in the prevention of colitis-associated tumorigenesis in a murine model of human IBD. We also found that
Celecoxib and some selective COX inhibitors prescribed for the treatment of arthritis, have been tested in human chemoprevention trials . However, long-term use of COX-2 selective coxib drugs has caused some adverse effects on the cardiovascular system and unsatisfactory responsiveness. While short-term administration of celecoxib markedly inhibited adenoma growth in animal tumor models, uninterrupted long-term celecoxib administration to
It has also been reported that COX-2 deletion in myeloid and endothelial cells, but not in epithelial cells, exacerbates DSS-induced murine colitis and exhibited greater weight loss, increased clinical scores, and decreased epithelial cell proliferation than control littermates . Such dual functions of COX-2 may account for a double-edged nature of NSAIDs . Thus, it is likely that selective COX-2 inhibition is not necessarily satisfactory for the prevention of colitis-associated carcinogenesis, and coordinated targeting of COX together with other pro-inflammatory molecules should be considered in order to better achieve CRC chemoprevention.
The authors thank Dr. Robert Langenbach for his generous supply of
No potential conflicts of interest were disclosed.
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