The role of cyclooxygenase-2 (COX-2) in two different morphological stages of intestinal polyps in APC
474 knockout mice
Ken-ichi Sunayama1,3,
Hiroyuki Konno1,
Toshio Nakamura1,
Hidehumi Kashiwabara1,
Tsuyoshi Shoji1,
Toshihiro Tsuneyoshi2 and
Satoshi Nakamura1
1 2nd Department of Surgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192, Japan and
2 Department of Materials Science, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi-Shi, Shizuoka, 437-8555, Japan
 |
Abstract
|
---|
The expression of COX-2 participates strongly in polyp formation of adenomatous polyposis coli (APC)-mutated mice. However, the mechanism of growth inhibition by COX-2 inhibition remains unclear. The aims of this study were to assess the role of COX-2 during the process of polyp formation in APC
474 knockout mice. Starting at 4 weeks of age, the treated group (T group) were given a diet containing JTE-522, a selective COX-2 inhibitor, and the control group (C group) were given a control diet. At 12 weeks of age, mice were killed and polyps located in a proximal 10 cm of the small intestine were classified into two morphological stages: large adenomas (>300 µm in diameter) which lacked normal villous structure, and small adenomas (
300 µm) covered with normal villous epithelia. In both classes, after counting the incidence, adenomas were examined for vascularity, expression of COX-2 and VEGF protein, labeling proliferating cell nuclear antigen (PCNA) and apoptosis with the TdT-mediated dUTP nick end labeling method, including expression of Bcl-2 and Bcl-X. JTE-522 significantly reduced the incidence of large adenomas, but not of small adenomas. Although it did not affect the proliferating potential of adenomas, the apoptosis index increased significantly in the T group accompanied by a reduction in Bcl-X expression in both small and large adenomas. In the C group, macrophages with both COX-2 and VEGF expression were observed in the submucosa of large adenomas, where some large vessels were also observed. JTE-522 inhibited the VEGF expression of these macrophages, resulting in a decrease in vascular area. In conclusion, macrophages with COX-2 and VEGF expression in the submucosal layer are responsible for angiogenesis in large adenomas, and a selective COX-2 inhibitor reduced the growth of adenoma mainly by its inhibitory effect on angiogenesis.
Abbreviations: APC, adenomatous polyposis coli; PCNA, proliferating cell nuclear antigen; TUNEL, TdT-mediated dUTP nick end labeling
 |
Introduction
|
---|
Epidemiological studies have demonstrated a 4050% decrease in the relative risk of colorectal cancer with the use of non-steroidal anti-inflammatory drugs (NSAIDs) (13). Cyclooxygenase-2, an inducible isozyme of COX, has been considered to be the major target molecule in cancer chemoprevention induced by NSAIDs because of its expression in colon neoplasms (47). Furthermore, rodent studies of colon carcinogenesis have reported the preventive and/or therapeutic efficacy by COX-2 inhibitors (811). Thus, cancer chemoprevention with COX-2 inhibitors is regarded as a potent anticancer strategy (12).
Some animal models with adenomatous polyposis coli (APC) mutations have been developed including APCMin (7,10,1315), APC
716 (8,1618), APC1309 (19) and APC
474 (9), which are useful as a model not only of familial adenomatous polyposis (FAP), but also of carcinogenesis in the adenoma-carcinoma sequence (20,21). The importance of COX-2 in polyp formation in these models was demonstrated by the elevation of COX-2 levels in polyps (7,8) and the reduction of tumor development by inhibition of COX-2 (9,10,17,22,23).
Inhibition of apoptosis (13,23), an increase in cell proliferation (23) and angiogenesis (24) were suggested as carcinogenic properties of COX-2. However, it is not considered that these mechanisms are uniform throughout the course of carcinogenesis. The aims of this study were to elucidate the role of COX-2 in the proliferation and apoptosis of epithelial cells, and in angiogenesis in two different stages of polyp development.
 |
Material and methods
|
---|
Animals
Experiments were approved by the Hamamatsu University School of medicine animal use committee. Male APC
474 knockout mice were obtained from the Central Pharmaceutical Institute of Japan Tobacco (Osaka, Japan) (9). Animals were housed to breed in groups of one male APC
474(+/) mouse and two to four C57BL/6J females. Progeny were genotyped by PCR assay to determine whether they were heterozygous type for APC or homozygous wild-type. Some APC
474(+/) male progeny were used to maintain the pedigree of APC
474 mice.
Genotyping
The newborn APC
474 mouse offspring were selected by mutant allele-specific PCR (MAS-PCR) of genomic DNA extracted from a small tissue fragment of the tail or ear. As the mutant allele was constructed (9) from a pMC-1/neo.polA vector (Stratagene, La Jolla, CA) and a BlueScript KS(+) vector (Stratagene), oligonucleotide primers flanking the neo cassette and KS vector sequence were used for the MAS-PCR. A negative control tube without DNA sample was carefully included in each PCR to confirm a normal reaction without contamination.
Drug treatments
Four weeks after birth, mice were randomly assigned after weaning to each of the experimental groups, maintaining a gender balance in each group. They were treated until 12 weeks after birth. JTE-522, a selective COX-2 inhibitor, which was reported to be a chemopreventive agent for polyposis in APC-mutated mice (9), was purchased from the Central Pharmaceutical Institute of Japan Tobacco. Mice were fed ad libitum with Certified Diet-MF (Oriental Yeast, Tokyo, Japan) as a control diet in the control group (C group) or a 0.01% JTE-522 concentration mixed with Certified Diet-MF diets prepared by Oriental Yeast in the JTE-522-treated group (T group). The T group (15 mice) received the JTE-522 diet from 4 to 12 weeks of age, and the C group (15 mice) received a control diet the same as the T group.
Tissue samples
Animals were killed by diethyl ether asphyxiation at 12 weeks of age. After laparotomy, the small intestine was removed. Segments of 10 cm in length from the pylorus ring, which was harvested for scoring of tumors and histological study, was washed with normal saline and half-fixed with 10% neutral buffered formalin for 30 min. After half-fixation, this segment of proximal small intestine was opened longitudinally, cut into five 2 cm segments, piled and crucified on rubber sheets and then fixed with 10% neutral buffered formalin for 24 h. After embedding in paraffin, serial 4 µm longitudinal sections of the piled specimens were made at intervals of 500 µm, and stained with hematoxylineosin. The remaining small intestine was opened longitudinally. Then, the polyp mucosa was harvested from the small intestine using a stereomicroscope for northern blotting.
Histological polyp scoring
The hematoxylineosin stained serial sections were observed microscopically. The adenomas were classified as follows and counted. (i) Small adenoma: diameter <300 µm and the superficial normal epithelia are intact; (ii) large adenoma: the diameter is 300 µm or more, and adenomatous epithelial cells replaced the normal epithelia as the surface of the polyp. Oshima et al. (16) described the former as `nascent polyps and `uni-villous polyps, and the latter as `multivillous polyps. In addition, the number of large adenomas, which extended across a few slices, was corrected by comparing adjacent sections. Two slices, which included more than one from the center of each large adenoma were chosen randomly from each mouse for cell death labeling, proliferating cell nuclear antigen (PCNA) labeling and immunohistochemistry. Totally, the number of evaluated large adenomas, small adenomas and normal crypts were 38, 45 and 75 in the C group, and 32, 45 and 75 in the T group, respectively.
PCNA labeling and TUNEL index
PCNA immunohistochemical detection was performed to determine the proliferating cells using a commercial kit (Zymed, San Francisco, CA). To determine the cells undergoing cell death, we used an in situ TdT-mediated dUTP nick end labeling (TUNEL) technique with an in situ Cell Death Detection Kit, POD (Roche Diagnostics, Mannheim, Germany) according to the manufacturers instructions.
Immunohistochemistry
Immunohistochemical staining for Bcl-2, Bcl-X, Bax, factor VIII-related antigen, COX-2, VEGF and F4/80 in the specimens of the upper small intestine was performed using the avidinbiotin complex technique. The sections of piled upper small intestine were deparaffinized and dehydrated, then placed in a 10 mM citrate buffer, pH 6.0, and heated for 20 min in a microwave oven at 500 W. Endogenous peroxidase activity was blocked by incubation with 0.3% H2O2 for 15 min. After washing with PBS, the sections were treated with 10% blocking serum for 20 min. The sections were then incubated with the first antibody overnight at 4°C as in Table I
. They were then washed, treated with a biotinylated secondary antibody for 45 min and treated with a streptavidin HRP-conjugated reagent (Nichirei, Tokyo, Japan) for 20 min at room temperature. Trypsin pre-treatment (1 mg/ml trypsin, 4 mM CaCl2 in 200 mM Tris, pH at 37°C for 15 min) was performed with staining of factor VIII-related antigen and F4/80. After reaction with 0.2% w/v diamino-benzidine·HCl for 5 min, the sections were counterstained with hematoxylin and mounted. Negative controls were similarly processed using normal IgG as the primary antibody.
Evaluation of staining
Each sections undergoing TUNEL, PCNA labeling and Bcl-X staining had its labeling index calculated as follows. After the staining procedure, two peripheral fields from each adenoma ranging from 290x220 µm were captured and digitized to image files using a microscope charge coupling device camera system; HC-2000 (Olympus, Tokyo, Japan) at 2.5x40 magnification. Small adenomas were also captured as above. Then, the positive and negative cells were counted on a computer screen. The positive cells were determined by comparing them with the normal epithelial cells in adjacent normal villi. These labeling indices were analyzed by the ratio of labeled cells to total cells for all adenomatous epithelial cells in small adenomas and over 500 cells in the periphery site of large adenomas. Also, for the evaluation of VEGF, the ratio of VEGF-positive cells was evaluated in interstitial cells in the submucosal layer.
Evaluation of angiogenesis
Angiogenesis of adenomas was assessed as follows. The blood vessels in the submucosa were identified immunohistochemically and captured to image files as above. The total sectional areas of vessels and the longitudinal length of the submucosal baseline were measured with NIH image 1.62. Then, the evaluation of submucosal vessel area was calculated using the total area of vessels (µm2)/submucosal length (µm).
Northern blot analysis (Bcl-2, Bcl-xL, Bax and VEGF)
All samples were frozen in liquid nitrogen and stored at 80°C for analysis of Bcl-2, Bcl-xL, Bax and VEGF mRNAs. Total cellular RNA was extracted from the frozen tumor samples using TRIZOL reagent (Sigma, St Louis, MO) according to the manufacturers protocol. Briefly, 100 mg of each tissue were homogenized in 1 ml of TRIZOL. Subsequently, 0.2 ml chloroform was added and the mix was centrifuged (8000 r.p.m., 30 min, 4°C). This separated the solution into an aqueous phase containing RNA. The aqueous layer was carefully aspirated and added to 0.5 ml of isopropanol for RNA precipitation. Following this, the solution was centrifuged (8000 r.p.m., 30 min, 4°C) and the pellet was washed with 75% ethanol and centrifuged (5000 r.p.m., 5 min, 4°C). After that, RNA was placed into DEPC-treated water. The RNA solution was kept frozen at 80°C until analysis. Then, 20 mg of total RNA was resolved in each lane, and transferred to Hybond-N nylon membranes which were then UV-irradiated for 6 min with a CL-1000 Ultraviolet Crosslinker (UVP, Upland, CA). Purified cDNA fragments of Bcl-2 (2.7 kb; Calbiochem, San Diego, CA), Bcl-xL (702 bp; R&D Systems, Mineapolis, MN), BAX (0.57 kb; Cayman Chemical, Ann Arbor, MI), COX-2 open reading frame (1.8 kb), VEGF (573 bp) and ß-actin were labeled with and a Megaprime DNA labeling system kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Probes were purified with nick columns and used at 1x106 c.p.m./ml in a hybridization solution. Northern blots were quantified with NIH image 1.62.
Statistical analysis
Statistical analysis was performed using a statistical software package (Statview 4.5, Abacus Concepts, Cary, NC). The data were analyzed using the MannWhitney U test to compare the C and T groups. The KruskalWallis test, analysis of variance (ANOVA) and the Scheffes post hoc test were used in multiple comparisons. Differences were considered significant when P values were <0.05.
 |
Results
|
---|
No mice in either group died during the experiment.
Tumor multiplicity
Histological examination revealed that most polyps were present in the small intestine and all tumors were diagnosed as adenomas. The number of large adenomas in the proximal intestine in the T group was significantly smaller than that of C group (P < 0.01), however, there was no significant difference in the incidence of small adenomas between the two groups (Table II
).
Cell death and cell proliferation index
As for the PCNA labeling index, there was no significant difference among the groups in large adenomas, small adenomas and normal crypts. In both the T and C groups, the TUNEL index in small adenomas was
3.5-fold that in large adenomas. The TUNEL index in both small and large adenomas in the T group was greater than that in the C group (P < 0.01) (Table III
). However, in normal crypts there was no significant difference in the TUNEL index between the C and T groups.
Immunohistochemical expression of apoptosis-related protein
Bcl-2 expression of the adenomatous epithelial cells was absent in both small and large adenomas, while the normal crypt cells were stained moderately and interstitial cells in the lamina propria were stained strongly (Figure 1A
).

View larger version (151K):
[in this window]
[in a new window]
|
Fig. 1. Immunohistochemical staining of the Bcl family in small and large adenomas. (A) Absence of staining of Bcl-2 in adenomatous epithelial cells despite staining of lamina propria cells and normal epithelial cells. (B) Immunoreaction of Bcl-X is shown in a large adenoma with heterogeneity. (C) Bcl-X immunostaining in a peripheral region of a large adenoma in the C group and (D) in the T group. The proportion of Bcl-X-positive epithelial cells in the T group was lower than that in the C group. (E) Staining of Bcl-X is shown in adenomatous epithelial cells of a small adenoma. (F) Negative staining for Bcl-X staining. (G and H) Bax was stained diffusely in adenomatous epithelial cells, as strong as in the normal epithelium. (AH) Bar = 100 µm.
|
|
In the small and large adenomas, the expression of Bcl-X was present with heterogeneity, which ranged from absence to a strong immunoreaction (Figure 1B, C and E
). The proportions of Bcl-X-positive cells to total epithelial cells in the T group were smaller than the C group in both the small and large adenomas (Figure 1C and D
and Table IV
). Immunostaining of Bcl-X was found diffusely with moderate strength in normal epithelial cells.
Bax immunoreactivity was detected diffusely in the small and large adenomas as strong as epithelial cells in normal villi (Figure 1G and H
), and there was no significant difference in the immunoreaction of Bax between the two groups.
mRNA expression of apoptosis-related protein
Bcl-2 mRNA expression was not detected in adenomas by northern blot analysis. The level of Bcl-xL expression of large adenomas in the T group was less than that in the C group (P < 0.05). There was no significant difference in expression of Bax mRNA between the two groups in large adenomas (Figure 2
).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2. Northern blot analysis of Bcl-xL and Bax. Bcl-xL mRNA expression of large adenomas in the T group was less than that in the C group. *P < 0.05 (n = 5). However, there was no significant difference in the expression of Bax mRNA.
|
|
Angiogenesis of adenomas
In small adenomas, the blood vessels were involved by occupying aberrant crypts with near density vessels to normal villi (Figure 3A
). There was no difference in the vascular network in the lamina propria or vessels in the submucosa between adenomas and normal villi. JTE-522 did not affect the vascularity of small adenomas. In large adenomas, vascular development was observed in the submucosal layer (Figure 3B
). The capillary network in the lamina propria, which is similar to normal villi and small adenomas, was observed in the whole lesion of adenomas (Figure 3C
). Submucosal vascular development was shown in large adenomas, but not in small adenomas. The vessel area in the submucosal layer below large adenomas was greater in the C group than the T group (Figure 3B and D
) (P < 0.01) (Table V
), whereas there were no differences in the vascular network located in the lamina propria between the two groups (data not shown).

View larger version (153K):
[in this window]
[in a new window]
|
Fig. 3. The blood vessels were labeled by immunohistochemical staining of Factor VIII-related antigen. (A) Normal villi and a small adenoma in the C group. In the small adenoma, blood vessels were involved, being occupied by aberrant crypts (arrows). Several vessels were scattered in the submucosal layer in the base of the small adenoma, the same as in normal villi (arrowheads). (B) A large adenoma in the C group. Some large vessels, which seem to feed the adenoma, were recognized in the submucosal layer (arrows). (C) The population of vessels in the lamina propria of the large adenoma was similar to that of normal villi and small adenomas. (D) A large adenoma in the T group. Some vessels were recognized in the submucosal layer (arrows); however, the vessel area was less than that of the C group (compare with B). (AD) Bar = 100 µm.
|
|
Expression of VEGF and COX-2
In both groups, immunohistochemistry for VEGF were found in interstitial cells in the lamina propria and submucosal layer of adenomas (Figure 4A
). Prominent expression of VEGF was shown in mononuclear cells in the submucosa below large adenomas in the C group (Figure 4B
). The assembly of VEGF-positive cells was not observed under small adenomas (Figure 4C
). The proportions of VEGF-positive cells to total interstitial cells in the submucosal layer under the large adenomas in the T group were smaller than those in the C group (Table VI
). Furthermore, the VEGF mRNA expression in large adenomas in the T group was significantly less than that in the C group (Figure 5
).

View larger version (147K):
[in this window]
[in a new window]
|
Fig. 4. Immunohistochemical staining of VEGF, F4/80 and COX-2. (A and B) A large adenoma in the C group. VEGF was localized predominantly to mononuclear cells in the lamina propria and submucosal layer. In the adenomatous epithelial cells, the VEGF expression was shown weakly. The diffuse staining below the erosion might be non-specific. (C) Immunohistochemical staining for VEGF in a small adenoma in the C group. (D) Negative staining for VEGF. (E) Immunohistochemical staining for F4/80 in an adjacent slice to (B). (F) Base of an adenoma with staining for VEGF in the T group. The population of VEGF-immunoreactive cells was smaller than that of the C group (compare with B). (G) Immunohistochemical staining for F4/80 in an adjacent slice to (F). (H) Immunohistochemical staining for COX-2 in an adjacent slice to (E). COX-2 was also localized predominantly to submucosal mononuclear cells. (AH) Bar = 100 µm.
|
|

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 5. Northern blot analysis of VEGF. The expression of VEGF mRNA of large adenomas in T group was less than that in C group. *P < 0.05 (n = 5).
|
|
F4/80 immunohistochemical staining revealed that the majority of cells containing immunoreactive VEGF were macrophages (Figure 4E
). The number of macrophages in the T group was similar to the C group (Figure 4E and G
), despite the fact that the expression of VEGF in submucosal macrophages decreased (Figure 4F
). COX-2 was also stained predominantly in the interstitial cells in the lamina propria and submucosa (Figure 4H
). There was no significant difference in the proportions of COX-2-positive cells between the two groups.
 |
Discussion
|
---|
The clinical evidence suggests that selective COX-2 inhibitors reduce the incidence and mortality from intestinal tumors in FAP (25). Both selective or non-selective COX inhibitors reduced the growth of adenomas in APC-mutated mice (810,17), but they did not decrease the incidence of tiny adenomas (9,14). The mechanisms by which COX-2 deficiency decreases tumorigenesis of intestinal adenomas are still unclear.
The role of COX-2 in tumorigenesis has been reported as follows: enhancement of cell proliferation (23,26,27), resistance to apoptosis (2730), enhancement of angiogenesis (24,3133), enhancement of invasion (34) and suppression of host immunity (35). In the current study, we focused on the role of COX-2 in the development of adenomas in terms of proliferation, apoptosis and angiogenesis in adenomas using APC
474 mice.
COX-2 is regarded as an angiogenic factor. Modulation of VEGF by prostaglandin products due to COX-2 (24,31,36) and/or induction of endothelial migration by COX-2 (37,38) may contribute to tumor angiogenesis. Recently, Seno et al. (39) reported the importance of COX-2 and VEGF in the development of the large adenomas in APC-mutated mice, and it was suggested that elevated prostaglandin E2 that stimulate cell surface receptor EP2 play an important role in angiogenesis. JTE-522 was shown to decrease the incidence of large adenomas through the inhibition of VEGF expression of interstitial cells, but not of small adenomas.
Bergers et al. (40) reported that an angiogenesis inhibitor in the pre-malignant stage suppressed rapid tumor expansion and the progression to cancer. Aotake et al. (41) reported the progression of angiogenesis as a step up in the carcinogenesis of human colorectal adenomas and carcinomas. It is probable that the inhibitory effect of JTE-522 on the angiogenesis of large adenomas is beneficial, not only in the inhibition of the rapid growth of adenomas, but also in cancer prevention.
The accumulation of macrophages with VEGF expression and development of vascularity were observed in the submucosal layer of large adenomas, and administration of JTE-522 significantly reduced both VEGF expression and vascularity. COX-2 in colorectal adenomas has been reported to be expressed more dominantly in interstitial cells than in epithelial cells (42). Sonoshita et al. (18) reported that the deficiency of COX-2 or prostaglandin E2 receptor suppressed the VEGF expression of interstitial cells in APC
716 mice, and suggested that COX-2 played an important role in tumorigenesis and angiogenesis in adenomas of APC-mutated mice. Hull et al. (15) also reported that the COX-2 expression was localized to macrophages in the lamina propria and submucosa in the adenomas of APC-mutated mice. Seno et al. (39) observed that stromal expression of COX-2 in large adenomas was responsible to up-regulation of VEGF that caused tumor angiogenesis. In this study, we demonstrated that the accumulation of macrophages with VEGF and COX-2 expression in submucosa was observed only in large adenomas, resulting in sumucosal angiogenesis. Thus, the accumulation of macrophages with both VEGF and COX-2 expression in the submucosa of adenomas may be responsible for switching on angiogenesis and a prompt for unrestrained growth of adenomas.
COX-2 expression in adenomatous epithelial cells was very weak in this model, whereas overexpression of COX-2 was observed in colorectal cancer epithelial cells. COX-2 expression in interstitial cells, but not in epithelial cells, may be crucial for adenomas to induce angiogenesis. Additional studies are required to clarify the mechanisms for macrophage assemblage in the submucosa below large adenomas.
NSAIDs suppressed the proliferation (18) and induced the apoptosis (13) of adenomatous cells in APC-mutated mice. However, in this study, administration of JTE-522 did not reduce the cell proliferation of adenomatous epithelial cells, but did increase the apoptotic index. Nishimura et al. (27) reported that induction of apoptosis was noted after treatment with a COX-2 inhibitor at a lower concentration than for the suppression of cell proliferation in a cancer xenograft model. Although the dose of JTE-522 might not be enough to suppress cell proliferation, it is probable that the influence of COX-2 inhibition is more dominant in the induction of apoptosis than cell proliferation in this model.
A lot of evidence supports the idea that COX-2 inhibitors induce apoptosis in tumor cells (28,29,43,44). The down-regulation of Bcl-2 by COX-2 inhibitor treatment in a fibroblast cell line (45) and induction of Bcl-2 expression by prostaglandin E2 in a colon cancer cell line (43) were reported previously. In this study, Bcl-X, but not Bcl-2 was expressed in adenomatous epithelial cells, and JTE-522 decreased the Bcl-X expression of both small and large adenomas. Further studies are needed to clarify the role of the Bcl-family in the apoptosis induction by selective COX-2 inhibitors. Both inhibition of angiogenesis and induction of apoptosis by JTE-522 may exert synergistically to suppress the tumorigenesis of adenomas. However, the current results indicate that the anti-angiogenic effect of JTE-522 is essential to inhibit the growth of adenomas, since JTE-522 induced the apoptosis of both small and large adenomas.
In conclusion, macrophages with expression of COX-2 and VEGF protein in the submucosal layer are responsible for angiogenesis in large adenomas, which is essential for the growth of adenomas in APC
474 mice. A selective COX-2 inhibitor, JTE-522, reduces the growth of adenomas via its inhibitory effect on angiogenesis, as well as induction of apoptosis.
 |
Notes
|
---|
3 To whom correspondence should be addressed Email: suna{at}hama-med.ac.jp 
 |
Acknowledgments
|
---|
We thank Dr Hitoshi Sasai and Dr Katsuyuki Nagai for providing APC
474 mice and JTE-522, and Dr Takanori Sakaguchi for providing the VEGF cDNA probe.
 |
References
|
---|
- Rosenberg,L., Palmer,J.R., Zauber,A.G., Warshauer,M.E., Stolley,P.D. and Shapiro,S.A. (1991) Hypothesis: non-steroidal anti-inflammatory drugs reduce the incidence of large bowel cancer. J. Natl Cancer Inst., 83, 355358.[Abstract]
- Thun,M.J., Namboodiri,M.M and Heath,C.W. Jr (1991) Aspirin use and reduce risk of fatal colon cancer. N. Engl. J. Med., 325, 15931596.[Abstract]
- Dubois,R.N., Giardello,F.M. and Smalley,W.E. (1996) Non-steroidal anti-inflammatory drugs, eicosanoids and colorectal cancer prevention. Gastroenterol. Clin. North Am., 25, 773791.[ISI][Medline]
- Sano,H., Kawahito,Y., Wilder,R.L., Hashiramoto,A., Mukai,S., Asai,K., Kimura,S., Kato,H., Kondo,M. and Hla,T. (1995) Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res., 55, 37853789.[Abstract]
- Williams,C.S. and Dubois,R.N. (1996) Prostaglandin endoperoxide synthase: why two isoforms? Am. J. Physiol., 270, G393G400.[Abstract/Free Full Text]
- Eberhart,C.E., Coffey,R.J., Radhika,A., Giardiello,F.M., Ferrenbach,S. and DuBois,R.N. (1994) Up-regulation of cyclooxygenase-2 gene expression in human colorectal adenoma and adenocarcinoma. Gastroentelology, 107, 11831188.
- Williams,C.S., Luongo,C., Radhika,A., Zhang,T., Lamps,L.W., Nanney,L.B., Beauchamp,R.D. and Dubois,R.N. (1996) Elevated cyclooxygenase-2 levels in Min mouse adenomas. Gastroenterology, 111, 11341140.[ISI][Medline]
- Oshima,M., Dinchuk,J.E., Kargman,S.L., Oshima,H., Hancock,B., Kwong,E., Trzaskos,J.M., Evans,J.F. and Taketo,M.M. (1996) Suppression of intestinal polyposis in APC
716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87, 803809.[ISI][Medline]
- Sasai,H., Masaki,M. and Wakitani,K. (2000) Suppression of polypogenesis in a new mouse strain with a truncated Apc
474 by a novel COX-2 inhibitor, JTE-522. Carcinogenesis, 21, 953958.[Abstract/Free Full Text]
- Jacoby,R.F., Seibert,K., Cole,C.E., Kelloff,G. and Lubet,R.A. (2000) The cyclooxygenase-2 inhibitor Celecoxib is a potent preventive and therapeutic agent in the Min mouse model of adenomatous polyposis. Cancer Res., 60, 50405044.[Abstract/Free Full Text]
- Reddy,B.S., Hirose,Y., Lubet,R., Steele,V., Kelloff,G., Paulson,S., Seibert,K. and Rao,C.V. (2000) Chemoprevention of colon cancer by specific cycooxygenase-2 inhibitor, celecoxib, administered during different stage of carcinogenesis. Cancer Res., 60, 293297.[Abstract/Free Full Text]
- Chapple,K.S., Cartwright,E.J., Hawcroft,G. et al. (2000) Localization of cyclooxygenase-2 in human sporadic colorectal adenomas. Am. J. Pathol., 156, 545553.[Abstract/Free Full Text]
- Boolbol,S.K., Dannenberg,A.J., Chadburn,D.A. et al. (1996) Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res., 56, 25562560.[Abstract]
- Torrance,C.J., Jackson,P.E., Montgomery,E.M., Kinzler,K.W., Vogelstein,B., Wissner,A., Nunes,M., Frost,P. and Discafani,C.M. (2000) Combinatorial chemoprevention of intestinal neoplasia. Nature Med., 6, 10241028.[ISI][Medline]
- Hull,M.A., Booth,J.K., Tisbury,A., Scott,N., Bonifer,C., Markham,A.M. and Coletta,P.L. (1999) Cyclooxygenase 2 is up-regulated and localized to macrophages in the intestine of Min mice. Br. J. Cancer, 79, 13991405.[ISI][Medline]
- Oshima,H., Oshima,M., Kobayashi,M., Tsutsumi,M. and Taketo,M. (1997) Morphological and molecular processes of polyp formation in Apc
716 knockout mice. Cancer Res., 57, 16441649.[Abstract]
- Oshima,M., Murai,N., Kargman,S., Arguello,M., Luk,P., Kwong,E., Taketo,M.M. and Evans,J.F. (2001) Chemopreventioon of intestinal polyposis in the APC
716 mouse by refecoxib, a specific cyclooxygenase-2 inhibitor. Cancer Res., 61, 17331740.[Abstract/Free Full Text]
- Sonoshita,M., Takaku,K., Sasaki,N., Sugimoto,Y., Ushikubi,F., Narumiya,S., Oshima,M. and Taketo,M.M. (2001) Acceleration of intestinal polyposis through prostaglandin receptor EP2 in knockout mice. Nature Med., 7, 10481051.[ISI][Medline]
- Quesada,C.F., Kimata,H., Mori,M., Nishimura,M., Tsuneyoshi,T. and Baba,S. (1998) Piroxicam and acarbose as chemopreventive agents for spontaneous intestinal adenomas in APC gene 1309 knockout mice. Jpn. J. Cancer Res., 89, 392396.[ISI][Medline]
- Vogelstein,B., Fearon,E.R., Hamilton,S.R., Kern,S.E., Preisinger,A.C., Leppert,M., Nakamura,Y., White,R., Smits,A.M. and Bos,J.L. (1988) Genetic alterations during colorectal-tumor development. N. Engl. J. Med., 319, 525532.[Abstract]
- Powell,S.M., Zilz,N., Beazer-Barclay,Y., Bryan,T.M., Hamilton,S.R., Thibodeau,S.N., Vogelstein,B. and Kinzler,K.W. (1992) APC mutations occur early during colorectal tumorigenesis. Nature, 359, 235237.[ISI][Medline]
- Kawamori T., Rao,C.V., Seibert,K. and Reddy,B.S. (1998) Chemopreventative activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res., 58, 409412.[Abstract]
- Sawaoka,H., Kawano,S., Tsuji,S., Tsujii,M., Gunawan,E.S., Takei,Y., Nagano,K. and Hori,M. (1998) Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice. Am. J. Physiol., 274 (6 Pt 1), G1061G1067.[Abstract/Free Full Text]
- Tsujii,M., Kawano,S., Tsuji,S., Sawaoka,H., Hori,M. and DuBois,R.N. (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell, 93, 705716.[ISI][Medline]
- Steinbach,G., Lynch,P.M., Phillips,K.S. et al. (2000) The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N. Engl. J. Med., 342, 19461952.[Abstract/Free Full Text]
- Tsuji,M., Kawano,S., Sawaoka,H., Takei,Y., Kobayashi,I., Nagano,K., Fusamoto,H. and Kamada,T. (1996) Evidence for involvement of cyclooxygenase-2 in proliferation of two gastrointestinal cancer cell lines. Prostaglandins Leukot. Essential Fatty Acids, 55, 179183.[ISI][Medline]
- Nishimura,G., Yanoma,S., Mizuno,H., Kawakami,K. and Tsukuda,M. (1999) A selective cyclooxygenase-2 inhibitor suppresses tumor growth in nude mouse xenografted with human head and neck squamous carcinoma cells. Jpn. J. Cancer Res., 90, 11521162.[ISI][Medline]
- Hara,A., Yoshimi,N., Niwa,M., Ino,N. and Mori,H. (1997) Apoptosis induced by NS-398, a selective cyclooxygenase-2 inhibitor, in human colorectal cancer cell lines. Jpn. J. Cancer Res., 88, 600604.[ISI][Medline]
- Elder,D.J.E., Halton,D.E., Hague,A. and Paraskeva,C. (1997) Induction of apoptotic cell death in human colorectal carcinoma cell lines by a cyclooxygenase-2 (COX-2)-selective nonsteroidal anti-inflammatory drug: independence from COX-2 protein expression. Clin. Cancer Res., 3, 16791683.[Abstract]
- Elder,D.J.E., Halton,D.E., Crew,T.E. and Paraskeva,C. (2000) Apoptosis induction and cyclooxygenase-2 regulation in human colorectal adenoma and carcinoma cell lines by the cyclooxygenase-2-selective non-steroidal anti-inflammatory drug NS-398. Int. J. Cancer, 86, 553560.[ISI][Medline]
- Masferrer,J.L., Leahy,K.M., Koki,A.T., Zweifel,B.S., Settle,S.L., Woerner,B.M., Edwards,D.A., Flickinger,A.G., Moore,R.J. and Seibert,K. (2000) Antiangiogeninc and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res., 60, 13061311.[Abstract/Free Full Text]
- Liu,X.H., Kirschenbaum,A., Yao,S., Lee,R., Holland,J.F. and Levine,A.C. (2000) Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J. Urol., 164, 820825.[ISI][Medline]
- Sawaoka,H., Tsuji,S., Tsujii,M., Gunawan,E.S., Sasaki,Y., Kawano,S. and Hori,M. (1999) Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab. Invest., 79, 14691477.[ISI][Medline]
- Dohadwala,M., Luo,J., Zhu,L., Lin,Y., Dougherty,G.J., Sharma,S., Huang,M., Pold,M., Batra,R.K. and Dubinett,S.M. (2001) Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44. J. Biol. Chem., 276, 2080920812.[Abstract/Free Full Text]
- Huang,M., Stolina,M., Sharma,S., Mao,J.T., Zhu,L., Miller,P.W., Wollman,J., Herschman,H. and Dubinett,S.M. (1998) Non-small cell lung cancer cyclooxygenase-2-dependent regulation of cytokine balance in lymphocytes and macrophages: up-regulation of interleukin 10 and down-regulation of interleukin 12 production. Cancer Res., 58, 12081216.[Abstract]
- Pourtau,J., Mirshahi,F., Li,H., Muraine,M., Vincent,L., Tedgui,A., Vannier,J.P., Soria,J., Vasse,M. and Soria,C. (1999) Cyclooxygenase-2 activity is necessary for the angiogenic properties of oncostatin M. FEBS Lett., 459, 453457.[ISI][Medline]
- Daniel,T.O., Liu,H., Morrow,J.D., Crews,B.C. and Marnett,L.J. (1999) Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis. Cancer Res., 59, 45744577.[Abstract/Free Full Text]
- Dormond,O., Foletti,A., Paroz,C. and Rüegg,C. (2001) NSAIDs inhibit
Vß3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis. Nature Med., 7, 10411047.[ISI][Medline]
- Seno,H., Oshima,M., Ishikawa,T., Oshima,H., Takaku,K., Chiba,T., Narumiya,S. and Taketo,M.M. (2002) Cyclooxygenase 2- and prostaglandin E2 receptor EP2-dependent angiogenesis in Apc
716 mouse intestinal polyps. Cancer Res., 62, 506511.[Abstract/Free Full Text]
- Bergers,G., Javaherian,K., Lo,K.M., Folkman,J. and Hanahan,D. (1999) Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science, 284, 808812.[Abstract/Free Full Text]
- Aotake,T., Lu,C.D., Chiba,Y., Muraoka,R. and Tanigawa,N. (1999) Changes of angiogenesis and tumor cell apoptosis during colorectal carcinogenesis. Clin. Cancer Res., 5, 135142.[Abstract/Free Full Text]
- Bamba,H., Ota,S., Kato,A., Adachi,A., Itoyama,S. and Matsuzaki,F. (1999) High expression of cyclooxygenase-2 in macrophages of human colonic adenoma. Int. J. Cancer, 83, 470475.[ISI][Medline]
- Sheng,H., Shao,J., Morrow,J.D., Beauchamp,R.D. and Dubois,R.N. (1998) Modulation of apoptosis and bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res., 58, 362366.[Abstract]
- Hsu,A.L., Ching,T.T., Wang,D.S., Song,X., Rangnekar,V.M. and Chen,C.S. (2000) The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of bcl-2. J. Biol. Chem., 275, 1139711403.[Abstract/Free Full Text]
- Liu,X.H., Yao,S., Kirschenbaum,A. and Levine,A.C. (1998) NS-398, a selective cyclooxygenase-2 inhibtor, induces apoptosis and down-regulates bcl-2 expression in LNCap cells. Cancer Res., 58, 42454249.[Abstract]
Received February 14, 2002;
revised April 29, 2002;
accepted April 30, 2002.