Expression of cyclooxygenase-2 parallels expression of interleukin-1beta, interleukin-6 and NF-kappaB in human colorectal cancer

Christian Maihöfner1,5, Michalis Panayiotou Charalambous2, Upinder Bhambra2, Tracy Lightfoot3, Gerd Geisslinger4, Nigel J. Gooderham2 and The Colorectal Cancer Group

1 Department of Neurology, University of Erlangen-nuremberg, Schwabachanlage 6, D-91054 Erlangen, Germany
2 Molecular Toxicology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK
3 JBUEC, Department of Biology, University of York, York YO1 5DD, UK
4 Centre for Clinical Pharmacology, Kinikum der Johann Wolfgang Goethe Universität, Frankfurt, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany

5 To whom correspondence should be addressed at: Institute of Physiology and Experimental Pathophysiology, Universitätsstrasse 17, D-91054 Erlangen, Germany Email: maihoefner{at}physiologie1.uni-erlangen.de


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Elevated expression of cyclooxygenase-2 (COX-2), the inducible isoform of prostaglandin H synthase, has been found in several human cancers, including colorectal cancer (CRC). This appears as a rationale for the chemopreventive effects of non-steroidal anti-inflammatory drugs in CRC. However, the reason for COX-2 overexpression is not fully understood. In cell culture experiments, COX-2 can be induced by proinflammatory cytokines, such as interleukin (IL)-1beta and IL-6. A crucial step in this signalling pathway is thought to be activation of transcription factor NF-{kappa}B. Based on these findings, we hypothesized an association between COX-2 overexpression and expression of IL-1beta, IL-6 and the NF-{kappa}B subunit p65 in human CRC. To test the hypothesis, we performed immunohistochemistry for the respective antigens on colorectal cancer specimens, obtained by surgical resections from 21 patients with CRC. Immunohistochemical results were confirmed by examination of protein levels in tissue lysates and nuclear extracts using western blotting. Non-neoplastic tissue specimens resected well outside the tumour border served as controls. COX-2 expression was found to be markedly enhanced in the neoplastic epithelium compared with controls. This was paralleled by a significantly higher expression of IL-1beta, IL-6 and p65. Serial sections revealed consistent cellular colocalizations of respective antigens in the neoplastic epithelium. Statistically, a significant correlation between expression of COX-2 and IL-1beta, IL-6 and p65 was found. Comparable results were obtained for stromal cells like macrophages and myofibroblasts. Further examination of nuclear extracts from CRC-specimens by western blotting confirmed a higher content of p65 protein compared with non-neoplastic control tissues. Therefore, our study provides evidence for an association between expression of COX-2 and IL-1beta, IL-6 and p65 in human CRC. The results are consistent with the thesis that proinflammatory cytokines such as IL-1beta and IL-6 may be accountable for the overexpression of COX-2 in CRC. Finally, the study corroborates a role for NF-{kappa}B in the control of COX-2 gene transcription in CRC. Given an antiapoptotic role for COX-2 in tumour cells, inhibition of NF-{kappa}B may offer an important strategy to interfere with the development and progression of CRC.

Abbreviations: CRC, colorectal cancer; COX-2, cyclooxygenase-2; IL, interleukin; FAP, familial adenomatous polyposis; NSAIDs, non-steroidal anti-inflammatory drugs; NF-{kappa}B, nuclear factor {kappa}B.


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Colorectal cancer (CRC) is the second leading cause of death in western countries (1,2). Despite this high prevalence the underlying pathological mechanisms remain largely unknown. Diet is thought to contribute to the incidence of the disease (3,4), and genetic traits have also been identified (5,6). Epidemiological studies demonstrated the capability of non-steroidal anti-inflammatory drugs (NSAIDs) to reduce the risk of CRC (7,8). Data from human trials suggest that NSAIDs such as sulindac are able to reduce the size and number of polyps in individuals with familial adenomatous polyposis (FAP) (9,10). However, the mode of action of these compounds has remained unclear. The pharmacodynamic target for NSAIDs is cyclooxygenase (COX), a key enzyme in the formation of prostaglandins (PGs) (11). Since 1992 it has been known that two isoenzymes of this enzyme exist, termed COX-1 and COX-2 (12). It is now generally thought that COX-1 is constitutively expressed in most tissues, and displays the characteristics of a ‘housekeeping-enzyme’. By contrast, COX-2 is the product of an ‘immediate-early-gene’ that is rapidly inducible and tightly regulated. Under normal conditions, COX-2 expression is highly restricted to certain organs including the central nervous system (CNS) (13,14), the kidney (15) and the eye (16), but COX-2 expression can be dramatically increased in various tissues following initiation of transcription by activating factors including different proinflammatory cytokines, growth factors or tumour promoters (12). Interestingly, COX-2 was found to be highly expressed in tumour specimens of patients with CRC (1719). Inhibition of COX-2 activity is mainly thought to account for the chemopreventative activity of NSAIDs against CRC and FAP in humans (20,21). However, the reason for enhanced COX-2 expression in CRC is not known. Therefore, it remains unclear if COX-2 plays a crucial role in colorectal tumorigenesis, or if it is an epiphenomenon of cancer. It is important to note that some of the cytokines involved in COX-2 induction have been found to be expressed in CRC, particularly IL-1beta and IL-6 (22,23). When bound to their respective receptors, both cytokines can contribute to the nuclear translocation of nuclear factor {kappa}B (NF-{kappa}B), where this transcription factor binds to distinct consensus sequences of promoters. Activation of NF-{kappa}B has been shown to be crucial for COX-2 induction in various cell types. However, immunohistochemical expression of NF-{kappa}B has not been investigated in human CRC so far. Based on these in vitro findings, we hypothesized a correlation between expression of COX-2 and IL-1beta, IL-6 and the p65 subunit of NF-{kappa}B in human CRC.

We report here an overexpression of COX-2, IL-1beta, IL-6 and p65 in human CRC compared with controls. Enhanced expression of COX-2 was significantly linked to high expression of IL-1beta, IL-6 and p65.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
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Tissue specimens
Informed consent to all tissue donations was obtained and the study adhered to the tenets of the Declaration of Helsinki. Ethical approval for the study was granted from the Human Research Ethics Committee at York District Hospital. We had access to 21 colorectal cancer specimens, obtained by surgical resection, from 21 patients (14 men and 7 women; aged 55–84 years, mean age 66.3 years ± 8.7 SEM). The clinical and pathological characteristics of the patients are given in Table I. The entire study was carried out blind using coded tissue sections. Histopathological evaluation of haematoxylin and eosin-stained slides revealed 10 well-differentiated, five moderately and six poorly differentiated adenocarcinomas. Tumours were classified according to the Duke's classification (see Table I). Twelve of the cancers were located in the colon and 9 in the rectum.


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Table I. Synopsis of the epidemiological and pathological data of the patients included in this study

 
Light microscopic immunohistochemistry
For immunohistochemistry, goat polyclonal antisera raised against human COX-2-protein (Santa Cruz Biotechnology, Santa Cruz, CA), human p65-protein (Santa Cruz Biotechnology, Santa Cruz, CA), human IL-1beta protein and human IL-6-protein (R & D Systems, Abingdon, UK) were employed. Tissue specimens were fixed in 4% v/v paraformaldehyde and embedded in paraffin wax. Six micrometer thick sections were cut, placed on glass slides and sequentially deparaffinized and rehydrated through xylene and graded alcohol solutions. The labelled Streptavidin–Biotin method using the LSAB Plus-kit (DAKO, Glostrup, Denmark) was applied according to the manufacturer's instructions. After quenching endogenous peroxidase activity of the tissue with 3% hydrogen peroxide in distilled water for 30 min, sections were incubated with proteinase K (0.02 mg/ml in Tris-buffered saline, pH 8) for 20 min to unmask antigens. After rinsing, sections were incubated overnight at 4°C with the primary anti-COX-2, p65, IL-1beta and IL-6 antibodies used at respective dilutions of 1:1500, 1:1500, 1:1000 and 1:1000. Afterwards, sections were washed with TBS for 15 min and probed with the biotinylated antibody and the HRP-conjugated Streptavidin, 30 min each. Staining was developed by immersing the slides in a solution of hydrogen peroxide, 3,3-diaminobenzidine (DAKO) in TBS for 4–5 min. Sections were successively immersed into haematoxylin, acid alcohol and Scott's tap water to counterstain. Finally, specimens were dehydrated and mounted with Aquatex (Merck, Darmstadt, Germany). After staining procedures, slides were coded and assessed observer-blinded for immunoreactivity (IR).

Immunohistochemical evaluation
In order to assess and grade intensity and distribution of the respective IR in the colonic epithelium, we used a scoring method that has been previously described by Yukawa and colleagues (24). The distribution was scored according to the numbers of positive cells: none (not stained), 0; focal (<one-third of cells stained), 1; multifocal (<two-thirds of cells stained), 2; and diffuse (most cells stained), 3. The staining intensity was scored as: none (not stained), 0; mild (between 0 and 2), 1; and strong, 2. The distribution and intensity scores were added to produce the following grades for the staining: 0, negative; 2, intermediate; and 3, 4 and 5, positive.

Additionally, we assessed the staining of stromal cells in the subepithelial layers as: negative, 0; intermediate, 1; positive, 2; and strong positive, 3.

Negative controls included incubation of colonic tissues with non-immunized goat serum, omitting the primary antibody and incubation of negative control tissues (muscle). Positive tissue controls included sections of brain, kidney and uterus (data not shown).

Western blotting experiments
Tumour and control specimens from four patients (patient numbers 4, 7, 14, 18, see Table I) that were also included in our immunohistochemical studies were homogenized in lysis buffer, containing 0.15 M NaCl, 0.1 M TBS, 50 mM diethyldithiopyrocarbonate, 1% (vol/vol) Tween-20, and 10 mM phenylmethylsulfonyl fluoride and evaluated by western blotting (13,16,25). Briefly, protein (50 µg/lane) was loaded on a 10% SDS–polyacrylamide gel and electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Membranes were probed with the primary anti-COX-2, p65, IL-1beta and IL-6 antibodies used at a dilution of 1:1000 each, followed by a horseradish peroxidase (HRP)-linked donkey anti-goat IgG secondary antibody (diluted 1:1000; Santa Cruz Biotechnology). Blots were developed with enhanced chemiluminescence (ECL) detection reagents (Amersham, Arlington Heights, IL).

Preparation of nuclear extracts
To assess the potential nuclear translocation of the NF-{kappa}B subunit p65, we analysed nuclear extracts from control non-neoplastic and CRC specimens using western blotting. Extraction of nuclear proteins was adapted from a method described by Tegeder et al. (26). Frozen tissue specimens were homogenized in 3 vol of buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 1 mg/ml leupeptin and aprotinin, pH 7.9) with a loose homogenizer and were placed on ice for 10 min and centrifuged at 3300 g for 15 min. The pellet was suspended in 3 ml of buffer B (20 mM HEPES, 20 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 0.2 mM PMSF, 1 mg/ml leupeptin and aprotinin and 25% glycerol, pH 7.9). One millilitre of buffer C (20 mM HEPES, 1.2 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 1 mg/ml leupeptin and aprotinin, pH 7.9) was then added and mixed. The sample was placed on ice then centrifuged at 15 000 g for 30 min at 4°C. The supernatant was filtered and dialyzed against buffer D (20 mM HEPES, 100 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, and 20% glycerol, pH 7.9) at 4°C overnight.

Fifty µg protein per lane was loaded on a 10% SDS–polyacrylamide gel and processed for NF-{kappa}B subunit p65 as described above.

Statistical analysis
The Wilcoxon-signed-ranked test was used to compare the scoring of the respective immunoreactivity for the antigens (COX-2, p65, IL-1beta and IL-6) between CRC and control tissues. Pearson's product-moment correlation analysis was employed to assess the correlation between COX-2 expression and p65, IL-1beta, IL-6.


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 Patients and methods
 Results
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 References
 
Immunohistochemical expression of COX-2, IL-1beta, IL-6 and p65 in normal colonic tissue
Cyclooxygenase-2
In sections from normal human colon (resected well outside the tumour border), weak positive COX-2-IR was occasionally observed in the cytoplasm of superficial epithelial cells (4/21). In most cases, COX-2-IR was restricted to a few positive scattered cells in the lamina propria and submucosa with the morphological characteristics of macrophages and myofibroblasts (11/21) (Figure 1a). By contrast, in none of our controls was COX-2-IR observed in the muscular layer. The mean rating scores for the epithelium and stroma were 1.47 and 0.84, respectively.



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Fig. 1. Expression of COX-2, IL-1beta, IL-6 and p65 in normal non-neoplastic colonic tissue (a–d) and colorectal cancer specimens (e–h). Positive immunoreactivity appears as reddish-brown staining. (a) COX-2-IR was infrequently observed in the epithelium of matched non-neoplastic control tissues. However, COX-2-IR was seen in scattered cells of the lamina propria and submucosa with the morphological features of macrophages and myofibroblasts (arrow). (b) Absent IL-1beta-IR in normal non-neoplastic colonic tissue. (c) Absent IL-6-IR in normal non-neoplastic colonic tissue. (d) p65-IR was occasionally observed in cells of the lamina propria and submucosa with the morphological features of macrophages and myofibroblasts (arrow). (e–h) Strong expression of COX-2 (e), IL-1beta (f), IL-6 (g) and p65 (h) in colorectal cancer specimens. The figures are of serial sections of a representative case. Insets are high power magnifications from the area indicated in Figure 1e (arrow). Consistently, cellular colocalizations were found for the four antigens. p65 was also detected endonuclear (inset in h, arrow).

 
Interleukin-1beta
In most of our control sections (17/21) IL-1beta-IR was absent (Figure 1b). Nevertheless, in 4 sections there was weak IL-1beta-IR in some epithelial cells or scattered stroma cells. Overall, the mean scores for the epithelium and stroma were 0.31 and 0.21, respectively.

Interleukin-6
IL-6 was absent in most of the control sections (15/21) (Figure 1c). In 6/21 cases IL-6-IR was restricted to some weakly positive macrophages and epithelial cells. The mean score for the epithelium was 0.21, for the lamina propria and submucosa 0.16.

p65
Few p65 immunoreactive macrophages and myofibroblasts were detected in the lamina propria and submucosa (Figure 1d). The staining was cytoplasmic and no staining was observed inside the nuclei. Occasionally, p65-IR was observed in the colonic epithelium of control sections. The staining was more accentuated in the superficial portion of the colonic crypts. The mean rating scores for the epithelium and stroma were 2.2 and 0.85, respectively.

Immunohistochemical expression of COX-2, IL-1beta, IL-6 and p65 in colorectal cancer specimens
Immunohistological detection of COX-2-protein showed strong cytoplasmic staining in the colonic carcinoma cells (Figure 1e). In the surrounding stroma there were numerous inflammatory cells, vascular endothelium, and fibroblasts that expressed COX-2-protein. Most of our CRC specimens (18/21) were positive for COX-2-IR. The staining index was 4.5 for the epithelium and 2.05 for the stroma, respectively. There was also a markedly higher expression of p65, IL-1beta and IL-6 in the neoplastic epithelium compared with normal colonic tissue (Figure 1f–h). Statistical analysis demonstrated a significantly higher rating of the respective intensity scores for the epithelium in CRC-cases compared with control sections (Figure 2a) (Wilcoxon-signed-ranked test; P = 0.012 for COX-2, P = 0.001 for IL-1beta, P = 0.001 for IL-6 and P = 0.03 for p65). Consistently, serial sections revealed numerous cellular colocalizations of COX-2 with the IL-1beta-, IL-6- and p65-protein (compare insets in Figure 1e–h). In agreement with this histological finding, there was a statistically significant correlation between COX-2-, IL-1beta-, IL-6- and p65-expression in the epithelium (Figure 2b; Pearson's product-moment correlation analysis, P < 0.05; r = 0.92 for COX-2 and IL-1beta; r = 0.91 for COX-2 and IL-6, r = 0.83 for COX-2 and p65).



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Fig. 2. (a) Diagram demonstrating significantly higher values for the respective intensity scores in the neoplastic epithelium (solid bars) compared with matched non-neoplastic controls (open bars). Values are given as mean ± standard error of the mean. Stars indicate significant differences (Wilcoxon-signed-ranked test; P = 0.012 for COX-2, P = 0.001 for IL-1beta, P = 0.001 for IL-6 and P = 0.03 for p65). (b) Relationship between COX-2 expression and expression of IL-1beta, IL-6 and p65 for all specimens (epithelium). As assessed by Pearson's product-moment correlation analysis, expression of COX-2 was significantly correlated with expression of IL-1beta, IL-6 and p65, respectively (P < 0.05; r = 0.92 for COX-2 and IL-1beta; r = 0.91 for COX-2 and IL-6, r = 0.83 for COX-2 and p65).

 
In contrast to control sections, p65-IR was not only found in the cytoplasm, but also consistently in the nucleus (Figure 1 h). Furthermore, intensity of IL-1beta-, IL-6- and p65-IR was clearly enhanced in numerous cells of the lamina propria and submucosa. The detected stromal cells showed the morphological characteristics of myofibroblasts and macrophages (Figure 1e–h). The respective intensity scores were significantly higher compared with the values of control specimens (Figure 3a). Again, a significant correlation between the expression of COX-2, IL-1beta, IL-6 and p65 in these cells could be demonstrated (Figure 3b; Pearson's product-moment correlation analysis, P < 0.05; r = 0.76 for COX-2 and IL-1beta; r = 0.81 for COX-2 and IL-6, r = 0.74 for COX-2 and p65). Comparison of the staining indices for COX-2, IL-1beta, IL-6 and p65 with the respective Duke's stages of the tumour revealed no statistical association (Pearson's product-moment correlation analysis, P < 0.05, r = 0.34). Furthermore, there was no statistical relationship between the staining indices and the alcohol consumption in units per week or tobacco use.



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Fig. 3. (a) Diagram demonstrating significantly higher values for the respective intensity scores in the stroma of CRC cases (solid bars) compared with matched non-neoplastic controls (open bars). Values are given as mean ± standard error of the mean. Stars indicate significant differences (Wilcoxon-signed-ranked test; P = 0.03 for COX-2, P = 0.02 for IL-1beta, P = 0.02 for IL-6 and P = 0.03 for p65). (b) Relationship between COX-2 expression and expression of IL-1beta, IL-6 and p65 for all specimens (stromal cells). As assessed by Pearson's product-moment correlation analysis, expression of COX-2 was significantly correlated with expression of IL-1beta, IL-6 and p65, respectively (P < 0.05; r = 0.76 for COX-2 and IL-1beta; r = 0.81 for COX-2 and IL-6, r = 0.74 for COX-2 and p65).

 
Western blotting analysis of normal and colorectal cancer specimens
To corroborate our immunohistochemical results, we performed western blotting experiments (Figure 4) for the respective antigens on protein extracts from four matched non-neoplastic control and CRC specimens (patient numbers 4, 7, 14, 18, see Table I). Immunoblotting (Figure 4) revealed already a weak expression of COX-2, IL-1beta, IL-6 and p65 protein in some of the control tissue samples. In CRC, the expression of COX-2, IL-1beta, IL-6 and p65 proteins was markedly increased compared with controls. Furthermore, to assess a potential nuclear translocation of p65 in CRC, we looked for the respective antigen in nuclear extracts of both non-neoplastic control and matched CRC tissue. Figure 5 illustrates the increased levels of p65 in nuclear extracts of CRC as compared with matched non-neoplastic tissue.



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Fig. 4. The expression of COX-2, IL-1beta, IL-6 and p65 proteins in non-neoplastic control (lanes 1, 2, 3 and 4) and CRC (lanes 5, 6, 7, and 8) tissue specimens determined by western blotting. Note the increased expression of COX-2, IL-1beta, IL-6 and p65 proteins in CRC.

 


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Fig. 5. The expression of p65 protein in nuclear extracts of non-neoplastic control (lanes 1, 2, 3) and CRC (lanes 4, 5, 6) tissue.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In the current study we demonstrated that COX-2, IL-1beta, IL-6 and the p65 subunit of NF-{kappa}B are strongly expressed in CRC. Using immunohistochemistry, we were able to show the in situ localization of the respective antigens. Expression of COX-2 was significantly linked to the expression of IL-1, IL-6 and p65, both in the neoplastic epithelium and the stroma. Furthermore, increased levels of p65 were found in nuclear extracts of CRC specimens.

Expression of COX-2 in colorectal cancer has been previously described (18,19,27) and is currently of great pharmacological interest with regard to the recent development of drugs that are able to preferentially inhibit this COX-isoform (7). The results of this study strongly corroborate the previous findings that COX-2 is highly expressed in CRC. There are several lines of evidence that COX-2 might be a rate-limiting step in colon carcinogenesis. Firstly, COX-2 appears to inhibit apoptosis in colon tumour cell lines and malignant colon tissue. COX-2 inhibitors exert their anti-proliferative effects in mouse models for CRC and in colon tumour cell lines most likely via induction of apoptosis (2832). Secondly, inhibition of COX-2 in mice with a defective APC tumour suppressor gene, protects against the development of intestinal tumours (33). Thirdly, epidemiological evidence shows that individuals who take NSAIDs have a markedly reduced risk of developing CRC and its presumed non-malignant precursor, the adenomatous polyp (7,8). Finally, expression of COX-2 was found to correlate with an unfavourable progression of the disease (34).

In this present study we further extended these findings with an immunohistochemical grading of COX-2 expression. However, no significant correlation was found between the level of COX-2 expression and the Duke's stage of the tumour. This might be due to the small case numbers available (n = 21) and confirmation of this will require a larger study. However, this result may imply that COX-2 expression occurs early in CRC tumorigenesis and is involved in the pathogenesis of tumour progression. This is also in line with findings in animal models for CRC, e.g. APC knockout mice, where COX-2 expression occurs in early stages of tumorigenesis (33).

Nevertheless, the cause of high COX-2 expression in human CRC is not fully understood. The human COX-2 promotor contains a TATA box and a number of transcriptional elements that are common in highly regulated genes, particularly those involved in inflammation (12). An essential transcription factor for COX-2 induction is NF-{kappa}B. Sequence analysis of the 5'-flanking region of the COX-2 gene shows two NF-{kappa}B binding sites (35). Recently, inhibition of the NF-{kappa}B pathway has been shown to attenuate COX-2 expression (31), indicating that NF-{kappa}B may play an important role in COX-2 induction in CRC. However, to the best of our knowledge, there are no immunohistochemical studies that describe the expression of NF-{kappa}B in CRC despite its reputed involvement (36). We here provide immunohistochemical evidence that the p65 subunit of NF-{kappa}B is highly expressed in both the malignant epithelium and the tumour surrounding stromal cells in human CRC. Furthermore, high expression of p65 was significantly correlated with COX-2 expression and increased levels of p65 were found in nuclear extracts of CRC tissue.

NF-{kappa}B factors are dimers of Rel family proteins (37). They are complexed with inhibitory {kappa}B proteins (i{kappa}B) in the cytoplasm. In response to different stimuli, i{kappa}B proteins become phosphorylated by i{kappa}B-kinases. This results in liberation of NF-{kappa}B allowing this transcription factor to accumulate in the nucleus where it binds to specific gene targets, such as consensus sequences within the COX-2 promotor. There is accumulating evidence indicating that NF-{kappa}B plays an important role in the development of cancer and metastasis (36). In fact, NF-{kappa}B acts as an anti-apoptotic factor and its activation is required to protect cells from the apoptotic cascade induced by TNF and other stimuli (38). Of significant interest is the observation that NF-{kappa}B can antagonize p53 protein, a key factor for apoptosis, possibly through the cross-competition for transcriptional co-activators (39). In this context it is important to note that in non-neoplastic control sections p65-IR was predominantly cytoplasmic. In contrast, as a consistent finding in CRC, there was an increase of both the p65-IR in the cytoplasm and the nucleus. These qualitative morphological results were also corroborated by the demonstration of increased nuclear levels of the p65 protein in CRC by means of western blotting analysis. It remains to be demonstrated that this translocated transcription factor is indeed able to bind to target promotor regions and may initiate transcription of COX-2 protein in CRC. However, these findings imply an increase of both NF-{kappa}B gene expression and activation of the latent cytoplasmic protein into an active endonuclear form in neoplastic colorectal epithelial. Therefore, our results provide a morphological and biochemical basis for interactions between NF-{kappa}B and COX-2 gene transcription in human CRC.

Among different proinflammatory cytokines, IL-1beta and IL-6 were shown to be potent activators of NF-{kappa}B in normal cell types and colon cancer cell types (12,35,37). Induction of COX-2 protein by IL-1beta in RAW macrophages, murine 3T3 fibroblasts, hepatocytes and rheumatoid synoviocytes has been demonstrated to be NF-{kappa}B dependent (12,35). In the present study we found a significant correlation between IL-1beta and COX-2 expression in CRC. This hints at a potential role for IL-1beta in the regulation of expressed COX-2 protein in human CRC. This is in line with other studies, as IL-1beta has been shown to mediate a variety of effects in cancer and is thought to potentiate invasiveness. Etoh and colleagues described the capability of IL-1beta to induce angiogenin m-RNA, the protein product of which is potently angiogenic for the neovascularization of cancer tissue (40). IL-1beta has stimulating effects on metalloproteinases which are thought to be involved in the mediation of metastasis (41), and it has also been shown to have effects on tumour growth (42,43). To our knowledge, the cellular distribution of IL-1beta protein in CRC has not been described. We provide here evidence for an enhanced IL-1beta expression both in the malignant epithelium and the stroma surrounding the tumour. In the latter it might have a role in the mediation of metastasis based on the above mentioned results of Etoh and colleagues and in the former it might act as a regulator of tumour growth. Induction of the IL-1 receptor antagonist is assumed to contribute to the antiproliferative effects of cytokines, such as interferon-{gamma} and tumour necrosis factor (44). Clinical trials with an attempt to block the effects of IL-1 are underway (45).

IL-1beta was also found to induce IL-6 via activation of NF-{kappa}B (46). We provide evidence for expression of IL-6 in both the neoplastic epithelium and the surrounding stroma cells. Yet in matched non-neoplastic control tissue, expression of IL-6 protein was only occasionally observed and staining was very weak. Since IL-6 plays a central role as a differentiation and growth factor of tumour cells, detection of its immunoreactive protein in CRC is highly relevant and supports the finding of high IL-6-mRNA levels in CRC (22). Furthermore, Kinoshita and colleagues suggested that serum IL-6 level may reflect the tumour proliferative activity in patients with CRC (23) and reported a correlation between serum IL-6 concentration, liver metastasis, tumour size and the Ki-67 labelling index. Interestingly, the human COX-2 promotor contains a binding site for the nuclear factor of IL-6 (NF-IL-6) (12,35) and recently, mutation of NF-IL-6 element has been shown to diminish COX-2 promotor activity in two colorectal cancer cell lines (47). Thus, NF-IL-6 binding site appears to be crucial for COX-2 upregulation in CRC. Additionally, IL-6 itself can also lead to an activation of the NF-{kappa}B pathway (48,49) and thereby induction of COX-2. These in vitro findings fit well with the cellular colocalization of COX-2, p65 and IL-6 in CRC.

In summary, this study describes the expression of COX-2, p65, IL-1beta and IL-6 in human CRC. COX-2 overexpression appears as a rationale for the chemopreventive effects of NSAIDs in CRC, however, the reason for COX-2 overexpression is not known. Based on immunohistochemical and biochemical investigations, we provide a potential explanation for this phenomenon. Expression of COX-2 parallels expression of IL-1beta and IL-6, two well-known inductors of COX-2, and expression of transcription factor NF-{kappa}B. These observations suggest that immunomodulatory approaches and/or modulation of NF-{kappa}B expression and activation could be possible pharmacological strategies to interfere with the development and progression of CRC.


    Notes
 
The Colorectal Cancer Study Group: Dr J.Barrett, Professor D.T.Bishop, Professor A.R.Boobis, U.Bhambra, Professor D.Forman, Professor R.C.Garner, Dr N.J.Gooderham, Dr T.J.Lightfoot, Dr C.Sachse, Dr G.Smith, Dr R.Waxman, Professor C.R.Wolf. Back


    Acknowledgments
 
This work was supported by the United Kingdom Food Standards Agency.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received December 16, 2002; revised January 2, 2003; accepted January 13, 2003.