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Cyclooxygenase-2 protein varies with tumor progression in experimental colon cancer
April 2000
AGAA53
gastric cancer. COX-2 promoter hypomethylation does not occur frequently in gastritis or in gastric tumors without MSI.
Frequency ofhypomethylation of COX-2 promoter ingastric cancers vs. adjacent normal tissue Tissue Type
Hypometh
%
P*
7/14 2/6 9/20 1/20 0/3
50 33 45 5 0
0004
MSI.high Iumors MSI·low tumors MSltotal MSI·neg Gastritis
NS
0008 NS
'Fisher's exact test, vs. MSI-neg
COX-I is constitutively expressed in colonocytes. We hypothesized that COX-2 expression would be upregulated during tumor initiation and promotion, while constitutive COX-I would remain unchanged. Methods: For tumor initiation F344 rats, induced with 20 mgfkg ip azoxyrnethane(AOM), were harvested after 5 days. For tumor promotion, harvest was 8 weeks after the intitial injection of 2 weekly AOM doses. Tissue was stained for COX-2 and COX-l by IHC. Results: COX-2 was not seen in vehicle-treated rats(data not shown). Data are mean +1- SEM(n);like superscripts, p<0.05, unpaired t-test.(table) Conclusion: Increased COX-2 protein during tumor initiation may relfect a generalized inflammatory or carcinogen activation response, which also occurs to a lesser degree with COX-I. The high rates of COX-2 staining in proximal colon at tumor initiation had resolved by tumor promotion. At tumor promotion, increased COX-2 in distal versus proximal colon is associated with increased aberrant crypt foci (ACF) and typical of the location of colon tumors in this model and human sporadic colon cancer. The increased ACF may reflect COX-2 tumorigenic effects. We conclude that COX-2 expression may serve different functions in the same tissue site during colon cancer progression.
476 EXPRESSION OF CYCLO-OXYGENASE-2 PROTEIN IS ASSOCIATED WITH DYSPLASIA, K-RAS MUTATION AND EXPRESSION OF P53 IN COLORECTAL ADENOMAS. Robert Benamouzig, Elisabeth Longchampt, Helen Yoon, Eric Jullian, Antoine Martin, Thierry Coste, Daniel Couturier, Jacques Rautureau, Stanislas Chaussade, Hosp Avicenne, Bobigny, France; Hosp Cochin, Paris, France. Cyclo-oxygenase-2 (COX-2) is an inducible enzyme that catalyzes the conversion of arachidonic acid to prostaglandins and thromboxanes. COX-2 protein expression is observed in colorectal neoplasms and may be a target for anticolorectal cancer activity of NSAIDs and aspirin in humans. The purpose of this study was to evaluate COX-2 protein hyperexpression in 182 adenomas obtained from the 116 first consecutive patients (73 males, 43 females, age 58 +1- 9 years) included in a large prospective multi-center randomised study aimed to evaluate the effect of long term daily use of low dose aspirin in reducing the occurence of new adenomatous polyps. All patients have had colonoscopy to the cecum with adequate preparation resulting in clearance of either a single adenoma> 10 mm in size or 3 adenomas of any size. Methods : All biopsy specimens were blindly assessed for architectural pattern and dysplasia by 2 independent pathologists. Immunohistochemistry was carried out on formalin-fixed paraffin-embedded sections with specific anti-COX-2 antibody and antiP53 antibody. Staining intensity was scored 0-3 by two blinded independent observers. After DNA extraction from these specimens, PCR amplification and sequencing were performed to detect K-ras mutation. Results : COX-2 epithelial cells staining was heterogenous both between crypts and inside a crypt. Superficial COX-2 staining was also observed in interstitial cells. An high to moderate hyperexpression of COX-2 was observed in 36% of adenomas (score 2 and 3), COX-2 hyperexpression was more frequent in adenomas exhibiting high grade dysplasia (p<0,05) and intense p53 immunostaining (p
477 CYCLOOXYGENASE-2 PROTEIN VARIES WITH TUMOR PROGRESSION IN EXPERIMENTAL COLON CANCER. Charlene Compher, John L. Rombeau, Noel N. Williams, Univ of Pennsylvania, Philadelphia, PA. Introduction: Cyclooxygenase-2(COX-2)is upregulated in colon tumors and cell lines, and may be an early event in carcinogenesis progression.
(% total epithelial cells I section) Initiation
COX-2 (14) COX·1 (8)
proximal
%-t(;ells/crypt 12.2+1-0.67'
7.7+/-0.76<.1
middle
distal
totalcolon
9.82+/-0.6 5.15+/-0.36
8.17+/-0.6 6.07+/-0.3'·,
9.66+/-0.44b 6.1 +/-0.32'·h
4.66+/-0.31 4.34+/-0.27 4.94+/-0.23
6.35+/-0.37' 3.88+/-0.13' 3.25+/-0.33'·, 6.22+1-0.1 (38)'
3.67+/-019b 3.24+/-0 15' 3.89+/-025 h
Promotion
COX·2 (16) COX·1 (9)
Qa,i
1.08+/-0.17' Vehicle Control COX·1 (5) 3.09+/·0.6' ACF(nlcm'l 2.18+/-0.04(30)'
478 INVOLVEMENT OF MAPK PATHWAYS IN INDUCED APOPTOSIS AND THE UPREGULATION OF CYCLOOXYGEANASE-2 BY THE COX-2 SELECTIVE INHmITOR NS-398 IN HUMAN COLORECTAL TUMOR CELL LINES. Douglas J. Elder, Dawn E. Halton, Christos Paraskeva, Univ of Bristol, Bristol, United Kingdom. Background: Non-steroidal antiinflammatory drugs (NSAIDs) are chemopreventive for colorectal cancer. An important component of their antineoplastic action is the ability to inhibit proliferation and to induce apoptosis of colorectal tumor cells. The signalling pathways by which these occur require further definition. We have previously shown the COX-2 selective NSAID NS-398 to induce COX-2 protein expression and apoptosis in the HT29 colorectal carcinoma cell line. The MAPK pathways have been shown to positively regulate COX-2 protein expresssion. Aim: To investigate the involvement of MAPK pathways in the regulation of COX-2 expression and induction of apoptosis by NS-398. Methods: Subconfluent cultures of human colorectal tumor cells were treated with the COX-2 selective inhibitor NS-398 (20·IOOJ.LM) and/or inhibitors of the kinases p38 (SB203580, 5J.LM) and MEK (UOI26, 1-1OJ.LM) for up to 96h. Following treatment, the cell yield and extent of apoptosis was determined in control (vehicle) and treated cultures. Cell lysates were analysed by Western blotting. MEK activity was assessed by determining the phosphorylation of ERK using polyclonal phospho-specific antibody. COX-2 expression was determined using a polyclonal antibody to COX-2. Results: NS-398 upregulated COX-2 protein expression in a dose-dependent manner in the colorectal adenoma cell lines AAICI and RRlCI as well as in the carcinoma line HT29. This was further examined in HT29 cultures where the presence of SB203580 or UOl26 completely inhibited the upregulation of COX-2 by NS-398. At a concentration of I J.LM, U1026 had no effect on the proliferation (determined by the cell yield) or apoptosis of HT29 cells and, although it did not fully inhibit MEK activity, inhibited the antiproliferative effect of NS-398 (40J.LM) by 53± II %. This was associated with the inhibition of NS-398-induced apoptosis. Conclusions: The upregulation of COX-2 protein expression by the COX-2 selective inhibitor NS-398 in human colorectal tumor cells involves the ERK and p38 signalling pathways. The inhibition of NS-398-induced apoptosis by blockade of the ERK pathway suggests an important role for this pathway in signalling apoptosis induced by this class of NSAID.
AGAA53
gastric cancer. COX-2 promoter hypomethylation does not occur frequently in gastritis or in gastric tumors without MSI.
Frequency ofhypomethylation of COX-2 promoter ingastric cancers vs. adjacent normal tissue Tissue Type
Hypometh
%
P*
7/14 2/6 9/20 1/20 0/3
50 33 45 5 0
0004
MSI.high Iumors MSI·low tumors MSltotal MSI·neg Gastritis
NS
0008 NS
'Fisher's exact test, vs. MSI-neg
COX-I is constitutively expressed in colonocytes. We hypothesized that COX-2 expression would be upregulated during tumor initiation and promotion, while constitutive COX-I would remain unchanged. Methods: For tumor initiation F344 rats, induced with 20 mgfkg ip azoxyrnethane(AOM), were harvested after 5 days. For tumor promotion, harvest was 8 weeks after the intitial injection of 2 weekly AOM doses. Tissue was stained for COX-2 and COX-l by IHC. Results: COX-2 was not seen in vehicle-treated rats(data not shown). Data are mean +1- SEM(n);like superscripts, p<0.05, unpaired t-test.(table) Conclusion: Increased COX-2 protein during tumor initiation may relfect a generalized inflammatory or carcinogen activation response, which also occurs to a lesser degree with COX-I. The high rates of COX-2 staining in proximal colon at tumor initiation had resolved by tumor promotion. At tumor promotion, increased COX-2 in distal versus proximal colon is associated with increased aberrant crypt foci (ACF) and typical of the location of colon tumors in this model and human sporadic colon cancer. The increased ACF may reflect COX-2 tumorigenic effects. We conclude that COX-2 expression may serve different functions in the same tissue site during colon cancer progression.
476 EXPRESSION OF CYCLO-OXYGENASE-2 PROTEIN IS ASSOCIATED WITH DYSPLASIA, K-RAS MUTATION AND EXPRESSION OF P53 IN COLORECTAL ADENOMAS. Robert Benamouzig, Elisabeth Longchampt, Helen Yoon, Eric Jullian, Antoine Martin, Thierry Coste, Daniel Couturier, Jacques Rautureau, Stanislas Chaussade, Hosp Avicenne, Bobigny, France; Hosp Cochin, Paris, France. Cyclo-oxygenase-2 (COX-2) is an inducible enzyme that catalyzes the conversion of arachidonic acid to prostaglandins and thromboxanes. COX-2 protein expression is observed in colorectal neoplasms and may be a target for anticolorectal cancer activity of NSAIDs and aspirin in humans. The purpose of this study was to evaluate COX-2 protein hyperexpression in 182 adenomas obtained from the 116 first consecutive patients (73 males, 43 females, age 58 +1- 9 years) included in a large prospective multi-center randomised study aimed to evaluate the effect of long term daily use of low dose aspirin in reducing the occurence of new adenomatous polyps. All patients have had colonoscopy to the cecum with adequate preparation resulting in clearance of either a single adenoma> 10 mm in size or 3 adenomas of any size. Methods : All biopsy specimens were blindly assessed for architectural pattern and dysplasia by 2 independent pathologists. Immunohistochemistry was carried out on formalin-fixed paraffin-embedded sections with specific anti-COX-2 antibody and antiP53 antibody. Staining intensity was scored 0-3 by two blinded independent observers. After DNA extraction from these specimens, PCR amplification and sequencing were performed to detect K-ras mutation. Results : COX-2 epithelial cells staining was heterogenous both between crypts and inside a crypt. Superficial COX-2 staining was also observed in interstitial cells. An high to moderate hyperexpression of COX-2 was observed in 36% of adenomas (score 2 and 3), COX-2 hyperexpression was more frequent in adenomas exhibiting high grade dysplasia (p<0,05) and intense p53 immunostaining (p
477 CYCLOOXYGENASE-2 PROTEIN VARIES WITH TUMOR PROGRESSION IN EXPERIMENTAL COLON CANCER. Charlene Compher, John L. Rombeau, Noel N. Williams, Univ of Pennsylvania, Philadelphia, PA. Introduction: Cyclooxygenase-2(COX-2)is upregulated in colon tumors and cell lines, and may be an early event in carcinogenesis progression.
(% total epithelial cells I section) Initiation
COX-2 (14) COX·1 (8)
proximal
%-t(;ells/crypt 12.2+1-0.67'
7.7+/-0.76<.1
middle
distal
totalcolon
9.82+/-0.6 5.15+/-0.36
8.17+/-0.6 6.07+/-0.3'·,
9.66+/-0.44b 6.1 +/-0.32'·h
4.66+/-0.31 4.34+/-0.27 4.94+/-0.23
6.35+/-0.37' 3.88+/-0.13' 3.25+/-0.33'·, 6.22+1-0.1 (38)'
3.67+/-019b 3.24+/-0 15' 3.89+/-025 h
Promotion
COX·2 (16) COX·1 (9)
Qa,i
1.08+/-0.17' Vehicle Control COX·1 (5) 3.09+/·0.6' ACF(nlcm'l 2.18+/-0.04(30)'
478 INVOLVEMENT OF MAPK PATHWAYS IN INDUCED APOPTOSIS AND THE UPREGULATION OF CYCLOOXYGEANASE-2 BY THE COX-2 SELECTIVE INHmITOR NS-398 IN HUMAN COLORECTAL TUMOR CELL LINES. Douglas J. Elder, Dawn E. Halton, Christos Paraskeva, Univ of Bristol, Bristol, United Kingdom. Background: Non-steroidal antiinflammatory drugs (NSAIDs) are chemopreventive for colorectal cancer. An important component of their antineoplastic action is the ability to inhibit proliferation and to induce apoptosis of colorectal tumor cells. The signalling pathways by which these occur require further definition. We have previously shown the COX-2 selective NSAID NS-398 to induce COX-2 protein expression and apoptosis in the HT29 colorectal carcinoma cell line. The MAPK pathways have been shown to positively regulate COX-2 protein expresssion. Aim: To investigate the involvement of MAPK pathways in the regulation of COX-2 expression and induction of apoptosis by NS-398. Methods: Subconfluent cultures of human colorectal tumor cells were treated with the COX-2 selective inhibitor NS-398 (20·IOOJ.LM) and/or inhibitors of the kinases p38 (SB203580, 5J.LM) and MEK (UOI26, 1-1OJ.LM) for up to 96h. Following treatment, the cell yield and extent of apoptosis was determined in control (vehicle) and treated cultures. Cell lysates were analysed by Western blotting. MEK activity was assessed by determining the phosphorylation of ERK using polyclonal phospho-specific antibody. COX-2 expression was determined using a polyclonal antibody to COX-2. Results: NS-398 upregulated COX-2 protein expression in a dose-dependent manner in the colorectal adenoma cell lines AAICI and RRlCI as well as in the carcinoma line HT29. This was further examined in HT29 cultures where the presence of SB203580 or UOl26 completely inhibited the upregulation of COX-2 by NS-398. At a concentration of I J.LM, U1026 had no effect on the proliferation (determined by the cell yield) or apoptosis of HT29 cells and, although it did not fully inhibit MEK activity, inhibited the antiproliferative effect of NS-398 (40J.LM) by 53± II %. This was associated with the inhibition of NS-398-induced apoptosis. Conclusions: The upregulation of COX-2 protein expression by the COX-2 selective inhibitor NS-398 in human colorectal tumor cells involves the ERK and p38 signalling pathways. The inhibition of NS-398-induced apoptosis by blockade of the ERK pathway suggests an important role for this pathway in signalling apoptosis induced by this class of NSAID.