Cyclooxygenase-2 expression in hamster and human pancreatic neoplasia

Abstract

Cyclooxygenase-2 (COX-2) has been implicated in the development of gastrointestinal malignancies. The aim of the present study was to determine COX-2 expression/activity throughout stages of experimental and human pancreatic neoplasia. COX-2 immunohistochemistry was performed in pancreata of hamsters subjected to the carcinogen N-nitrosobis-(2-oxopropyl)amine (BOP) and in human pancreatic tumors. COX-2 activity was determined by prostaglandin E2 assay in tumor versus matched normal pancreatic tissues. The activity of the COX inhibitor sulindac was tested in the PC-1 hamster pancreatic cancer model. COX-2 expression was elevated in all pancreatic intraepithelial neoplasias (PanINs) and adenocarcinomas. In BOP-treated hamsters, there were significant progressive elevations in COX-2 expression throughout pancreatic tumorigenesis. In human samples, peak COX-2 expression occurred in PanIN2 lesions and remained moderately elevated in PanIN3 and adenocarcinoma tissues. COX-2 activity was significantly elevated in hamster and human pancreatic cancers compared to pair-matched normal pancreas. Furthermore, hamster pancreatic tumor engraftment/formation in the PC-1 hamster pancreatic cancer model was reduced 4.9-fold by oral administration of sulindac. Increased COX-2 expression is an early event in pancreatic carcinogeneses. The BOP-induced hamster carcinogenesis model is a representative model used to study the role of COX-2 in well-differentiated pancreatic tumorigenesis. COX inhibitors may have a role in preventing tumor engraftment/formation.

Figure 1

(A) H&E-stained normal hamster pancreas, hamster pancreatic intraepithelial neoplasms (PanIN1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma. (B) COX-2 immunohistochemistry in normal hamster pancreas, hamster pancreatic intraepithelial neoplasms (PanIN1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma. (C) H&E-stained normal human pancreas, human pancreatic intraepithelial neoplasms (PanIN 1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma. (D) COX-2 immunohistochemistry in normal human pancreas, human pancreatic intraepithelial neoplasms (PanIN1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma.

Figure 1

(A) H&E-stained normal hamster pancreas, hamster pancreatic intraepithelial neoplasms (PanIN1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma. (B) COX-2 immunohistochemistry in normal hamster pancreas, hamster pancreatic intraepithelial neoplasms (PanIN1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma. (C) H&E-stained normal human pancreas, human pancreatic intraepithelial neoplasms (PanIN 1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma. (D) COX-2 immunohistochemistry in normal human pancreas, human pancreatic intraepithelial neoplasms (PanIN1, PanIN2, and PanIN3), and invasive pancreatic adenocarcinoma.

Figure 2

(A) COX-2 expression (average score) in hamster pancreatic neoplasms. Hamster pancreatic cancer was initiated at time 0 with the chemical carcinogen BOP. On week 42, all hamsters were euthanized, and lesions were scored by neoplastic stage. COX-2 expression was measured by immunohistochemistry in the most advanced lesion in each pancreas, and each lesion was assigned a value of 0 (absent/weak staining), 1 (intermediate staining), or 2 (intense staining). The data represent the mean ± SEM. ANOVA indicated that COX-2 expression significantly (P < .001) increased with each stage of neoplastic progression, except PanIN2 to PanIN3. (B) COX-2 expression in human pancreatic neoplasms. COX-2 expression was measured by immunohistochemistry. The COX-2 average expression score for each ductule, duct, PanIN1-3, and invasive cancer was the multiplicative factor of the intensity (0–3) times the frequency (0–4) of cells stained for COX-2. The data represent mean ± SEM. Analysis of variance indicated that COX-2 expression was significantly different in all stages of neoplasia compared to normal ductules and normal ducts (P < .001) and increased with each stage of neoplastic progression up to the PanIN2 stage (P < .05).

Figure 3

(A) COX-2 expression in human and hamster pancreatic neoplasms. Pair-matched samples of human or hamster invasive pancreatic cancer versus normal tissue were cryopreserved and then analyzed for COX-2 expression by Western blot analysis. A representative Western blot is shown of both human and hamster pancreatic neoplasms (T, tumor) compared to pair-matched normal control tissues from the same patient or hamster (NL, normal). Actin, which is known to be elevated in tumor samples, is also shown. Gels stained with Coomassie blue confirmed an equivalent overall protein loading of the samples (data not shown). (B) COX-2 activity in human and hamster pancreatic neoplasms. Pair-matched samples of human or hamster invasive pancreatic cancer versus normal tissue were cryopreserved and then analyzed for COX-2 activity by PGE₂ assay. The assay is based on competition between unlabeled PGE₂ and a fixed quantity of peroxidase-labeled PGE₂ for binding to a PGE₂-specific antibody bound to a plate coated with goat antimouse immunoglobulin. The human tumor PGE₂ level [4.7 ± 0.8pg/µg total protein (mean ± SEM)] was significantly higher than that of normal tissue [1.1 ± 0.3pg/µg total protein (mean ± SEM), n = 3, t-test, P < .05]. Similarly, the hamster tumor PGE₂ level [1.4 ±0.2 pg/µg total protein (mean ± SEM)] was significantly higher than that in normal tissue [0.1 ±0.0 pg/µg total protein (mean ± SEM), n = 7, t-test, P < .05].

Figure 4

Inhibition of PC-1 hamster pancreatic cancer engraftment/formation with the nonselective COX inhibitor sulindac. Male Syrian golden hamsters were fed either control or 0.01% (wt/wt) sulindac diet starting 5 days before the injection of hamster PC-1 pancreatic adenocarcinoma cells. The data represent the percentage of 23 control and 12 sulindac animals with tumor 4 weeks after PC-1 cell injection (*P <. 0005 vs control by chi-square analysis).