Strong Cyclooxygenase-2 Expression Research Articles

Collagen modifications predictive of lymph node metastasis in dogs with carcinoma in mixed tumours.

Mixed tumours in the canine mammary gland are the most common histological type in routine diagnosis. In general, these neoplasms have a favourable prognosis that does not evolve into metastatic disease. However, some cases develop into lymph node metastases and are associated with worse patient survival rates. Here is a retrospective study of 46 samples of primary mixed tumours of the canine mammary gland: 15 cases of benign mixed tumours (BMT), 16 cases of carcinoma in mixed tumours without lymph node metastasis (CMT), and 15 cases of carcinomas in mixed tumours with lymph node metastasis (CMTM). In addition, we selected 23 cases of normal mammary glands (NMT) for comparison. The samples were collected from biopsies performed during nodulectomy, simple mastectomy, regional mastectomy, or unilateral/bilateral radical mastectomy. We used multiphoton microscopy, second harmonic generation, and two-photon excited fluorescence, to evaluate the characteristics of collagen fibres and cellular components in biopsies stained with haematoxylin and eosin. We performed Ki67, ER, PR, and HER-2 immunostaining to define the immunophenotype and COX-2. We showed that carcinomas that evolved into metastatic disease (CMTM) present shorter and wavier collagen fibres as compared to CMT. When compared to NMT and BMT the carcinomas present a smaller area of fibre coverage, a larger area of cellular coverage, and a larger number of individual fibres. Furthermore, we observed a correlation between the strong expression of COX-2 and a high rate of cell proliferation in carcinomas with a smaller area covered by cell fibres and a larger number of individual fibres. These findings highlight the fundamental role of collagen during tumour progression, especially in invasion and metastatic dissemination.

Abstract 2863: Interferon-gamma induces NOS2 and COX2 in ER- breast cancer that drives increase metastatic potential

Abstract NOS2 and COX2 expression is significantly correlated with poor prognosis in ER- breast cancer. Multiple pro-oncogenic pathways involving metastasis, proliferation, angiogenesis, immunosuppression, and chemoresistance are stimulated in a synergistic manner by NOS2 and COX2, resulting in much more aggressive cancers. In this study, our single-cell RNAseq data demonstrated that certain combinations of cytokines, IFNg with TNFA or IL1B, drove NOS2 and COX2 expressions in vitro in the MDA-MB-231 cell line after 48 hours. Analysis of t-SNE plots indicates that the clustering patterns of NOS2 and COX2 are also related to cytokine expression. Moreover, according to our spatial proteomic analysis, CD8 that are associated with IFNg are more likely to have a positive response, indicating a dichotomy in the requirement for IFNg. To better understand this mystery, the spatial distribution of NOS2 and COX2 with CD8 cells in 21 ER- breast tumors was evaluated. In the presence of tumor-excluded CD8, high expression and clustering of NOS2-positive cells were seen near the tumor-stromal interface, showing that NOS2-positive cells were excluded from the tumor. Within the tumor nest, strong expression and population clustering of COX2 reached into the immune desert. In contrast, tumor-penetrant CD8 cells were few and poorly expressed. Metastasis and chemoresistance were among the leading causes of death in this cohort, and it was shown in vitro that IFNg/IL1B or TNFA increased tumor cell migration, suggesting that the interface of tumor with stromal restricted lymphoid aggregates provides the unique spatial environment to increase NOS2 expression and increase metastatic potential. The spatial study of the TME (tumor microenvironment) provides vital insight into how NOS2 and restricted CD8 lead to a higher probability of poor survival. Citation Format: Yuk Sing Robert Cheng, Lisa Ridnour, Abigail Walke, Noemi Kedei, Adelaide Wink, Elijah Edmonston, Donna Butcher, Tiffany Dorsey, William Heinz, Richard Bryant, Robert Kinders, Stanley Lipkowitz, Stephen Wong, Milind Pore, Stephen Hewitt, Daniel McVicar, Stephen Anderson, Jenny Chang, Stefan Ambs, Stephen Lockett, David Wink. Interferon-gamma induces NOS2 and COX2 in ER- breast cancer that drives increase metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2863.

First study on the immunohistochemical expression of cyclooxygenase-2 and clinicopathological association in canine hepatoid gland neoplasms.

Hepatoid gland neoplasms (HGNs) constitute one of the most common cutaneous tumors that arise from perianal glands in dogs and are clinically characterized by rapid growth. Cyclooxygenase-2 (COX-2), the inducible form of the enzyme, is associated with several hallmarks of tumorigenesis. Its expression has been confirmed in several human and animal neoplastic tissues, but there are no reports in hepatoid gland tissues. Therefore, this study aimed to investigate COX-2 immunoexpression in canine HGNs, compare the expression among groups of normal hepatoid glands, hepatoid gland adenomas (HGAs), hepatoid gland epitheliomas (HGEs), and hepatoid gland carcinomas (HGCs), and assess the association of the COX-2 expression with clinicopathological features. Sixty-one formalin-fixed paraffin-embedded canine hepatoid gland tissues (20 samples of HGAs, 16 of HGEs, 15 of HGCs, and 10 of normal hepatoid glands) were analyzed for COX-2 expression using immunohistochemistry with scoring for percentage positivity and intensity. Multiple comparisons of COX-2 expression among normal and neoplastic hepatoid glands and the associations between COX-2 expression and clinicopathological features were analyzed. Cyclooxygenase-2 expression was not detected in 60% of normal hepatoid glands and 25% of HGAs. Seventy-five percent of HGAs had a weak expression, while 43.7% and 56.3% of HGEs showed weak and moderate expression, respectively. The expression of HGCs ranged from weak (13.3%) to moderate (33.3%) and strong (53.3%). The immunoreactivity score of COX-2 labeling was significantly different among the normal and neoplastic hepatoid glands (p < 0.0001). The highest score was observed in the HGCs. Only in HGCs, the strong COX-2 expression was significantly associated with some clinicopathological features, including tissue invasion (p = 0.007) and necrosis (p = 0.029). These results suggest that COX-2 may play a role in the modulation of neoplastic cell growth. These preliminary data lead to further investigation on the potential of COX-2 expression as a prognostic indicator and COX-2 inhibitors for canine HGCs treatment.

Biomarker Profiling Revealed Carcinoembryonic Antigen as a Target of Artesunate in a Ductal Breast Cancer Patient.

Patients with metastatic tumors commonly have a poor prognosis. Frequently, patients suffering from progressive tumors have a high willingness for the compassionate use of non-approved medications. One of these medications is the antimalarial drug artesunate (ART) which also showed profound anticancer activity in vitro, in vivo, and in preliminary clinical pilot studies. Herein, we report on the compassionate use of ART in a patient with metastatic breast cancer. The clinical course of a Caucasian female who was diagnosed with ductal breast cancer at the age of 33 is described. Tumor markers in the blood have been measured, and tumor-associated protein expression has been determined by immunohistochemistry. Microscale thermophoresis and molecular docking in silico were used to study protein-drug interactions. The tumor responded to ART administered at doses of 150-300 mg daily, and the patient experienced a stabilization of her disease for 1.5 years. ART treatment caused no or minimal side-effects (headache, dizziness, slight tachycardia, slight stomach upset, slight fatigue). Tumor marker determination in the blood of the patient revealed a reduction of carcinoembryonic antigen (CEA), but not CA 27.29 or CA 15.3 levels. We hypothesized that the reduction of CEA levels might be due to binding of ART to this protein. Microscale thermophoresis with recombinant CEA indeed showed binding of ART to this protein in vitro. This result was verified by molecular docking in silico. Immunohistochemical biomarker profiling and computerbased quantification of biomarker expression in a tumor biopsy revealed strong expression of COX2, GRP78, CD71, GSTP1, and c-MYC but weak or minimal expression of VEGFR, P-glycoprotein, survivin, and LOX1. Among a panel of tumor-related proteins tested, the interaction with CEA may have contributed to the anticancer activity of ART in this patient. It deserves further investigation whether CEA represents not only a valuable biomarker but also a treatment target. ART might be useful for the individualized treatment of metastatic breast tumors.

Особливості експресії СОХ-2 в різних молекулярних підтипах інвазивного протокового раку грудної залози

The purpose of the study was to determine the role of COX-2 in the development and progression of molecular subtypes of invasive ductal breast cancer by comparing COX-2 expression level between different clinical and pathological prognostic parameters. Materials and methods. We studied 193 cases of invasive ductal breast cancer using comprehensive morphological, including immunohistochemical methods. General histological processing of samples was performed according to standard methods. Immunohistochemical studies for COX-2, ER, PR, c-erbB2, Ki-67 were performed according to the manufacturer's protocol with the control of samples. The grade of malignancy was determined according to the modified scheme of P. Scarff, H. Bloom and W. Richardson. COX-2 expression level was quantified using the Histoscore counting system in 86 cases. Immunoreactive index was defined as the product of positive cells and color intensity with a value from 0 to 12. The distribution of tumors with weak or strong expression of COX-2 was determined at the level of limit 6. Comparison of COX-2 expression at different clinical and pathological parameters was evaluated using the criterion Pearson χ2. For all types of analysis, differences were considered significant at p &lt;0.05. Results and discussion. Immunohistochemical studies showed overexpression of COX-2 tumor cells in 53.49 [42.95-63.87] % of cases of invasive ductal breast cancer, mainly in premenopausal patients. Increased expression of COX-2 was determined in tumors with metastases to regional lymph nodes (53.66 66 [38.46-68.52] % - 63.64 (34.52-88.13] %), large size (73, 81 (59.63-85.82] % - 85.71 [52.74-99.97] %), running clinical stage (65.12 [54.78-74.78] %), with a low degree of differentiation G3 (80.95 [61.88-94.45] %), negative ER status and overexpression of HER-2/neu. Overexpression of COX-2 prevailed in patients under the age of 50 years (69.23 [54.04-82.54] %), in patients with triple-negative carcinoma phenotype (75.00 [54.26-91.01] %) and HER-2/neu + phenotype of invasive ductal breast cancer (73.68 [52.19-90.47] %). Conclusion. Overexpression of COX-2 has been associated with adverse prognosis factors such as young age, metastatic lymph node involvement, large tumor size, G3 poorly differentiation (high grade), negative ER status and HER-2/neu overexpression, and has been associated with an increased risk of recurrence of the disease, progression of the tumor process, increased risk of death. Overexpression of COX-2 may be a sign of an aggressive phenotype with metastatic potential, depend on hormonal status and be useful as a prognostic biomarker of invasive ductal breast cancer

The pro-inflammatory and anti-inflammatory role of hyaluronic acid in endometriosis

ObjectiveEndometriosis is a bothersome disease affected women worldwide, the mechanism of disease development is still under investigation. Several inflammatory responses after clinical hyaluronic acid (HA) use were reported. Cyclooxygenase (COX)-2 mediated inflammation pathway is involved in the pathogenesis of endometriosis. Thus, we tried to investigate the inflammatory role of hyaluronic acid in endometriosis. Materials and methodsPeritoneal fluid was collected in endometriosis and disease-free patients for the measurement of HA. Endometriotic stromal cells were treated with IL-1β and HA and expression of COX-2 was evaluated. Mice model of endometriosis was established and treated with fluid or gel form of HA. Endometriotic lesion size and weight were recorded and level of COX-2 was evaluated by immunohistochemistry staining. ResultsThe level of HA in the peritoneal fluid had no statistically significant difference between normal, early and advanced stage endometriosis patients. The overexpression of COX-2 protein was detected when treating endometriotic stromal cell with HA in the presence of IL-1β (P < 0.001). The endometriotic lesion size was reduced in mice model when treated with higher concentration gel form HA. It further showed less proportion of strong COX-2 expression compare of gel form HA to fluid form treatment in COX-2 expression score of endometriosis lesion. ConclusionBoth proinflammatory evidence, elevated COX-2 expression, and anti-inflammatory result, reduced endometriosis lesion size and COX-2 expression score, were noted in our study after treating HA in in vivo and in vitro models. We hypothesized it is possible that HA may induce an acute proinflammatory response followed by chronic anti-inflammatory reaction in the formation of endometriosis.

Streptococcus suis Induces Expression of Cyclooxygenase-2 in Porcine Lung Tissue.

Streptococcus suis is a common pathogen colonising the respiratory tract of pigs. It can cause meningitis, sepsis and pneumonia leading to economic losses in the pig industry worldwide. Cyclooxygenase-2 (COX-2) and its metabolites play an important regulatory role in different biological processes like inflammation modulation and immune activation. In this report we analysed the induction of COX-2 and the production of its metabolite prostaglandin E2 (PGE2) in a porcine precision-cut lung slice (PCLS) model. Using Western blot analysis, we found a time-dependent induction of COX-2 in the infected tissue resulting in increased PGE2 levels. Immunohistological analysis revealed a strong COX-2 expression in the proximity of the bronchioles between the ciliated epithelial cells and the adjacent alveolar tissue. The morphology, location and vimentin staining suggested that these cells are subepithelial bronchial fibroblasts. Furthermore, we showed that COX-2 expression as well as PGE2 production was detected following infection with two prevalent S. suis serotypes and that the pore-forming toxin suilysin played an important role in this process. Therefore, this study provides new insights in the response of porcine lung cells to S. suis infections and serves as a basis for further studies to define the role of COX-2 and its metabolites in the inflammatory response in porcine lung tissue during infections with S. suis.

Helicobacter pylori outer membrane vesicles inhibit human T cell responses via induction of monocyte COX-2 expression.

The modulation of T cell responses by Helicobacter pylori is thought to potentiate both H. pylori persistence and development of gastric pathologies including cancer. Release of outer membrane vesicles (OMV) by H. pylori provides a potential vehicle for modulation of the immune system. Although OMV are thought to have T cell suppressive activity, this has not yet been demonstrated. Their suppressive activity was investigated in this study using the responses of peripheral blood mononuclear cells (PBMC) to T cell stimuli as a readout. We demonstrate that addition of OMV to PBMC significantly inhibits subsequent T cell proliferation in a cyclo-oxygenase-2 (COX-2)-dependent manner. Addition of OMV did not significantly modulate PBMC apoptosis, but induced strong expression of COX-2 by the monocytes present and significantly increased levels of PGE2 and IL-10. These effects were independent of vacuolating cytotoxin expression. Together, these findings demonstrate that OMV can suppress human T cell responses and that the predominant mechanism is not through a direct effect on the T cells but results from the induction of COX-2 expression in monocytes. This increased COX-2 activity may modulate not only H. pylori-directed immune responses but also wider immune responses.

Expression of COX-2 and p53 in juvenile polyposis coli and its correlation with adenomatous changes.

Gastrointestinal polyps commonly affect the pediatric population. The commoner variety amongst these is the solitary rectal polyp. Juvenile polyposis coli (JPC) is rare, characterized by multiple polyps occurring throughout the gut. The role of cyclooxygenase-2 (COX-2) has been implicated in gastrointestinal tumorigenesis. We aimed to look at the clinicopathological spectrum of solitary vs juvenile polyposis and compare their differences in expression of COX-2 and p53. We studied 38 polyps from eight cases of JPC, collected over the past 10 years along with 40 solitary rectal polyps (SRP). The size of polyps was significantly more in cases of JPC compared to SRP. Adenomatous change was observed significantly more often in JPC. COX-2 expression was also significantly higher in the JPC group compared to SRPs. All cases of JPC polyps with adenomatous change showed strong COX-2 expression. There was no significant difference in expression of p53 in the JPC and SRP groups. We observed significantly higher COX-2 expression in JPC. Establishment of the role of COX-2 in JPC will help us formulate chemopreventive therapies as an adjunct to its surgical management.

English

Inflammation plays an important role to the process of prostate carcinogenesis by increasing the rate of cell proliferation, which contributes to an aggressive tumour phenotype. Cyclooxygenase-2 (COX-2) has been found overexpressed in various types of cancer cells including prostate. The aim of this study was to investigate the COX-2 expressions in different types of human prostate tissues. Paraffin-embedded prostate tissues from 263 samples were examined for the expression of COX-2 marker by immunohistochemistry method. COX-2 was found highly expressed in prostate adenocarcinoma (p=0.001) as compared to benign and normal tissues. The score of COX-2 expressions in most of normal prostate was weak 49 (77.8%), while only 16 (16%) of BPH showed strong expression. 56 cases (56%) prostate cancer showed strong COX-2 expression. Prostate cancer cases showed significant differences in staining patterns as tumour grade increased. In addition, COX-2 expression was significantly correlated with Gleason score in cancerous tissues. This study suggests that COX-2 overexpression is associated with prostate cancer and higher grade tumour.

The SK-N-AS human neuroblastoma cell line develops osteolytic bone metastases with increased angiogenesis and COX-2 expression

Neuroblastoma (NB), which arises from embryonic neural crest cells, is the most common extra-cranial solid tumor of childhood. Approximately half of NB patients manifest bone metastasis accompanied with bone pain, fractures and bone marrow failure, leading to disturbed quality of life and poor survival. To study the mechanism of bone metastasis of NB, we established an animal model in which intracardiac inoculation of the SK-N-AS human NB cells in nude mice developed osteolytic bone metastases with increased osteoclastogenesis. SK-N-AS cells induced the expression of receptor activator of NF-κB ligand and osteoclastogenesis in mouse bone marrow cells in the co-culture. SK-N-AS cells expressed COX-2 mRNA and produced substantial amounts of prostaglandin E2 (PGE2). In contrast, the SK-N-DZ and SK-N-FI human NB cells failed to develop bone metastases, induce osteoclastogenesis, express COX-2 mRNA and produce PGE2. Immunohistochemical examination of SK-N-AS bone metastasis and subcutaneous tumor showed strong expression of COX-2. The selective COX-2 inhibitor NS-398 inhibited PGE2 production and suppressed bone metastases with reduced osteoclastogenesis. NS-398 also inhibited subcutaneous SK-N-AS tumor development with decreased angiogenesis and vascular endothelial growth factor-A expression. Of interest, metastasis to the adrenal gland, a preferential site for NB development, was also diminished by NS-398. Our results suggest that COX2/PGE2 axis plays a critical role in the pathophysiology of osteolytic bone metastases and tumor development of the SK-NS-AS human NB. Inhibition of angiogenesis by suppressing COX-2/PGE2 may be an effective therapeutic approach for children with NB.

Immunohistochemical analysis of cyclooxygenase-2 and brain fatty acid binding protein expression in grades I-II meningiomas: correlation with tumor grade and clinical outcome after radiotherapy.

This study was done to evaluate the association of cyclooxygenase 2 (COX-2) and brain fatty acid binding protein (BFABP) with tumor grade and outcome of grades I-II meningiomas treated with radiotherapy. From 1996 to 2008, 40 patients with intracranial grades I-II meningiomas were treated with radiotherapy. Immunohistochemical staining for COX-2 and BFABP were performed on formalin-fixed paraffin-embedded tissues. COX-2 expression was significantly associated with BFABP status and both COX-2 (P < 0.01) and BFABP (P = 0.01) expression were stronger in the grade II meningiomas than in grade I tumors. Among the clinicopathologic factors, age and COX-2 status were prognostic in progression-free survival. Patients with moderate or strong COX-2 expression had worse outcome than those with negative or weak COX-2 expression (P = 0.03) after controlling for potential confounders. Our results suggest that the molecular biomarker COX-2 has prognostic significance in intracranial grades I-II meningiomas following radiotherapy.

Expression of HuR and Cyclooxygenase-2 in Nodular Fasciitis and Low-Grade Sarcoma: An Immunohistochemical Study.

BackgroundNodular fasciitis is the most common reactive mesenchymal lesion to be misidentified as a type of sarcoma. HuR is an mRNA-binding protein that can stabilize cyclooxygenase-2 (COX-2) mRNA leading to COX-2 overexpression. The aim of this study is a comparison of the expressions of COX-2 and HuR and the relationships between their expressions and the clinicopathological parameters in nodular fasciitis and low-grade sarcoma.MethodsWe measured the expression of HuR and COX-2 in 21 cases of nodular fasciitis and 37 cases of low-grade sarcoma using immunohistochemistry.ResultsThe frequency of cytoplasmic immunoreactivity for HuR was 5 of 21 cases of nodular fasciitis (23.8%) and 23 of 37 cases of low-grade sarcoma (62.1%) (p=.013). COX-2 expression was moderate or strong in nodular fasciitis (12/21, 57.1%) and in low-grade sarcoma (29/37, 78.4%) (p=.034). In addition, a significant difference existed between these two entities in terms of the relationship between moderate or strong COX-2 expression and HuR cytoplasmic immunoreactivity (p=.009). Moderate or strong COX-2 immunoreactivity correlated with nuclear (p=.016) or cytoplasmic HuR (p=.024) expression in low-grade sarcoma but not in nodular fasciitis.ConclusionsThis study suggests that HuR and COX-2 expression may be useful to differentiate nodular fasciitis from low-grade sarcoma.

Virtual Screening for Finding Novel COX-2 Inhibitors as Antitumor Agents

The cyclooxygenase-2 (COX-2) enzyme binds to arachidonic acid resulting in the release of metabolites that induce pain and inflammatory responses. Recent studies have shown that strong COX-2 expression is highly correlated with increased tumor risk. Therefore, the development of potent COX-2 inhibitors to relieve pain and treat cancers requires further investigation. We used virtual screening to find three COX-2 inhibitors (Phar-95239, T0511-4424 and Zu- 4280011) from a huge zinc database containing 2000000 compounds. The effects of the compounds on COX-2 were compared to those on COX-1 using a colorimetric COX (ovine) screening assay kit. The selectivity index, the ratio of IC50 for COX-1 inhibition to that of COX-2, calculated were MTT assay was used to evaluate the cytotoxic activity of the compounds using different dilutions. The IC50 values were calculated. Based on the results of the MTT assay, the IC50 values for compounds Phar-95239, T0511-4424 and Zu-4280011 were 178.52, 143 and 97.61 µM, respectively, and the selectivity indices of the compounds were 11.36, 12.20 and 20.03, respectively. These results indicated a relationship between the selectivity index and anticancer activity. Zu-4280011 displayed the highest selectivity index and the best results in the MTT assay among selected componds.

야콘(Smallanthus sonchifolius) 추출물이 흰쥐의 췌장 섬유화에 미치는 영향

Yacon has been used in folk medicines as a medicinal tea for hypoglycemia treatment. In a recent study described herein, antioxidative, antibacterial, antifungal activities, and cell-protective functions of yacon leaves have been reported. To evaluate the effects on fibrosis on pancreatitis, the efficacy of 1% of yacon extract (YE) on dibutyltin dichloride (DBTC) (8 mg/kg)-induced pancreatitis in rats was examined. On the 21st day after the DBTC treatment, a large increase in collagen was observed in the pancreas in the DBTC-treatment group (DT). But this was noticeably decreased with YE. In relation to the expression of COX-2, there was no response or a very weak response in the pancreas of the control group (CON). However, in DT, strong expression of COX-2 was observed in the pancreas on the 14th day, and COX-2 was present in inflammatory cells in the pancreas of the DT, especially on the 21st day. The expression was decreased for YE compared with DT. A remarkable increase in TGF-β1 expression was observed in inflammatory cells in the pancreas in DT on the 21st day, whereas the expression was not found in YE after 21 days. However, on the 21th day, TGF-β1 expression was increased in acinar cells of YE compared with DT. VEGF expression was very similar to the expression of in the pancreas. These results suggest that YE has an inhibitory effect on DBTC-induced pancreatic fibrosis.

881 5-Aminosalicylic Acid Mediates Expression of Cyclooxygenase-2 and 15-Hydroxyprostaglandin Dehydrogenase to Suppress Tumorigenesis in C-HA-RAS Transgenic Mice

Background: Cyclooxygenase-2 (COX-2) is a key enzyme that produces prostaglandin E2 (PGE2) and plays an important role in colorectal tumor growth. Several studies showed that COX-2 inhibiting medications are useful for prevention of colorectal cancer (CRC), however, due to possible adverse events, these medications are not routinely recommended for the prevention. In addition to inhibition of COX-2 activity, recent focus is on 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which degrades PGE2, as another mechanism to regulate PGE2. Some studies reported that 5-aminosalicylic acid (5-ASA), the most widely used antiinflammatory medication for inflammatory bowel diseases with demonstrated long term safety, is associated with reduction in risk of CRC, but the mechanism of action is still unclear. To explore the potential of 5-ASA as a safe preventive agent for CRC, we determined the effect of 5-ASA on COX-2 and 15-PGDH expression. We also investigated the preventive effect of 5-ASA in c-Ha-ras transgenic mice. Materials and Methods: To examine whether 5-ASA affects the expression of COX-2 and 15-PGDH, HT29 cells were cultured in the absence or presence of 5-ASA. To investigate the preventive effect of 5-ASA In Vivo, we used c-Ha-ras transgenic mice, which highly mimics human sporadic colorectal tumor development following injection of 1,2dihydrochloride. The transgenic mice were randomized to Ras or Ras+5ASA group. Mice assigned to the Ras+5-ASA group were treated orally with 5-ASA. The dose of 5-ASA used in the experiments were chosen carefully to reflect efficacy and safety for use in humans. Total protein was extracted from HT-29 cells and from murine colons after excluding sites of tumors and Western blotting analysis was performed for on COX-2 and 15-PGDH. The expression of these proteins were quantified and normalized by beta-actin expression. We examined the number and size of tumors in colon specimens, and performed histopathological analysis and immunohistochemistry. Result: 5-ASA significantly suppressed COX-2 and induced 15-PGDH expression in HT29 cells. In the transgenic mice, we confirmed that tumors in this model were sporadic and there was no inflammation in background mucosa and detected strong expression of COX2 in tumor lesions. The expression of 15-PGDH was remarkably weak regardless of colonic area. Oral 5-ASA intake reduced incidence of colorectal tumor formation from 83% to 40% (P = 0.048) and tumor size from 1.9 ± 1.1mm to 1.1 ± 0.2mm (P = 0.015). Furthermore, we observed down-regulation of COX-2 (P = 0.017) and up-regulation of 15-PGDH (P = 0.048) in the colons. Conclusion: We provide evidence that 5-ASA exerts a chemopreventive effect against colorectal tumor development in c-Ha-ras transgenic mice through mediation of COX-2 and 15-PGDH expression.

882 Omega-3 Polyunsaturated Fatty Acids and Low Fat Diet Prevent Colorectal Cancer by Inhibiting COX-2 in Colonic Mucosa and by Increasing Adiponectin in Adipose Tissue

Background: Cyclooxygenase-2 (COX-2) is a key enzyme that produces prostaglandin E2 (PGE2) and plays an important role in colorectal tumor growth. Several studies showed that COX-2 inhibiting medications are useful for prevention of colorectal cancer (CRC), however, due to possible adverse events, these medications are not routinely recommended for the prevention. In addition to inhibition of COX-2 activity, recent focus is on 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which degrades PGE2, as another mechanism to regulate PGE2. Some studies reported that 5-aminosalicylic acid (5-ASA), the most widely used antiinflammatory medication for inflammatory bowel diseases with demonstrated long term safety, is associated with reduction in risk of CRC, but the mechanism of action is still unclear. To explore the potential of 5-ASA as a safe preventive agent for CRC, we determined the effect of 5-ASA on COX-2 and 15-PGDH expression. We also investigated the preventive effect of 5-ASA in c-Ha-ras transgenic mice. Materials and Methods: To examine whether 5-ASA affects the expression of COX-2 and 15-PGDH, HT29 cells were cultured in the absence or presence of 5-ASA. To investigate the preventive effect of 5-ASA In Vivo, we used c-Ha-ras transgenic mice, which highly mimics human sporadic colorectal tumor development following injection of 1,2dihydrochloride. The transgenic mice were randomized to Ras or Ras+5ASA group. Mice assigned to the Ras+5-ASA group were treated orally with 5-ASA. The dose of 5-ASA used in the experiments were chosen carefully to reflect efficacy and safety for use in humans. Total protein was extracted from HT-29 cells and from murine colons after excluding sites of tumors and Western blotting analysis was performed for on COX-2 and 15-PGDH. The expression of these proteins were quantified and normalized by beta-actin expression. We examined the number and size of tumors in colon specimens, and performed histopathological analysis and immunohistochemistry. Result: 5-ASA significantly suppressed COX-2 and induced 15-PGDH expression in HT29 cells. In the transgenic mice, we confirmed that tumors in this model were sporadic and there was no inflammation in background mucosa and detected strong expression of COX2 in tumor lesions. The expression of 15-PGDH was remarkably weak regardless of colonic area. Oral 5-ASA intake reduced incidence of colorectal tumor formation from 83% to 40% (P = 0.048) and tumor size from 1.9 ± 1.1mm to 1.1 ± 0.2mm (P = 0.015). Furthermore, we observed down-regulation of COX-2 (P = 0.017) and up-regulation of 15-PGDH (P = 0.048) in the colons. Conclusion: We provide evidence that 5-ASA exerts a chemopreventive effect against colorectal tumor development in c-Ha-ras transgenic mice through mediation of COX-2 and 15-PGDH expression.

Effects of silencing cyclooxygenase-2 expression via RNA interference on the tumorigenicity of the SMMC-7721 human hepatocarcinoma cell line

We constructed a vector carrying a shRNA sequence against cyclooxygenase-2 (COX-2) that was subsequently transfected into the human hepatocarcinoma cell line SMMC‑7721. Furthermore, we established a COX-2-deficient stable cell line and a model of tumor-shRNA transplantation in nude mice. Negative shRNA was used as the control. The tumor volume in the experimental group was smaller compared to that in the control group. Hematoxylin and eosin staining indicated that the cells in the experimental group differentiated better than those in the control group. The COX-2 mRNA level in the tumor tissues injected with SMMC-7721/COX-2i was markedly downregulated compared to that in the tumor tissues injected with SMMC-7721/negative shRNA. The inhibition rate reached 68.6%. Immunohistological study showed a significantly strong COX-2 expression in the control group tumor cells, whereas the experimental group exhibited moderate expression, indicating the inhibition of COX-2 expression after transfection of cells with shRNA against COX-2. Western blot analysis further proved the inhibition of COX-2 expression. In conclusion, RNAi-mediated regulation of COX-2 expression could efficiently inhibit liver-transplanted tumor growth in BALB/c nude mice.

Induction of COX-2 Enzyme and Down-regulation of COX-1 Expression by Lipopolysaccharide (LPS) Control Prostaglandin E2 Production in Astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.

Expression levels of cyclooxygenase-2, tumor necrosis factor-α and inducible NO synthase in placental tissue of normal and preeclamptic pregnancies

Objective: Although preeclampsia (PE) is one of the most important problems affecting pregnant women, etiologic factors in its development are still unclear. We aimed to investigate the expression levels of cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and inducible NO synthase (iNOS) in preeclamptic and healthy control placentas. Patients and methods: Placental tissue samples were obtained after delivery from patients diagnosed with PE and from normal-term pregnants and analyzed for COX-2, TNF-α and iNOS expression by immunohistochemistry. Results: A strong expression of COX-2 was observed in syncytiotrophoblast cells of preeclamptic placentas, which was significantly higher than that of normal placentas (p = 0.005). A mild expression of TNF-α in both normal and preeclamptic syncytiotrophoblasts was seen (p = 0.435). In addition, a strong expression of iNOS in normal syncytiotrophoblasts was found, but the intensity of the iNOS expression was highly reduced in preeclamptic placentas (p = 0.001). No correlation was detected between COX-2, TNF-α and iNOS expression levels. Conclusion: The findings of a decrease of iNOS expression and an increase of COX-2 expression in placenta suggest the existence of functional roles of iNOS and COX-2 in the pathophysiology of PE, probably by contributing to the reduced placental blood flow and increased resistance to flow in the fetomaternal circulation.