Systematic study of constitutive cyclo-oxygenase-2 expression: role of NFκB and NFAT transcriptional pathways

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system

Reduction in the risk of human breast cancer by selective cyclooxygenase-2 (COX-2) inhibitors

BACKGROUND: Epidemiologic and laboratory investigations suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive effects against breast cancer due to their activity against cyclooxygenase-2 (COX-2), the rate-limiting enzyme of the prostaglandin cascade. METHODS: We conducted a case control study of breast cancer designed to compare effects of selective and non-selective COX-2 inhibitors. A total of 323 incident breast cancer patients were ascertained from the James Cancer Hospital, Columbus, Ohio, during 2003–2004 and compared with 649 cancer free controls matched to the cases at a 2:1 ratio on age, race, and county of residence. Data on the past and current use of prescription and over the counter medications and breast cancer risk factors were ascertained using a standardized risk factor questionnaire. Effects of COX-2 inhibiting agents were quantified by calculating odds ratios (OR) and 95% confidence intervals. RESULTS: Results showed significant risk reductions for selective COX-2 inhibitors as a group (OR = 0.29, 95% CI = 0.14–0.59), regular aspirin (OR = 0.49, 95% CI = 0.26–0.94), and ibuprofen or naproxen (0.36, 95% CI = 0.18–0.72). Acetaminophen, a compound with negligible COX-2 activity and low dose aspirin (81 mg) produced no significant change in the risk of breast cancer. CONCLUSION: Selective COX-2 inhibitors (celecoxib and rofecoxib) were only recently approved for use in 1999, and rofecoxib (Vioxx) was withdrawn from the marketplace in 2004. Nevertheless, even in the short window of exposure to these compounds, the selective COX-2 inhibitors produced a significant (71%) reduction in the risk of breast cancer, underscoring their strong potential for breast cancer chemoprevention

Use of Aspirin postdiagnosis improves survival for colon cancer patients

Background: The preventive role of non-steroid anti-inflammatory drugs (NSAIDs) and aspirin, in particular, on colorectal cancer is well established. More recently, it has been suggested that aspirin may also have a therapeutic role. Aim of the present observational population-based study was to assess the therapeutic effect on overall survival of aspirin/NSAIDs as adjuvant treatment used after the diagnosis of colorectal cancer patients. Methods: Data concerning prescriptions were obtained from PHARMO record linkage systems and all patients diagnosed with colorectal cancer (1998-2007) were selected from the Eindhoven Cancer Registry (population-based cancer registry). Aspirin/NSAID use was classified as none, prediagnosis and postdiagnosis and only postdiagnosis. Patients were defined as non-user of aspirin/NSAIDs from the date of diagnosis of the colorectal cancer to the date of first use of aspirin or NSAIDs and user from first use to the end of follow-up. Poisson regression was performed with user status as time-varying exposure.Results:In total, 1176 (26%) patients were non-users, 2086 (47%) were prediagnosis and postdiagnosis users and 1219 (27%) were only postdiagnosis users (total n=4481). Compared with non-users, a survival gain was observed for aspirin users; the adjusted rate ratio (RR) was 0.77 (95% confidence interval (CI) 0.63-0.95; P=0.015). Stratified for colon and rectal, the survival gain was only present in colon cancer (adjusted RR 0.65 (95%CI 0.50-0.84; P=0.001)). For frequent users survival gain was larger (adjusted RR 0.61 (95%CI 0.46-0.81; P=0.001). In rectal cancer, aspirin use was not associated with survival (adjusted RR 1.10 (95%CI 0.79-1.54; P=0.6). The NSAIDs use was associated with decreased survival (adjusted RR 1.93 (95%CI 1.70-2.20; P<0.001). Conclusion: Aspirin use initiated or continued after diagnosis of colon cancer is associated with a lower risk of overall mortality. These findings strongly support initiation of a placebo-controlled trial that investigates the role of aspirin as adjuvant treatment in colon cancer patients

Therapeutic targeting of Krüppel-like factor 4 abrogates microglial activation

<p>Abstract</p> <p>Background</p> <p>Neuroinflammation occurs as a result of microglial activation in response to invading micro-organisms or other inflammatory stimuli within the central nervous system. According to our earlier findings, Krüppel-like factor 4 (Klf4), a zinc finger transcription factor, is involved in microglial activation and subsequent release of proinflammatory cytokines, tumor necrosis factor alpha, macrophage chemoattractant protein-1 and interleukin-6 as well as proinflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-treated microglial cells. Our current study focuses on finding the molecular mechanism of the anti-inflammatory activities of honokiol in lipopolysaccharide-treated microglia with emphasis on the regulation of Klf4.</p> <p>Methods</p> <p>For <it>in vitro </it>studies, mouse microglial BV-2 cell lines as well as primary microglia were treated with 500 ng/mL lipopolysaccharide as well as 1 μM and 10 μM of honokiol. We cloned full-length Klf4 cDNA in pcDNA3.1 expression vector and transfected BV-2 cells with this construct using lipofectamine for overexpression studies. For <it>in vivo </it>studies, brain tissues were isolated from BALB/c mice treated with 5 mg/kg body weight of lipopolysaccharide either with or without 2.5 or 5 mg/kg body weight of honokiol. Expression of Klf4, cyclooxygenase-2, inducible nitric oxide synthase and phospho-nuclear factor-kappa B was measured using immunoblotting. We also measured the levels of cytokines, reactive oxygen species and nitric oxide in different conditions.</p> <p>Results</p> <p>Our findings suggest that honokiol can substantially downregulate the production of proinflammatory cytokines and inflammatory enzymes in lipopolysaccharide-stimulated microglia. In addition, honokiol downregulates lipopolysaccharide-induced upregulation of both Klf4 and phospho-nuclear factor-kappa B in these cells. We also found that overexpression of Klf4 in BV-2 cells suppresses the anti-inflammatory action of honokiol.</p> <p>Conclusions</p> <p>Honokiol potentially reduces inflammation in activated microglia in a Klf4-dependent manner.</p

Potential use of COX-2–aromatase inhibitor combinations in breast cancer

Cyclooxygenase-2 (COX-2) is overexpressed in several epithelial tumours, including breast cancer. Cyclooxygenase-2-positive tumours tend to be larger, higher grade, node-positive and HER-2/neu-positive. High COX-2 expression is associated with poor prognosis. Cyclooxygenase-2 inhibition reduces the incidence of tumours in animal models, inhibits the development of invasive cancer in colorectal cancer and reduces the frequency of polyps in familial adenomatous polyposis (FAP). These effects may be as a result of increased apoptosis, reduced angiogenesis and/or proliferation. Studies of COX-2 inhibitors in breast cancer are underway both alone and in combination with other agents. There is evidence to suggest that combining COX-2 inhibitors with aromatase inhibitors, growth factor receptor blockers, or chemo- or radiotherapy may be particularly effective. Preliminary results from combination therapy with celecoxib and exemestane in postmenopausal women with advanced breast cancer showed that the combination increased the time to recurrence. Up to 80% of ductal carcinomas in situ (DCISs) express COX-2, therefore COX-2 inhibition may be of particular use in this situation. Cyclooxygenase-2 expression correlates strongly with expression of HER-2/neu. As aromatase inhibitors appear particularly effective in patients with HER-2/neu-positive tumours, the combination of aromatase inhibitors and COX-2 inhibitors may be particularly useful in both DCIS and invasive cancer

In Vivo, Multimodal Imaging of B Cell Distribution and Response to Antibody Immunotherapy in Mice

BACKGROUND: B cell depletion immunotherapy has been successfully employed to treat non-Hodgkin's lymphoma. In recent years, increasing attention has been directed towards also using B-cell depletion therapy as a treatment option in autoimmune disorders. However, it appears that the further development of these approaches will depend on a methodology to determine the relation of B-cell depletion to clinical response and how individual patients should be dosed. Thus far, patients have generally been followed by quantification of peripheral blood B cells, but it is not apparent that this measurement accurately reflects systemic B cell dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cellular imaging of the targeted population in vivo may provide significant insight towards effective therapy and a greater understanding of underlying disease mechanics. Superparamagnetic iron oxide (SPIO) nanoparticles in concert with near infrared (NIR) fluorescent dyes were used to label and track primary C57BL/6 B cells. Following antibody mediated B cell depletion (anti-CD79), NIR-only labeled cells were expeditiously cleared from the circulation and spleen. Interestingly, B cells labeled with both SPIO and NIR were not depleted in the spleen. CONCLUSIONS/SIGNIFICANCE: Whole body fluorescent tracking of B cells enabled noninvasive, longitudinal imaging of both the distribution and subsequent depletion of B lymphocytes in the spleen. Quantification of depletion revealed a greater than 40% decrease in splenic fluorescent signal-to-background ratio in antibody treated versus control mice. These data suggest that in vivo imaging can be used to follow B cell dynamics, but that the labeling method will need to be carefully chosen. SPIO labeling for tracking purposes, generally thought to be benign, appears to interfere with B cell functions and requires further examination

Genetic polymorphisms in the cyclooxygenase-2 gene, use of nonsteroidal anti-inflammatory drugs, and breast cancer risk

INTRODUCTION: The association between use of nonsteroidal anti-inflammatory drugs (NSAIDs) and breast cancer risk remains unclear. Inconsistencies in previously reported findings may be partly due to differences in expression of cyclooxygenase (COX)-2. We hypothesized that genetic polymorphisms (COX-2 .926, COX-2 .5209, and COX-2 .8473) may reduce overall breast cancer risk or risk for subtypes of breast cancer by modulating the inflammatory response and may interact with aspirin or any NSAID use. METHODS: We conducted a population-based, case-control study in which we genotyped 1,067 breast cancer cases and 1,110 control individuals included in the Long Island Breast Cancer Study Project. RESULTS: No major effects of the three COX-2 variant alleles on breast cancer risk were found. A total of eight distinct haplotypes and 18 diplotypes were observed in the population. Overall, no significant associations between COX-2 haplotypes/diplotypes and breast cancer risk were observed. Among women who used aspirin or any NSAID there was little evidence for an interaction with the at-risk COX-2 genotypes, with one exception. Among women with hormone receptor positive breast cancer, the reduced risk for any NSAID use was only evident among those who had at least one variant C allele of COX-2 .8473 (odds ratio = 0.7, 95% confidence interval = 0.5 to 1.0; P for the interaction = 0.02). There was no corresponding interaction for aspirin use, possibly because of limited power. CONCLUSION: These data provide modest evidence that the C allele of COX-2 .8473 may interact with NSAIDs to reduce risk for hormone receptor positive breast cancer

NGF and proNGF Regulate Functionally Distinct mRNAs in PC12 Cells: An Early Gene Expression Profiling

The biological activities of NGF and of its precursor proNGF are quite distinct, due to different receptor binding profiles, but little is known about how proNGF regulates gene expression. Whether proNGF is a purely pro-apoptotic molecule and/or simply a “less potent NGF” is still a matter of debate. We performed experiments to address this question, by verifying whether a proNGF specific transcriptional signature, distinct from that of NGF, could be identified. To this aim, we studied gene expression regulation by proNGF and NGF in PC12 cells incubated for 1 and 4 hours with recombinant NGF and proNGF, in its wild-type or in a furin-cleavage resistant form. mRNA expression profiles were analyzed by whole genome microarrays at early time points, in order to identify specific profiles of NGF and proNGF. Clear differences between the mRNA profiles modulated by the three neurotrophin forms were identified. NGF and proNGF modulate remarkably distinct mRNA expression patterns, with the gene expression profile regulated by NGF being significantly more complex than that by proNGF, both in terms of the total number of differentially expressed mRNAs and of the gene families involved. Moreover, while the total number of genes modulated by NGF increases dramatically with time, that by proNGFs is unchanged or reduced. We identified a subset of regulated genes that could be ascribed to a “pure proNGF” signalling, distinct from the “pure NGF” one. We also conclude that the composition of mixed NGF and proNGF samples, when the two proteins coexist, influences the profile of gene expression. Based on this comparison of the gene expression profiles regulated by NGF and its proNGF precursor, we conclude that the two proteins activate largely distinct transcriptional programs and that the ratio of NGF to proNGF in vivo can profoundly influence the pattern of regulated mRNAs

Explorative data analysis of MCL reveals gene expression networks implicated in survival and prognosis supported by explorative CGH analysis

<p>Abstract</p> <p>Background</p> <p>Mantle cell lymphoma (MCL) is an incurable B cell lymphoma and accounts for 6% of all non-Hodgkin's lymphomas. On the genetic level, MCL is characterized by the hallmark translocation t(11;14) that is present in most cases with few exceptions. Both gene expression and comparative genomic hybridization (CGH) data vary considerably between patients with implications for their prognosis.</p> <p>Methods</p> <p>We compare patients over and below the median of survival. Exploratory principal component analysis of gene expression data showed that the second principal component correlates well with patient survival. Explorative analysis of CGH data shows the same correlation.</p> <p>Results</p> <p>On chromosome 7 and 9 specific genes and bands are delineated which improve prognosis prediction independent of the previously described proliferation signature. We identify a compact survival predictor of seven genes for MCL patients. After extensive re-annotation using GEPAT, we established protein networks correlating with prognosis. Well known genes (CDC2, CCND1) and further proliferation markers (WEE1, CDC25, aurora kinases, BUB1, PCNA, E2F1) form a tight interaction network, but also non-proliferative genes (SOCS1, TUBA1B CEBPB) are shown to be associated with prognosis. Furthermore we show that aggressive MCL implicates a gene network shift to higher expressed genes in late cell cycle states and refine the set of non-proliferative genes implicated with bad prognosis in MCL.</p> <p>Conclusion</p> <p>The results from explorative data analysis of gene expression and CGH data are complementary to each other. Including further tests such as Wilcoxon rank test we point both to proliferative and non-proliferative gene networks implicated in inferior prognosis of MCL and identify suitable markers both in gene expression and CGH data.</p