Indirect effects of interference of two emerging environmental contaminants on cell health: Radiofrequency radiation and gold nanoparticles.

BACKGROUND: The global need for wireless technologies is growing rapidly. So, we have been exposed to a new type of environmental pollution: radiofrequency radiation (RFR). Recent studies have shown that RFR can cause not only direct effects but also indirect or non-targeted effects such as the bystander effect (BE). In this study, we investigated the BE induced by RFR in the present of gold nanoparticles (GNP). Moreover, we studied the expression of cyclooxygenase-2 (COX-2). METHODS: Non-toxic dose of 15-nm GNP was used to treat the Chinese Hamster Ovary (CHO) cells. After 48 h of incubation, cells were exposed to 900 MHz GSM RFR for 24 h. Then we collected the cell culture medium of these cells (conditioned culture medium, CCM) and transferred it to new cells (bystander cells). Cell deaths, DNA breaks, oxidative stress and COX-2 expression were analyzed in all groups. RESULTS: The results showed that RFR increased metabolic death in cells treated with GNP. Inversely, the colony formation ability was reduced in bystander cells and RFR exposed cells either in the presence or absence of GNP. Also, the level of reactive oxygen species (ROS) in GNP treated cells showed a significant reduction compared to those of untreated cells. However, RFR-induced DNA breaks and the frequencies of micronuclei (MN) were not significantly affected by GNP. The expression of COX-2 mRNA increased in RFR GNP treated cells, but the difference was not significant. CONCLUSION: Our results for the first time indicated that RFR induce indirect effects in the presence of GNP. However, the molecular mediators of these effects differ from those in the absence of GNP. Also, to our knowledge, this is the first study to show that COX-2 is not involved in the bystander effect induced by 900 MHz RFR.

Regulation of COX-2 expression by selected trace elements and heavy metals: Health implications, and changes in neuronal plasticity. A review.

Trace elements or trace metals are essential components of enzymes, proteins, hormones and play a key role in biochemical processes, cell growth and differentiation, as well as in neurotransmission, affecting human physiology. In nature there are also heavy metals that exhibit toxic effects on the human body, including the brain. The importance of trace elements has been established in neurodegenerative disorders, schizophrenia, depression among others. In parallel, an important regulatory element in the above diseases is cyclooxygenase-2 (COX-2), a modulator of the arachidonic acid (AA) pathway, and a cause of neuroinflammation, and glutamate (Glu) dysregulation, affecting calcium (Ca) metabolism in cells. This review presents the effects of major trace elements and heavy metals on COX-2 expression. Calcium (Ca), zinc (Zn), cadmium (Cd), vanadium (V), nickel (Ni), copper (Cu), and iron (Fe) can potentially increase COX-2 expression, inducing neuroinflammation and Glu excitotoxicity; while magnesium (Mg), lithium (Li), and selenium (Se) can potentially decrease COX-2 expression. The associated mechanisms are described in the article.

Glycolipid-enriched fraction of Osmanthus fragrans inhibits LPS-induced expression of inflammatory genes, COX-2, E-selectin, and Interleukin-8.

ETHNOPHARMACOLOGICAL RELEVANCE: Osmanthus fragrans Lour. is a small ornamental tree native to the Southeastern parts of China. It is mainly cultivated because of its characteristic fragrance, and used in the food and perfume industry. Besides, its flowers are used in traditional Chinese medicine to treat a variety of diseases including those related to inflammation. AIM OF THE STUDY: The aim of the study was to investigate in more detail the anti-inflammatory properties of O. fragrans flowers, and to characterize their active principles and mechanisms of action. MATERIALS AND METHODS: O. fragrans flowers were successively extracted with n-hexane, dichloromethane and methanol. The extracts were further fractionated by chromatographic separation. COX-2 mRNA expression in PMA-differentiated, LPS-stimulated THP-1 cells was used as lead assay for activity-guided fractionation. The most potent fraction was chemically analyzed by LC-HRMS. The pharmacological activity was also evaluated in other inflammation-related in-vitro models, such as analysis of IL-8 secretion and E-selectin expression in HUVECtert cells and selective inhibition of COX-isoenzymes. RESULTS: n-Hexane and dichloromethane extracts of O. fragrans flowers significantly inhibited COX-2 (PTGS2) mRNA expression. Additionally, both extracts inhibited COX-2 enzyme activity, whereas COX-1 enzyme activity was affected to a significantly lower extent. Fractionation of the extracts led to a highly active, glycolipid-containing fraction. In total, 10 glycolipids were tentatively annotated by LC-HRMS. This fraction also inhibited LPS-induced COX-2 mRNA expression, IL-8 secretion and E-selectin expression. The effects were limited to LPS-induced inflammation and not observed when inflammatory genes were induced by TNF-α, IL-1ß or FSL-1. Since all these inducers of inflammation act via different receptors, it is likely that the fraction interferes with the binding of LPS to the TLR4-receptor, which mediates pro-inflammatory effects of LPS. CONCLUSION: Taken together, the results demonstrate the anti-inflammatory potential of O. fragrans flower extracts in general, and of the glycolipid-enriched fraction in particular. The effects of glycolipid-enriched fraction are potentially mediated via the inhibition of the TLR4 receptor complex.

Celecoxib substituted biotinylated poly(amidoamine) G3 dendrimer as potential treatment for temozolomide resistant glioma therapy and anti-nematode agent.

Glioblastoma multiforme (GBM) is a one of the most widely diagnosed and difficult to treat type of central nervous system tumors. Resection combined with radiotherapy and temozolomide (TMZ) chemotherapy prolongs patients' survival only for 12 - 15 months after diagnosis. Moreover, many patients develop TMZ resistance, thus important is search for a new therapy regimes including targeted drug delivery. Most types of GBM reveal increased expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2), that are considered as valuable therapeutic target. In these studies, the anti-tumor properties of the selective COX-2 inhibitor celecoxib (CXB) and biotinylated third generation of the poly(amidoamine) dendrimer substituted with 31 CXB residues (G3BC31) on TMZ -resistant U-118 MG glioma cell line were examined and compared with the effect of TMZ alone including viability, proliferation, migration and apoptosis, as well as the cellular expression of COX-2, ATP level, and PGE2 production. Confocal microscopy analysis with the fluorescently labeled G3BC31 analogue has shown that the compound was effectively accumulated in U-118 MG cells in time-dependent manner and its localization was confirmed in lysosomes but not nuclei. G3BC31 reveal much higher cytotoxicity for U-118 MG cells at relatively low concentrations in the range of 2-4 µM with compared to CBX alone, active at 50-100 µM. This was due to induction of apoptosis and inhibition of proliferation and migration. Observed effects were concomitant with reduction of PGE2 production but independent of COX-2 expression. We suggest that investigated conjugate may be a promising candidate for therapy of TMZ-resistant glioblastoma multiforme, although applicable in local treatment, since our previous study of G3BC31 did not demonstrate selectivity against glioma cells compared to normal human fibroblasts. However, it has to be pointed that in our in vivo studies conducted with model organism, Caenorhabditis elegans indicated high anti-nematode activity of G3BC31 in comparison with CXB alone that confirms of usefulness of that organism for estimation of anti-cancer drug toxicity.

Tea polyphenols attenuate liver inflammation by modulating obesity-related genes and down-regulating COX-2 and iNOS expression in high fat-fed dogs.

BACKGROUND: Tea polyphenols (TPs) attenuate obesity related liver inflammation; however, the anti-obesity effects and anti-inflammatory mechanisms are not clearly understood. This study aimed to determine whether the anti-obesity and anti-inflammatory TPs mechanisms associated with cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression levels, and obesity-related gene response in dogs. RESULTS: Dogs fed TPs displayed significantly decreased (p < 0.01) mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6) compared to dogs that consumed high-fat diet (HFD) alone. TPs significantly (p < 0.01) inhibited COX-2 and iNOS expression level, and decreased liver fat content and degeneration. CONCLUSION: These results suggested that TPs act as a therapeutic agent for obesity, liver inflammation, and fat degeneration via COX-2 and iNOS inhibition, with TNF-α, IL-1ß, and IL-6 involvement.

Biotinylated PAMAM G3 dendrimer conjugated with celecoxib and/or Fmoc-l-Leucine and its cytotoxicity for normal and cancer human cell lines.

Tumors still remain one of the main causes of mortality due to the lack of effective anti-cancer therapy. Recently it has been shown, that overexpression of inducible cyclooxygenase-2 (COX-2) and decrease of peroxisome proliferator-activated receptor γ (PPARγ) expression accompany many malignances, therefore, it has been proposed, that COX-2 inhibitors and PPARγ agonists are potential candidates for anticancer therapy and their synergistic, antineoplastic action has been described. In the present study a COX-2 inhibitor (celecoxib) and/or PPARγ agonist (Fmoc-l-Leucine) were conjugated with the biotinylated G3 PAMAM dendrimer to form a three different constructs targeted to cells with increased biotin uptake. All conjugates were characterized by the NMR spectroscopy. Investigation of three types of human cells: normal skin fibroblasts (BJ), immortalized keratinocytes (HaCaT) and cancer lines: glioblastoma (U-118 MG) and squamous cell carcinoma (SCC-15) revealed similar biotin labeled ATTO590 accumulation (after 24 h), except for SCC-15 with significantly lower loading. Constitutive expression of COX-2 protein was confirmed in all tested cells with significantly higher levels (2-2.5 times) in both cancer lines. Comparison of cytotoxicity of the new synthetized dendrimers clearly documented the highest cytotoxicity of the G31B16C15L dendrimer conjugated with both drugs (1: 1) as compared with drugs alone and single conjugates. Additive effects of construct with both compounds were shown for fibroblasts and both cancer cell lines in the order BJ > U-118 MG > SCC-15 with IC50 in the range: 0.69, 1.44 and 2.22 µM, respectively and lowest cytotoxicity in HaCaT cells (IC50 = 2.88). Our results showed, that biotinylated G3 PAMAM dendrimers substituted with COX-2 inhibitor, celecoxib, and PPARγ agonist, Fmoc-l-Leucine (1:1) may be a good candidate for local therapy of glioblastoma but not a skin cancer. Since the effect of PPARγ agonists on COX-2 expression vary depending upon the cell type, specificity of used agonist and the presence of other environmental factors, it is necessary to carefully evaluate the response of chosen drugs on the target cells.

Resveratrol inhibits the proliferation of A549 cells by inhibiting the expression of COX-2.

PURPOSE: The aim was to investigate resveratrol effects on A549 cells proliferation. METHODS: A total of 104 lung adenocarcinoma tissues and nontumor tissues were collected. BEAS-2B cells were cultured in RPMI 1640 medium (group A). A549 cells were treated with RPMI 1640 medium containing different resveratrol concentrations. A549 cells were transfected and grouped as follows: blank group, siRNA-negative control group, siRNA-COX-2 group and resveratrol + siRNA-COX-2 group. qRT-PCR and Western blot were conducted to detect COX-2 expression. MTT assay, soft agar clone assay and flow cytometry were performed to assess proliferation and cell cycle. RESULTS: The relative expression of COX-2 mRNA was significantly increased in lung adenocarcinoma tissues (P<0.01) and it was closely related with clinical stages. Resveratrol at 60 µmol/L significantly inhibited A549 cells proliferation, S phase cells proportion and COX-2 expression (P<0.01). COX-2 expression in siRNA-COX-2 group was significantly lower than that in blank group and siRNA-negative control group (P<0.01). OD570 values, colony formation rate and S phase cells proportion of resveratrol + siRNA-COX-2 group were much lower than those of other groups (P<0.01). CONCLUSION: Resveratrol inhibits A549 cells proliferation by inhibiting COX-2 expression.

Rational drug design and synthesis of new α-Santonin derivatives as potential COX-2 inhibitors.

Sesquiterpene compounds are widely known for their numerous pharmacological activities. Herein the focus of the authors was on α-Santonin, a sesquiterpene lactone from the Artemisia genus: the aim was to determine whether α-Santonin could be considered in the treatment of inflammation and pain. To this purpose, a small series of derivatives was designed and screened in silico against the enzyme COX-2 along with the parent compound. Drug-likeness parameters were also assessed. The compounds were eventually synthesized, and few were tested to determine their efficacy in the inhibition of COX-2 activity and expression. Overall, compound A2 was the only one with a detectable inhibitory potential of COX-2 activity whilst two of its ether derivatives demonstrated improved ability in the inhibition of COX-2 expression.

Bisphenol A induces COX-2 through the mitogen-activated protein kinase pathway and is associated with levels of inflammation-related markers in elderly populations.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical, and it is one of the highest volume chemicals produced worldwide. Even though several in vivo and in vitro studies showed positive associations of BPA exposure with pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-6, the mechanism by which BPA induces inflammation is unclear. We investigated the mechanism by which BPA induces inflammation (expression of inflammation-related genes, changes in oxidative stress, and cell proliferation and migration) and evaluated the effect of BPA exposure on inflammation-related markers in epidemiologic studies using repeat urine and serum samples from elderly subjects. BPA induced COX-2 expression via nuclear translocation of NF-κB and activation of mitogen-activated protein kinase (MAPK) by phosphorylation of ERK1/2 and enhanced the migration of lung cancer A549 and breast cancer MDAMB-231 cells. In two epidemiologic studies, we detected associations of BPA with six inflammation-related markers (WBC, CRP, IL-10, ALT, AST, and γ-GTP levels). Our findings probably suggest that BPA exposure induces inflammation and exacerbates tumorigenesis.

Retrospective evaluation of COX-2 expression, histological and clinical factors as prognostic indicators in dogs with renal cell carcinomas undergoing nephrectomy.

Limited veterinary literature is available regarding prognostic markers for canine renal cell carcinoma (CRCC). We retrospectively evaluated COX-2 expression, histological and clinical features associated with prognosis of CRCC. Sixty-four cases post-nephrectomy were included, 54 had histopathological assessment and 30 had COX-2 immunostaining performed. Eight dogs (13%) had metastatic disease at initial diagnosis. Twenty-seven dogs (42%) received adjuvant therapy after nephrectomy. On univariate analysis, COX-2 expression, mitotic index (MI), histologic type, vascular invasion, neoplastic invasiveness and metastasis at diagnosis were significantly associated with overall median survival time (MST). COX-2 score (COX-2 score > 3 MST 420 days versus 1176 days if COX-2 score <3; P = 0.011) and MI (MI > 30 MST 120 days versus 540 days for MI < 30; P = 0.003) were the only variables associated with CRCC outcome on multivariate analysis. The addition of MI and COX-2 immunostaining to standard histopathological evaluation would help predicting outcome in CRCC patients.

Cyclodextrin-Based Nanohydrogels Containing Polyamidoamine Units: A New Dexamethasone Delivery System for Inflammatory Diseases.

Glucocorticoids are widely prescribed in treatment of rheumatoid arthritis, asthma, systemic lupus erythematosus, lymphoid neoplasia, skin and eye inflammations. However, well-documented adverse effects offset their therapeutic advantages. In this work, novel nano-hydrogels for the sustained delivery of dexamethasone were designed to increase both bioavailability and duration of the administered drug and reducing the therapeutic dose. Hydrogels are soft materials consisting of water-swollen cross-linked polymers to which the insertion of cyclodextrin (CD) moieties adds hydrophobic drug-complexing sites. Polyamidoamines (PAAs) are biocompatible and biodegradable polymers apt to create CD moieties in hydrogels. In this work, ß or γ-CD/PAA nanogels have been developed. In vitro studies showed that a pretreatment for 24⁻48 h with dexamethasone-loaded, ß-CD/PAA nanogel (nanodexa) inhibits adhesion of Jurkat cells to human umbilical vein endothelial cells (HUVEC) in conditions mimicking inflammation. This inhibitory effect was faster and higher than that displayed by free dexamethasone. Moreover, nanodexa inhibited COX-2 expression induced by PMA+A23187 in Jurkat cells after 24⁻48 h incubation in the 10-8⁻10-5 M concentration range, while dexamethasone was effective only at 10-5 M after 48 h treatment. Hence, the novel nanogel-dexamethasone formulation combines faster action with lower doses, suggesting the potential for being more manageable than the free drug, reducing its adverse side effects.

Optical imaging of COX-2: studies on an autofluorescent 2,3-diaryl-substituted indole-based cyclooxygenase-2 inhibitor.

This study aimed at in vivo visualization of cyclooxygenase-2 (COX-2) by optical imaging using a representative compound of a class of autofluorescent 2,3-diaryl-substituted indole-based selective COX-2 inhibitors (2,3-diaryl-indole coxibs). COX-2 was successfully visualized in mice models with phorbol myristate ester (TPA)-induced inflammation or bearing xenografted human melanoma cells by 2-[4-(aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-1H-indole (C1). COX-2 protein expression in both TPA-induced inflammatory sites and human melanoma xenografts was confirmed by immunoblotting. Control experiments using surrogate markers, sham injections, and non-COX-2 expressing melanoma cells further confirmed specificity of tissue association of C1. The merging of therapeutic and diagnostic properties of 2,3-diaryl-indole coxibs may widen the range of applications of COX-2-targeted treatment, e.g., for in situ-guided surgery and ex vivo diagnostics.

Assessment of cyclooxygenase-1 and 2 gene expression levels in chronic autoimmune thyroiditis, papillary thyroid carcinoma and nontoxic nodular goitre.

INTRODUCTION: The cyclooxygenases are a group of enzymes catalyzing the formation of prostaglandins from arachidonic acid. Cyclooxygenase-1 (COX-1) is a constitutive form, thought to be a "housekeeping gene", with constant levels of expression in most tissues. COX-1 expression in the thyroid gland, except for medullary thyroid carcinoma, has not been a subject of much interest. Cyclooxygenase-2 (COX-2) can be expressed in response to various stimuli, such as mitogens, hormones, cytokines, growth factors. The product of COX-2 activity has been implicated in carcinogenesis. Recent studies have shown that up-regulation of COX-2 is associated with numerous neoplasms. Hereby, we present a study analysing COX-1 and COX-2 expression in papillary thyroid carcinoma (PTC), Hashimoto thyroiditis (HT) and nontoxic nodular goitre (NNG) in fine needle aspiration biopsy (FNAB) washouts and in postoperative tissue. MATERIAL AND METHODS: Cytological specimens from 120 patients (105 females and 15 males) have been studied, including patients with HT, PTC and NNG. Moreover, we have examined postoperative tissue specimens from 51 patients with PTC and NNG. The methods of molecular analysis have included extraction of total RNA from FNAB cytological material and postoperative tissues, spectrophotometric assessment of the RNA purity, cDNA synthesis in reverse transcription reaction and an analysis of genes expression data by real-time PCR. RESULTS: The performed analysis has revealed statistically significant higher expression level of the COX-2 gene in PTC group, in comparison with HT and NNG groups (in both cytological and postoperative material). In PTC patients, COX-2 gene expression levels in the material obtained by FNAB were similar to those in the postoperative thyroid tissue. No correlations between COX-2 gene expression level and TNM staging in PTC samples have been observed. There were no correlations between COX-2 expression and anti-TPO antibodies level, or patient's sex or age in the studied groups. Also, there were no correlations of COX-1 gene expression level among PTC, HT and NNG groups. CONCLUSIONS: Our results suggest that COX-2 gene does not participate in the mechanisms involved in molecular association of HT with PTC. However, in case of PTC itself, it may play some role in neoplastic transformation.

High cyclooxygenase-2 expression is associated with advanced stages in colorectal cancer.

BACKGROUND: Despite compelling evidence from the genetic background and clinical studies indicating that cyclooxygenase-2 (COX2) up-regulation is a key step in carcinogenesis of colorectal carcinoma (CRC), controversy regarding its role as a prognostic factor exists. However, all evidence indicates that increased COX2 activity promotes progression of CRC. This study, aimed to evaluate the expression of COX2 in CRC, and correlate it with different patient clinicopathological data, emphasizing on the role of COX2 as a prognostic factor for CRC. MATERIALS AND METHODS: In the present study, archival samples from 145 patients with stage I, II, III, or IV CRC treated during 1981-1990 at the Turku University Hospital (Finland) were used (as microarray blocks) to analyze COX2 expression by immunohistochemistry (IHC). RESULTS: Higher levels of COX2 expression were associated with higher TNM class (p<0.06), and higher Dukes' stage (p<0.045). In contrast, there was no significant correlation with age, gender, tumor grade or lymph node status. However, univariate survival analysis of metastases showed borderline association with COX2 expression in that patients with metastases with COX2-positive tumors were alive for shorter periods of time compared with patients whose tumors had no COX2 expression (p<0.023, log-rank). CONCLUSION: COX-2 expression has shown a significant correlation with tumor stage and hence is assumed to be a prognostic factor in our cohort of colorectal cancer patients.