Cyclooxygenase-2 expression correlates with phaeochromocytoma malignancy.

Abstract Aims: Phaeochromocytomas are rare but potentially life-threatening neuroendocrine tumours of the adrenal medulla or sympathetic nervous system ganglia. There are no histological features which reliably differentiate benign from malignant phaeochromocytomas. The current study evaluated cyclooxygenase-2 (Cox-2) and Bcl-2 as tissue-based biomarkers of phaeochromocytoma prognosis. Methods and Results: Cox-2 and Bcl-2 expression were examined immunohistochemically in tissue from forty-one sporadic phaeochromocytoma patients followed up for a minimum of five years after diagnosis. There was a statistically significant association between Cox-2 histoscore (intensity x porportion) and the development of tumour recurrence or metastases (p=0.006). A significant relationship between the co-expression of Cox-2 and Bcl-2 in the primary tumour and the presence of recurrent disease was observed (p=0.034). A highly significant association was observed between, (i) the tumour-associated expression of these two oncoproteins (p=0.001) and, (ii) Cox-2 histoscore and the presence of Bcl-2 expression (p=0.002). Cox regression analysis demonstrated no significant relationship between, (i) the presence or absence of either Cox-2 or Bcl-2 and patient survival or, (ii) between Cox-2 histoscore and patient survival. Conclusions: These results suggest that Cox-2 and Bcl-2 may promote phaeochromocytoma malignancy and that these oncoproteins may be valuable surrogate markers of an aggressive tumour phenotype.

Bcl-2 expression in sequential biopsies of potentially malignant oral mucosal lesions assessed by immunocytochemistry

OBJECTIVE: To examine, for the first time Bcl-2 expression in sequential (autogenous) oral mucosal biopsies taken from the same sites in a gender, risk-factor matched, Caucasoid sample, over a 21-year period,

Glucocorticoid receptor β and histone deacetylase 1 and 2 expression in the airways of severe asthma.

Rationale Upregulation of glucocorticoid receptor ß (GRß) has been implicated in steroid resistance in severe asthma, although previous studies are conflicting. GRß has been proposed as a dominant negative isoform of glucocorticoid receptor a (GRa) but it has also been suggested that GRß can cause steroid resistance via reduced expression of histone deacetylase 2 (HDAC2), a key regulator of steroid responsiveness in the airway.


Objectives To examine GRß, GRa, HDAC1 and HDAC2 expression at transcript and protein levels in bronchial biopsies from a large series of patients with severe asthma, and to compare the findings with those of patients with mild to moderate asthma and healthy volunteers.


Methods Bronchoscopic study in two UK centres with real-time PCR and immunohistochemistry performed on biopsies, western blotting of bronchial epithelial cells and immunoprecipitation with anti-GRß antibody.


Measurements and main results Protein and mRNA expression for GRa and HDAC2 did not differ between groups. GRß mRNA was detected in only 13 of 73 samples (seven patients with severe asthma), however immunohistochemistry showed widespread epithelial staining in all groups. Western blotting of bronchial epithelial cells with GRß antibody detected an additional ‘cross-reacting’ protein, identified as clathrin. HDAC1 expression was increased in patients with severe asthma compared with healthy volunteers.


Conclusions GRß mRNA is expressed at low levels in a minority of patients with severe asthma. HDAC1 and HDAC2 expression was not downregulated in severe asthma. These data do not support upregulated GRß and resultant reduced HDAC expression as the principal mechanism of steroid resistance in severe asthma. Conflicting GRß literature may be explained in part by clathrin cross-reactivity with commercial antibodies.

PTGS2 (Cyclooxygenase-2) expression and survival among colorectal cancer patients: A systematic review

Background: Studies have examined whether tumor expression of PTGS2 (also known as COX-2), an enzyme inhibited by nonsteroidal anti-inflammatory drugs such as aspirin, is associated with prognosis in patients with colorectal cancer. However, results to date have been mixed. Methods: Using terms for PTGS2 and colorectal cancer, the Medline, Embase, and Web of Science databases were systematically searched for studies published, in any language, until December 2011. Random effects meta-analyses were used to calculate pooled HRs [95% confidence intervals (CI)] for the association between PTGS2 expression and tumor recurrence, colorectal cancer-specific survival, and overall survival. Results: In total, 29 studies, which had prognostic data on 5,648 patients, met the inclusion criteria. PTGS2- positive patients were at an increased risk of tumor recurrence (n = 9 studies; HR, 2.79; 95% CI, 1.76-4.41; P <0.001) and had poorer colorectal cancer-specific survival (n = 7; HR, 1.36; 95% CI, 1.02-1.82; P = 0.04). However, there was funnel plot asymmetry, possibly due to publication bias, for the association with cancerspecific survival but less so for recurrence. PTGS2 expression was not associated with overall survival [(n= 16; pooled unadjusted HR, 1.30; 95% CI, 0.94-1.79; P=0.11) and (n=9; pooled adjusted HR, 1.02; 95% CI, 0.72-1.45; P = 0.91)]. Conclusions: PTGS2 expression was associated with an increased risk of tumor recurrence and poorer colorectal cancer-specific survival but not overall survival among patients with colorectal cancer. However, confounding by tumor characteristics such as tumor stage seems likely. Impact: There is insufficient evidence to recommend PTGS2 expression as a prognostic marker in patients with colorectal cancer. Furthermore, studies providing adjusted results are required. © 2013 AACR.

Protease activated receptor 2 expression and functionality in caries

Introduction: Protease activated receptors (PARs) are G-protein-coupled transmembrane receptors that are expressed on many cell types and implicated in various inflammatory processes in vivo. The induction of PAR2 as a result of the inflammatory response associated with dental caries remains to be determined. Objectives: The aim was to localise the expression of PAR2 in human dental pulp from carious teeth and to confirm receptor functionality using an in vitro assay. Methods: Dental pulp sections from decalcified carious teeth were examined by immunocytochemsitry. Membrane preparations from cultured pulp fibroblasts were subject to SDS-PAGE and immunoblotting to confirm fibroblast-associated immunoreactivity. The functionality of PAR2 on dental pulp fibroblasts was studied using calcium imaging in the presence of several potential activators including a PAR2 agonist (PAR2-AP), trypsin and pulpal enzymes from a carious tooth. Results: Immunocytochemistry revealed intense PAR2 immunoreactivity on pulpal fibroblasts subjacent to carious lesions but not in surrounding regions of the dental pulp. Pulp specimens from a dental injury model showed no expression of PAR2, suggesting its expression was related to cellular changes associated with ongoing caries. The localisation of PAR2 staining to pulpal fibroblasts in carious teeth was confirmed by Western blotting which revealed PAR2 immunoreactive bands in membrane fractions prepared from pulp fibroblasts. In functional studies, challenge of cultured pupal fibroblasts with PAR2-AP, trypsin and an extract of proteolytic enzymes from a carious dental pulp, showed specific activation of PAR2. Conclusions: This work demonstrates that PAR2 is functional and inducible in human dental pulp fibroblasts in response to caries and that endogenous pulpal enzymes can activate PAR2.

Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 mu g/ml) the number of cells in the G2/M phase increased to 51.82 +/- 2.69% relative to control cells (15.1 +/- 2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 mu g/ml) to exert rapid inhibition of p38 (38.7 +/- 4.7%) and CREB (28.6 +/- 5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9 +/- 9.3%). Our data suggest that olive oil polyphenols may exert chemo preventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development. (c) 2007 Elsevier Inc. All rights reserved.

CLEC-2 expression is maintained on activated platelets and on platelet microparticles

The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.

Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts

Yano Y, Cesar KR, Araujo M, Rodrigues Jr. AC, Andrade LC, Magaldi AJ. Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts. Am J Physiol Renal Physiol 296: F54-F59, 2009. First published October 1, 2008; doi: 10.1152/ajprenal.90367.2008.-It is well known that Glucagon (Gl) is released after a high protein diet and participates in water excretion by the kidney, principally after a protein meal. To study this effect in in vitro perfused inner medullary collecting ducts (IMCD), the osmotic water permeability (Pf; mu m/s) at 37 degrees C and pH 7.4 in normal rat IMCDs (n = 36) perfused with Ringer/HCO(3) was determined. Gl (10(-7) M) in absence of Vasopressin (AVP) enhanced the Pf from 4.38 +/- 1.40 to 11.16 +/- 1.44 mu m/s (P < 0.01). Adding 10(-8), 10(-7), and 10(-6) M Gl, the Pf responded in a dose-dependent manner. The protein kinase A inhibitor H8 blocked the Gl effect. The specific Gl inhibitor, des-His(1)-[Glu(9)] glucagon (10(-7) M), blocked the Gl-stimulated Pf but not the AVP-stimulated Pf. There occurred a partial additional effect between Gl and AVP. The cAMP level was enhanced from the control 1.24 +/- 0.39 to 59.70 +/- 15.18 fm/mg prot after Gl 10(-7) M in an IMCD cell suspension. The immunoblotting studies indicated an increase in AQP2 protein abundance of 27% (cont 100.0 +/- 3.9 vs. Gl 127.53; P = 0.0035) in membrane fractions extracted from IMCD tubule suspension, incubated with 10(-6) M Gl. Our data showed that 1) Gl increased water absorption in a dose-dependent manner; 2) the anti-Gl blocked the action of Gl but not the action of AVP; 3) Gl stimulated the cAMP generation; 4) Gl increased the AQP2 water channel protein expression, leading us to conclude that Gl controls water absorption by utilizing a Gl receptor, rather than a AVP receptor, increasing the AQP2 protein expression.

Insulin suppresses LPS-induced iNOS and COX-2 expression and NF-kappa B activation in alveolar macrophages

The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin downregulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E(2), on the expression of inducible nitric oxide synthase ( iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-kappa B) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-kappa B p65 activation and inhibitor of kappa B (I-kappa B) a phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE(2) by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE(2), and, in parallel, NF-kappa B p65 activation and cytoplasmic I-kappa B alpha phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE(2) production and Nuclear NF-kappa B p65 activation. Insulin also prevented cytoplasmic I-kappa Ba phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-kappa B, possibly by blocking I-kappa Ba degradation, and supresses the production of NO and PGE(2), two molecules that contribute to septic shock. Copyright (C) 2008 S. Karger AG, Basel.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04

Organotellurium(]V) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC50 for the compound after 24 h incubation was 6.8 and 0.35 mu M, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells. (C) 2008 Elsevier Ltd. All rights reserved.

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulurn Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PR,L) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to nonpregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (U). The expression of the sarcoendoplasmic reticulum Ca2+-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in P-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of PI 9 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

Changes in apoptosis and Bcl-2 expression in human hyperglycemic, term placental trophoblast

Apoptosis and its associated regulatory mechanisms are physiological events crucial to the maintenance of placental homeostasis; imbalance of these processes, however, such as occurs under various pathological conditions, may compromise placenta function and, consequently, pregnancy success. Increased apoptosis occurs in the placentas of pregnant women with several developmental disabilities, while increased Bcl-2 expression is generally associated with pregnancy-associated tumors. Herein, we tested the hypothesis that apoptosis-associated disturbs might be involved in the placental physiopathology subjected to different maternal hyperglycemic conditions.Thus, in the present study we investigated and compared the incidence of apoptosis using TUNEL reaction and Bcl-2 expression, in term-placentas of normoglycemic, diabetic and daily hyperglycemic patients. Tissue samples were collected from 37 placentas, being 15 from healthy mothers with normally delivered healthy babies, and 22 from mothers with glucose disturbances. From these latter 22 patients, 10 showed maternal daily hyperglycemia and 12 were clinically diabetics. Both Bcl-2 expression and apoptotic DNA fragmentation were established and quantified in the trophoblasts of healthy mothers. Compared to these reference values, a higher apoptosis index and lower Bcl-2 expression were disclosed in the placentas of the diabetic women, while in the daily hyperglycemic group, values were intermediate between the diabetic and normoglycemic patients. The TUNEL/Bcl-2 index ratio in the placentas varied from 0.02 to 0.09 for pregnant normoglycemic and diabetic women, respectively, revealing a predominance of apoptosis in the diabetic group. Our findings suggest that hyperglycemia may be a key factor evoking apoptosis in the placental trophoblast, and therefore, is relevant to diabetic placenta function. (c) 2006 Elsevier B.V.. All rights reserved.