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Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca 2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Recent studies indicate that androgen deprivation therapy (ADT), the main therapeutic approach for metastatic prostate cancer (PCa), accelerates PCa invasion and metastasis. Annexin A1 (ANXA1) is a Ca 2+ -regulated phospholipid-binding protein that can promote PCa migration and invasion., Aim of the Study: The aim of this study is to determine whether ANXA1 is regulated by ADT and participates in PCa progression after ADT, and to explore the possible mechanism of ANXA1-mediated PCa migration., Methods: Expression of ANXA1 and androgen receptor (AR) in PCa cell lines and tissues was detected, and the association between these two proteins were analyzed. Expression of ANXA1 was evaluated after AR knockdown or AR inhibition in PCa cell lines. Cell migration of PCa cell liness after ANXA1 knockdown or overexpression was determined by in vitro migration assay. Transcriptome analysis was used to explore the possible mechanism of ANXA1-mediated PCa migration., Results: ANXA1 expression in PCa cell lines and tissues was reversely associated with AR. In vitro studies revealed an increase in ANXA1 expression after AR knockdown or treatment with AR antagonist. Moreover, functional assays indicated that ANXA1 knockdown in PCa cells significantly inhibited cell migration, while ANXA1 overexpression in PCa cells significantly accelerated cell migration. Transcriptome analysis showed that ANXA1 regulated multiple genes involved in cell junction organization, such as CADM1, LIMCH1 and PPM1F., Conclusions: Our results indicate that ADT might accelerate PCa metastasis via ANXA1 expression and PCa cell migration., (Copyright © 2021 IMSS. Published by Elsevier Inc. All rights reserved.)

Astrocytomas represent the majority of cerebral gliomas. Studies show that the anti-inflammatory protein Annexin-A1 (ANXA1) is associated with the tumor invasion process and that its actions can be mediated by the receptor for formylated peptides (FPR). Therefore, we evaluated the expression of ANXA1, the receptor FPR2 and matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in brain astrocytomas. Detection of proteins was performed in sections of diffuse astrocytomas (grade II), anaplastic astrocytomas (grade III) and glioblastomas (GBM, grade IV) and quantifications were made by densitometry. Our analyses showed increased expression of ANXA1 in astrocytomas of all grades, but especially in GBM. The expression of FPR2 is similar to that found for ANXA1, being higher in GBM. Immunostaining for MMPs is also stronger as the degree of malignancy increases, especially with respect to MMP-9. The positive correlation between ANXA1/FPR2 and ANXA1/MMP-9 was observed in all tumors studied. The data indicate the possible action of ANXA1 and FPR2 on the development and progression of astrocytomas, related to increased expression of MMP-9. Thereby, ANXA1 and FPR2 are involved in the biology and malignancy of diffuse astrocytic tumors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mast cells (MCs) have important immunoregulatory roles in skin inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory protein that can be expressed by mast cells, neutrophils, eosinophils, monocytes, epithelial and T cells. This study investigated MCs heterogeneity and ANXA1 expression in human dermatoses with special emphasis in leprosy. Sixty one skin biopsies from 2 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 type 1 reaction/T1R, 11 type 2 reaction/T2R); 21 patients with other dermatoses. Tryptase/try+ and chymase/chy + phenotypic markers and toluidine blue stained intact/degranulated MC counts/mm 2 were evaluated. Try + /chy + MCs and ANXA1 were identified by streptavidin-biotin-peroxidase immunostaining and density was reported. In leprosy, degranulated MCs outnumbered intact ones regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), leprosy reactions (reactional/reaction-free) and type of reaction (T1R/T2R). Compared to other dermatoses, leprosy skin lesions showed lower numbers of degranulated and intact MCs. Try + MCs outnumbered chy + in leprosy lesions (reaction-free/reactional, particularly in T2R), but not in other dermatoses. Compared to other dermatoses, ANXA1 expression, which is also expressed in mast cells, was higher in the epidermis of leprosy skin lesions, independently of reactional episode. In leprosy, higher MC degranulation and differential expression of try + /chy + subsets independent of leprosy type and reaction suggest that the Mycobacterium leprae infection itself dictates the inflammatory MCs activation in skin lesions. Higher expression of ANXA1 in leprosy suggests its potential anti-inflammatory role to maintain homeostasis preventing tissue and nerve damage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Previous studies showed that ceftriaxone can cause acute kidney injury (AKI) in the pediatric population. This study proposed a cellular model of crystalline nephropathy in ceftriaxone-associated AKI and explored the related pathophysiology by using a proteomic approach., Methods: Ceftriaxone was crystallized with calcium in artificial urine. Madin-Darby Canine Kidney (MDCK) cells, a model of distal renal tubular cell, were cultured in the absence (untreated control) or presence of ceftriaxone crystals for 48-h (n = 5 each). MDCK cells were harvested and subsequently analyzed by proteomic analysis. Protein bioinformatics (i.e., STRING and Reactome) was used to predict functional alterations, and subsequently validated by Western blotting and cellular studies. p < 0.05 was considered statistically significant., Results: Phase-contrast microscopy showed increased intracellular vesiculation and cell enlargement as a result of ceftriaxone crystal exposure. Proteome analysis revealed a total of 20 altered proteins (14 increased, 5 decreased and 1 absent) in ceftriaxone crystal-treated MDCK cells as compared to untreated cells (p < 0.05). Protein bioinformatics and validation studies supported heat stress response mediated by heat shock protein 70 (Hsp70) and downregulation of annexin A1 as the proposed pathophysiology of crystalline nephropathy in ceftriaxone-associated AKI, in which impaired proliferation and wound healing of crystal-induced distal tubular cells were outcomes., Conclusions: This study, for the first time, used the in vitro model of crystalline nephropathy to investigate the underlying pathophysiology of ceftriaxone-associated AKI, which should be investigated in vivo for potential clinical benefits in the future., (© 2018 S. Karger AG, Basel.)

Epithelial to mesenchymal transition (EMT) is a critical step in the metastasis of epithelial cancer cells. Butyrate, which is produced from dietary fiber by colonic bacterial fermentation, has been reported to influence EMT. However, some studies have reported that butyrate promotes EMT, while others have reported an inhibitory effect. To clarify these controversial results, it is necessary to elucidate the mechanism by which butyrate can influence EMT. In this study, we examined the potential role of annexin A1 (ANXA1), which was previously reported to promote EMT in breast cancer cells, as a mediator of EMT regulation by butyrate. We found that ANXA1 mRNA and protein were expressed in highly invasive melanoma cell lines (A2058 and A375), but not in SK-MEL-5 cells, which are less invasive. We also showed that butyrate induced ANXA1 mRNA and protein expression and promoted EMT-related cell invasion in SK-MEL-5 cells. Downregulation of ANXA1 expression using specific small interfering RNAs in butyrate-treated SK-MEL-5 cells resulted in increased expression of the epithelial marker E-cadherin and decreased cell invasion. Moreover, overexpressing ANXA1 decreased the expression of the E-cadherin. Collectively, these results indicate that butyrate induces the expression of ANXA1 in human melanoma cells, which then promotes invasion through activating the EMT signaling pathway.

Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintenance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent anti-inflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

HIV-1 disease progression is paradoxically characterized by systemic chronic immune activation and gut mucosal immune dysfunction, which is not fully defined. Annexin A1 (ANXA1), an inflammation modulator, is a potential link between systemic inflammation and gut immune dysfunction during the simian immunodeficiency virus (SIV) infection. Gene expression of ANXA1 and cytokines were assessed in therapy-naïve rhesus macaques during early and chronic stages of SIV infection and compared with SIV-negative controls. ANXA1 expression was suppressed in the gut but systemically increased during early infection. Conversely, ANXA1 expression increased in both compartments during chronic infection. ANXA1 expression in peripheral blood was positively correlated with HLA-DR+CD4+ and CD8+ T-cell frequencies, and negatively associated with the expression of pro-inflammatory cytokines and CCR5. In contrast, the gut mucosa presented an anergic cytokine profile in relation to ANXA1 expression. In vitro stimulations with ANXA1 peptide resulted in decreased inflammatory response in PBMC but increased activation of gut lymphocytes. Our findings suggest that ANXA1 signaling is dysfunctional in SIV infection, and may contribute to chronic inflammation in periphery and with immune dysfunction in the gut mucosa. Thus, ANXA1 signaling may be a novel therapeutic target for the resolution of immune dysfunction in HIV infection.

BACKGROUND The endogenous protein annexin A1 (ANXA1) is an anti-inflammatory mediator in the brain that is thought to contribute to the progression of many neurological conditions. However, its exact role in temporal lobe epilepsy (TLE) remains unclear. We hypothesized that ANXA1 exerts negative actions on TLE by alleviating inflammatory damage in neurons. To identify the potential mechanism of TLE by assessing ANXA1 expression in TLE rats. MATERIAL AND METHODS TLE was induced in rats (n=70) via an intraperitoneal injection of lithium chloride (LiCl) and pilocarpine (PILO). The control group (n=10) received an injection of the equivalent amount of saline. ANXA1 expression was detected via immunohistochemistry and Western blotting. RESULTS Successful establishment of the TLE model in rats resulted in epileptic seizures. ANXA1 was immunohistochemically detected as brownish yellow particles in the dentate gyrus and the CA1 region of the door zone; this expression was predominantly localized to the cytoplasm of glia rather than neurons. ANXA1 expression was stronger in TLE rats compared with the control group. ANXA1 expression in TLE was also assessed via Western blotting, and compared between groups at various time points. ANXA1 expression was significantly increased in the acute (the first 24 h) and chronic (after 1 month) phases (P<0.001) but significantly decreased during the recovery phase (72 h, 1 week, and 2 weeks) (P<0.001). These findings suggest that ANXA1 expression is correlated with TLE activity. CONCLUSIONS Our data suggest that ANXA1 plays an important role in TLE by alleviating inflammatory damage and protecting neurons.

Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein, involved in anti-inflammatory effects, regulation of cellular differentiation, proliferation and apoptosis. While many studies have investigated the ANXA1 expression in various tumor types, the role of ANXA1 is not fully understood. Therefore, in the present study, we evaluated the ANXA1 expression in 211 breast cancer patients and compared the levels with clinicopathological factors. ANXA1 was positively expressed in 31 (14.7%) of the 211 cases in our cohort, and these positive cases were associated with triple-negative breast cancer (TNBC) (P=0.007) and venous invasion (P=0.028). The in vitro cell experiment found that the MDA-MB-231 cell line, which is a TNBC cell line, highly expressed ANXA1. Using this cell line, the functional role of ANXA1 in breast cancer was revealed and the knockdown of ANXA1 by specific siRNA demonstrated a significant reduction in cellular invasion. Further experiments indicated that ANXA1 was induced by hypoxia with hypoxia-inducible factor-1α induction. These results suggested that ANXA1, which enhanced breast cancer invasion and metastasis under hypoxia, were significantly associated with the worst patient outcome. This is particularly noted in TNBC, the group of breast cancer with the worst outcome for which new therapeutic implications are required.

Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Also, it has been reported to be associated with the onset of various cancers. An effective anti-inflammatory drug should be able to inhibit the development of inflammation without interfering in normal homeostasis. Current approaches to overcome the inflammation include the use of immune selective anti-inflammatory derivatives, selective glucocorticoid receptor agonist, resolvins and protectins and TNF inhibitors. A number of herbal drugs have been identified in the past that can target inflammatory cytokines. This review mainly focuses on the newer molecules to combat the inflammation and also emphasise on various studies carried out in the past. Thus, the high prevalence of inflammation obliges the development of new drugs; therefore, a safe and efficient drug molecule to confer protection against inflammation is urgently needed.

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Adenocarcinoma of lung has high incidence and a poor prognosis, woith chemotherapy as the main therapeutic tool, most commonly with cisplatin. However, chemotherapy resistance develops in the majority of patients during clinic treatment. Mechanisms of resistance are complex and still unclear. Although annexin play important roles in various tumor resistance mechanisms, their actions in cisplatin-resistant lung adenocarcinoma remain unclear. Preliminary studies by our group found that in cisplatin-resistant lung cancer A549 cells and lung adenocarcinoma tissues, both mRNA and protein expression of annexins A1, A2 and A3 is increased. Using a library of annexin A1, A2 and A3 targeting combined molecules already established by ourselves we found that specific targeting decreased cisplatin-resistance. Taken together, the underlined effects of annexins A1, A2 and A3 on drug resistance and suggest molecular mechanisms in cisplatin-resistant A549 cells both in vivo and in vitro. Furthermore, the study points to improved research on occurrence and development of lung adenocarcinoma, with provision of effective targets and programmes for lung adenocarcinoma therapy in the clinic.

Background: Malaria is the most prevalent parasitic disease in the world. In Brazil, the largest number of malaria cases (98%) is within the Legal Amazon region, where Plasmodium vivax is responsible for over 80% of diagnosed cases. The aim of this study was to investigate the annexin-A1 expression in CD4+, CD8+ T cells, regulatory T cells (Treg) and cytokine IL-10 quantification in plasma from patients with malaria caused by P. vivax., Methods: The quantification of the cytokine IL-10 of patients infected with P. vivax and healthy controls were evaluated by enzyme-linked immunosorbent assay (ELISA). The determination of the expression of annexin-A1 in lymphocytes from patients and healthy controls was determined by immunofluorescence staining. All results were correlated with the parasitaemia and the number of previous episodes of malaria., Results: The cytokine IL-10 plasma levels showed a significant increase in both patients with low (650.4 ± 59.3 pg/mL) and high (2870 ± 185.3 pg/mL) parasitaemia compared to the control (326.1 ± 40.1 pg/mL). In addition, there was an increase of this cytokine in an episode dependent manner (individuals with no previous episodes of malaria--primoinfected: 363.9 ± 31.1 pg/mL; individuals with prior exposure: 659.9 ± 49.4 pg/mL). The quantification of annexin-A1 expression indicated a decrease in CD4+ and CD8+ T cells and an increase in Treg in comparison with the control group. When annexin-A1 expression was compared according to the number of previous episodes of malaria, patients who have been exposed more than once to the parasite was found to have higher levels of CD4+ T cells (96.0 ± 2.5 A.U) compared to primoinfected (50.3 ± 1.7). However, this endogenous protein had higher levels in CD8+ (108.5 ± 3.1) and Treg (87.5 ± 2.5) from patients primoinfected., Conclusion: This study demonstrates that in the patients infected with P. vivax the release of immunoregulatory molecules can be influenced by the parasitaemia level and the number of previous episodes of malaria. annexin-A1 is expressed differently in lymphocyte sub-populations and may have a role in cell proliferation. Furthermore, annexin-A1 may be contributing to IL-10 release in plasma of patients with vivax malaria.

TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-β and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-β and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-β and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-β and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine and glucocorticoid (GC) counter-regulator, has emerged as an important modulator of inflammatory responses. However, the molecular mechanisms of MIF counter-regulation of GC still remain incomplete. In the present study, we investigated whether MIF mediated the counter-regulation of the anti-inflammatory effect of GC by affecting annexin 1 in RAW 264.7 macrophages. We found that stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) resulted in down-regulation of annexin 1, while GC dexamethasone (Dex) or Dex plus LPS led to significant up-regulation of annexin 1 expression. RNA interference-mediated knockdown of intracellular MIF increased annexin 1 expression with or without incubation of Dex, whereas Dex-induced annexin 1 expression was counter-regulated by the exogenous application of recombinant MIF. Moreover, recombinant MIF counter-regulated, in a dose-dependent manner, inhibition of cytosolic phospholipase A2α (cPLA2α) activation and prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ) release by Dex in RAW 264.7 macrophages stimulated with LPS. Endogenous depletion of MIF enhanced the effects of Dex, reflected by further decease of cPLA2α expression and lower PGE2 and LTB4 release in RAW 264.7 macrophages. Based on these data, we suggest that MIF counter-regulates Dex-induced annexin 1 expression, further influencing the activation of cPLA2α and the release of eicosanoids. These findings will add new insights into the mechanisms of MIF counter-regulation of GC., (© 2013 John Wiley & Sons Ltd.)

Background: The benefit of induction chemotherapy in locally advanced oral squamous cell carcinoma (OSCC) remains to be clearly defined. Induction chemotherapy is likely to be effective for biologically distinct subgroups of patients and biomarker development might lead to identification of the patients whose tumors are to respond to a particular treatment. Annexin A1 may serve as a biomarker for responsiveness to induction chemotherapy. The aim of this study was to investigate Annexin A1 expression in pre-treatment biopsies from a cohort of OSCC patients treated with surgery and post-operative radiotherapy or docetaxel, cisplatin and 5-fluorouracil (TPF) induction chemotherapy followed by surgery and post-operative radiotherapy. Furthermore we sought to assess the utility of Annexin A1 as a prognostic or predictive biomarker., Methods: Immunohistochemical staining for Annexin A1 was performed in pre-treatment biopsies from 232 of 256 clinical stage III/IVA OSCC patients. Annexin A1 index was estimated as the proportion of tumor cells (low and high, <50% and ≥50% of stained cells, respectively) to Annexin A1 cellular membrane and cytoplasm staining., Results: There was a significant correlation between Annexin A1 expression and pathologic differentiation grade (P=0.015) in OSCC patients. The proportion of patients with low Annexin A1 expression was significantly higher amongst those with moderate/poorly differentiated tumor (78/167) compared to those with well differentiated tumor (18/65). Multivariate Cox model analysis showed clinical stage (P=0.001) and Annexin A1 expression (P=0.038) as independent prognostic risk factors. Furthermore, a low Annexin A1 expression level was predictive of longer disease-free survival (P=0.036, HR=0.620) and locoregional recurrence-free survival (P=0.031, HR=0.607) compared to high Annexin A1 expression. Patients with moderate/poorly differentiated tumor and low Annexin A1 expression benefited from TPF induction chemotherapy as measured by distant metastasis-free survival (P=0.048, HR=0.373) as well as overall survival (P=0.078, HR=0.410)., Conclusions: Annexin A1 can be used as a prognostic biomarker for OSCC. Patients with moderate/poorly differentiated OSCC and low Annexin A1 expression can benefit from the addition of TPF induction chemotherapy to surgery and post-operative radiotherapy. Annexin A1 expression can potentially be used as a predictive biomarker to select OSCC patients with moderate/poorly differentiated tumor who may benefit from TPF induction chemotherapy.

Skeletal muscle complaints are a common consequence of cholesterol-lowering therapy. Transverse tubular (T-tubular) vacuolations occur in patients having statin-associated myopathy and, to a lesser extent, in statin-treated patients without myopathy. We have investigated quantitative changes in T-tubular morphology and looked for early indicators of T-tubular membrane repair in skeletal muscle biopsy samples from patients receiving cholesterol-lowering therapy who do not have myopathic side effects. Gene expression and protein levels of incipient membrane repair proteins were monitored in patients who tolerated statin treatment without myopathy and in statin-naive subjects. In addition, morphometry of the T-tubular system was performed. Only the gene expression for annexin A1 was up-regulated, whereas the expression of other repair genes remained unchanged. However, annexin A1 and dysferlin protein levels were significantly increased. In statin-treated patients, the volume fraction of the T-tubular system was significantly increased, but the volume fraction of the sarcoplasmic reticulum remained unchanged. A complex surface structure in combination with high mechanical loads makes skeletal muscle plasma membranes susceptible to injury. Ca(2+)-dependent membrane repair proteins such as dysferlin and annexin A1 are deployed at T-tubular sites. The up-regulation of annexin A1 gene expression and protein points to this protein as a biomarker for T-tubular repair.

Annexin A1 (AnxA1) is an important anti-inflammatory mediator during granulocytic differentiation in all trans-retinoic acid (ATRA) treated acute promyelocytic leukemic (APL) cells. Dexamethasone has been used successfully to prevent complications in ATRA-treated APL patients, although its mechanism of action is still not clear. In the present study, we have examined the effect of dexamethasone on the modulation of AnxA1 in ATRA-APL NB4 (ATRA-NB4) cells, ATRA-NB4 cells-derived microparticles (MPs) and its role during cell-cell interaction between ATRA-NB4 cells and endothelial cells. Our results have shown that dexamethasone can inhibit the percentage of ATRA-NB4 cells expressing surface AnxA1 and its receptor FPR2/ALX in a time-dependent manner based on flow cytometric analysis. However, dexamethasone treatment of ATRA-NB4 cells has no significant effect on the level of AnxA1 mRNA, the total cellular level of AnxA1 protein or the release of AnxA1 from these cells, as determined by RT-PCR, Western blotting, and ELISA, respectively. Further studies demonstrate that dexamethasone is able to significantly inhibit the adhesion of ATRA-NB4 cells to endothelial cells, and this anti-adhesive effect can be inhibited if the cells were pre-treated with a neutralizing antibody specific for AnxA1. Finally, dexamethasone also enhances the release of AnxA1-containing MPs from ATRA-NB4 cells which can in turn prevent the adhesion of the ATRA-NB4 cells to endothelial cells. We conclude that biologically active AnxA1 originating from dexamethasone-treated ATRA-APL cells and their MPs plays an anti-adhesive effect and this contributes to inhibit the adhesion of ATRA-APL cell to endothelial cells., (Copyright © 2012 Wiley Periodicals, Inc.)

Altered functions of the lung epithelial surface likely contribute to the respiratory morbidities in infants with bronchopulmonary dysplasia (BPD). Infants with BPD exhibit decreased expressions of secretoglobins (SCGBs), including Clara cell secretory protein (CCSP). Expression of lung SCGB and annexin A1 (ANXA1) is persistently altered in CCSP knockout mice suggesting that CCSP indirectly influences innate immune responses. The present studies tested the hypothesis that neonatal hyperoxic exposure induces deficits in CCSP expression that are associated with persistent alterations in lung SCGB and ANXA1 expression. Newborn C3H/HeN mice were exposed to room air (RA) or 85% O2 from birth and were sacrificed at 14 d or returned to RA for 14 d. Neonatal hyperoxia followed by RA recovery was associated with decreased lung CCSP and SCGB3A1 protein but not mRNA expression. Hyperoxia-induced alterations in the charge characteristics of ANXA1 were unchanged by RA recovery and were associated with elevated lung macrophage numbers. These findings support a model in which hyperoxia-induced alterations in Clara cell function influence lung innate immune function through effects on immunomodulatory proteins. Studies to determine the mechanism(s) by which CCSP alterations affect SCGBs, ANXA1, and innate immune responses in BPD are warranted.

Elevated cyclooygenase-2 (COX-2) expression is frequently observed in human non-small cell lung cancer (NSCLC) and associated with poor prognosis, indicating critical involvement of the inflammatory pathway in lung carcinogenesis. Recently, we found that green tea extract (GTE) induced Annexin-1 (ANX1) in the lung adenocarcinoma A549 cells. ANX1 is a glucocorticoid-inducible 37kDa protein involved in a wide range biological function and is an important anti-inflammatory mediator. The present study further examines the interplay between the expressions and production of ANX1, COX-2, phospholipase A(2) (cPLA(2)) and prostaglandin E(2) (PGE(2)) following the treatment of NSCLC cell lines with GTE. We found that GTE induced ANX1 and inhibited COX-2 expression in lung cancer A549, H157 and H460 cell lines. Addition of pro-inflammatory cytokine IL-1β diminished GTE-induced ANX1. Silence of ANX1 in cells abrogates the inhibitory activity on COX-2, indicating that the anti-inflammatory activity of GTE is mediated at least partially by the up-regulation of ANX1. However, differential pattern of inhibitory effects of ANX1 on cPLA(2) expression was observed among various cell types, suggesting that the anti-inflammatory activity mediated by ANX1 is cell type specific. Our study may provide a new mechanism of GTE on the prevention of lung cancer and other diseases related to inflammation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In aged humans, stroke is a major cause of disability for which no neuroprotective measures are available. In animal studies of focal ischemia, short-term hypothermia often reduces infarct size. Nevertheless, efficient neuroprotection requires long-term, regulated lowering of whole-body temperature. Previously, we reported that post-stroke exposure to hydrogen sulfide (H(2)S) effectively lowers whole-body temperature and confers neuroprotection in aged animals. In the present study using magnetic resonance imaging, electroencephalogram recording, DNA arrays, reverse transcriptase polymerase chain reaction, western blotting and immunofluorescence, we characterized the central nervous system response to H(2)S-induced hypothermia and report, for the first time, that annexin A1, a major pro-inflammatory protein that is upregulated after stroke, was consistently downregulated in polymorphonuclear cells in the peri-lesional cortex of post-ischemic, aged rat brain after 48 hours of hypothermia induced by exposure to H(2)S. Our data suggest that long-term hypothermia may be a viable clinical approach to protecting the aged brain from cerebral injury. Our findings further suggest that, in contrast to monotherapies that have thus far uniformly failed in clinical practice, hypothermia has pleiotropic effects on brain physiology that may be necessary for effective protection of the brain after stroke.

Angiogenesis and inflammation are important therapeutic targets in non-small cell lung cancer (NSCLC). It is well known that proteolysis mediated by matrix metalloproteinases (MMPs) promotes angiogenesis and inflammation in the tumor microenvironment. Here, the effects of the MMP inhibitor TIMP-2 on NSCLC inflammation and angiogenesis were evaluated in TIMP-2-deficient (timp2-/-) mice injected subcutaneously (SC) with Lewis lung carcinoma cells and compared with the effects on tumors in wild-type mice. TIMP-2-deficient mice demonstrated increased tumor growth, enhanced expression of angiogenic marker αvβ3 in tumor and endothelial cells, and significantly higher serum vascular endothelial growth factor-A levels. Tumor-bearing timp2-/- mice showed a significant number of inflammatory cells in their tumors, upregulation of inflammation mediators, nuclear factor-kappaB, and Annexin A1, as well as higher levels of serum interleukin (IL)-6. Phenotypic analysis revealed an increase in myeloid-derived suppressor cell (MDSC) cells (CD11b+ and Gr-1+) that coexpressed vascular-endothelial-growth factor receptor 1 (VEGF-R1) and elevated MMP activation present in tumors and spleens from timp2-/- mice. Furthermore, TIMP-2-deficient tumors upregulated expression of the immunosuppressing genes controlling MDSC growth, IL-10, IL-13, IL-11, and chemokine ligand (CCL-5/RANTES), and decreased interferon-γ and increased CD40L. Moreover, forced TIMP-2 expression in human lung adenocarcinoma A-549 resulted in a significant reduction of MDSCs recruited into tumors, as well as suppression of angiogenesis and tumor growth. The increase in MDSCs has been linked to cancer immunosuppression and angiogenesis. Therefore, this study supports TIMP-2 as a negative regulator of MDSCs with important implications for the immunotherapy and/or antiangiogenic treatment of NSCLC.

Our previous work has demonstrated that the cellular phenotype changes of human pulmonary artery smooth muscle cells (PASMCs) play an important role during pulmonary vascular remodelling. However, little is known about the role of PASMCs phenotype modulation in the course of hypoxia-induced migration and its behind molecular mechanisms. In this study, we have shown that cGMP-dependent protein kinase (PKG) Iα transfection significantly attenuated the hypoxia-induced down-regulation of the expressions of SM-α-actin, MHC and calponin. Hypoxia-induced PASMC migration was also suppressed by PKGIα overexpression. Furthermore, this overexpression attenuated ANX A1 upregulation under hypoxic conditions. All those effects were reversed by a PKG inhibitor KT5823. Our data indicate that manipulating upstream entity e.g., PKGIa, may have a potential therapeutic value to prevent hypoxia-associated pulmonary arterial remodeling for pulmonary hypertension development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical production at a higher concentration. In the present study, we have further characterised the effects of Ac2-26 on equine neutrophil function. In addition, as anti-inflammatory glucocorticoids are known to up-regulate annexin-1, we have examined the effects of dexamethasone on annexin-1 expression in equine leukocytes. The effects of Ac2-26 alone and on agonist (CXCL8, leukotriene (LT)B(4) and PAF)-induced adherence and migration were examined by measuring adhesion of neutrophils to serum-coated plastic and by use of a ChemoTx migration assay. The role of formyl peptide receptors (FPRs) in mediating the effects of Ac2-26 was examined using the pan-FPR antagonist, BOC-2. Flow cytometry was used to measure the effects of dexamethasone on annexin-1 expression. Pre-incubation with Ac2-26 (10(-5)M) significantly inhibited neutrophil adhesion and migration in response to other agonists but when used alone could also induce these responses. The stimulatory and inhibitory effects of Ac2-26 were reduced by BOC-2, indicating a dependency on FPR activation. Dexamethasone increased the percentage of annexin-1 positive neutrophils and mononuclear cells by 1h post treatment (from 45±5% to 93±1% and 62±14% to 87±9% for neutrophils and monocytes, respectively) but by 4h there was no difference from control cells. No difference was seen between the percentages of annexin-1 positive cells pre- and post-treatment in animals that had undergone a dexamethasone suppression test. The attenuation of agonist-induced adherence and migration by Ac2-26 may play a part in regulating recruitment of equine neutrophils in inflammatory conditions of the horse. However, if high concentrations are produced in vivo following release of annexin-1 from activated cells, direct stimulatory effects may occur which could be either beneficial or detrimental. The therapeutic efficacy of anti-inflammatory steroids in the horse may be mediated in part by increasing annexin-1 expression although this effect appears to be short-lived., (Copyright © 2011 Elsevier B.V. All rights reserved.)

One challenge in understanding the polygenic disease of hypertension is elucidating the genes involved and defining responses to environmental factors. Many studies focus on animal models of hypertension; however, this does not necessarily extrapolate to humans. Current technology and cost limitations are prohibitive in fully evaluating hypertension within humans. Thus, we have designed a single-array platform that allows direct comparison of genes relevant to hypertension in animal models and non-human primates/human hypertension. The custom array is targeted to 328 genes known to be potentially related to blood pressure control. Studies compared gene expression in the kidney from normotensive rats and baboons. We found 74 genes expressed in both the rat and baboon kidney, 41 genes expressed in the rat kidney that were not detected in the baboon kidney and 34 genes expressed in the baboon kidney that were not detected in the rat kidney. To begin the evaluation of the array in a pathological condition, kidney gene expression was compared between the salt-sensitive deoxycorticosterone acetate (DOCA) rat model of hypertension and sham animals. Gene expression in the renal cortex and medulla from hypertensive DOCA compared with sham rats revealed three genes differentially expressed in the renal cortex: annexin A1 (up-regulated; relative intensity: 1.316 ± 0.321 versus 2.312 ± 0.283), glutamate-cysteine ligase (down-regulated; relative intensity: 3.738 ± 0.174 versus 2.645 ± 0.364) and glutathione-S transferase (down-regulated; relative intensity: 5.572 ± 0.246 versus 4.215 ± 0.411) and 21 genes differentially expressed in the renal medulla. Interestingly, few genes were differentially expressed in the kidney in the DOCA-salt model of hypertension; this may suggest that the complexity of hypertension may be the result of only a few gene-by-environment responsive events.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), one of secondary metabolites of low molecular weight present in plant, has various important biological effects. It can induce apoptosis in human leukemia cell types in vitro, although the mechanism is not fully understood. In the present study, we demonstrated reduced viability and DNA synthesis, as well as increased proportion of the subdiploid cell population, in HL-60 cells as determined by cell cycle analysis with resveratrol. Resveratrol treatment resulted in a gradual time-dependent decrease in the expression of anti-apoptotic Bcl-2 and increase in that of Bax, annexin A1, growth arrest- and DNA damage-induced gene 45α (GADD45α), and cleaved caspase-3. In addition, resveratrol markedly increased caspase-3 activity in cells. Our results suggest that resveratrol could inhibit the proliferation and induce apoptosis of HL-60 cells through a GADD45α and annexin A1/caspase-3 pathway., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Metastasis is the most common cause of death in lung cancer patients and is a major obstacle to the successful treatment. To discover novel metastasis-related proteins in lung adenorcinoma (AdC), quantitative proteomic analysis was performed between primary lung AdC tissues with (LNM AdC) and without lymph node metastasis (non-LNM AdC). In this study, annexin A1 was identified to be significantly up-regulated in LNM AdC compared with non-LNM AdC. Immunohistochemistry showed that annexin A1 over-expression was frequently observed in LNM AdCs and matched lymph node metastases compared with non-LNM AdCs. Annexin A1 over-expression was significantly associated with advanced clinical stage (P < 0.05) and lymph node metastasis (P < 0.05) and increased relapse rate (P < 0.05) and decreased overall survival (P < 0.05) in lung AdCs. Cox regression analysis indicated annexin A1 over-expression was an independent prognostic factor. Furthermore, suppression of annexin A1 expression by siRNA interference significantly inhibited the invasion ability of lung adenocarcinoma cell A549 in vitro. In conclusion, annexin A1 expression correlated with tumor stage, lymph node metastasis, relapse, and patient survival. Annexin A1 is proposed to function importantly in the progression of lung AdC.

Oropharyngeal candidiasis (OPC), caused primarily by Candida albicans, is the most common oral infection in HIV(+) persons. Although Th1-type CD4(+) T cells are the predominant host defense mechanism against OPC, CD8(+) T cells and epithelial cells become important when blood CD4(+) T cells are reduced below a protective threshold during progression to AIDS. In an early cross-sectional study, OPC(+) tissue biopsied from HIV(+) persons had an accumulation of activated memory CD8(+) T cells at the oral epithelial-lamina propria interface, with reduced expression of the adhesion molecule E-cadherin, suggesting a protective role for CD8(+) T cells but a dysfunction in the mucosal migration of the cells. In a subsequent 1-year longitudinal study, OPC(-) patients with high oral Candida colonization (indicative of a preclinical OPC condition), had higher numbers of CD8(+) T cells distributed throughout the tissue, with normal E-cadherin expression. In OPC(+) patients, where lack of CD8(+) T cell migration was associated with reduced E-cadherin, subsequent evaluations following successful treatment of infection revealed normal E-cadherin expression and cellular distribution. Regarding epithelial cell responses, intact oral epithelial cells exhibit fungistatic activity via an acid-labile protein moiety. A proteomic analysis revealed that annexin A1 is a strong candidate for the effector moiety. The current hypothesis is that under reduced CD4(+) T cells, HIV(+) persons protected from OPC have CD8(+) T cells that migrate to the site of a preclinical infection under normal expression of E-cadherin, whereas those with OPC have a transient reduction in E-cadherin that prohibits CD8(+) T cells from migrating for effector function. Oral epithelial cells concomitantly function through annexin A1 to keep Candida in a commensal state but can easily be overwhelmed, thereby contributing to susceptibility to OPC.

Annexins are a structurally related family of calcium- and phospholipid-binding proteins that have been implicated in a broad range of molecular and cellular processes. The altered expression of individual annexins has been implicated in tumor development and progression. In this study the expression of annexin A1 and annexin A2 was studied by immunohistochemistry in intestinal-type sinonasal adenocarcinoma using a study set of 57 intestinal-type sinonasal adenocarcinomas represented on an intestinal-type sinonasal adenocarcinoma tissue microarray to assess its potential role in the pathogenesis of these tumors. Our results showed that annexin A1 expression was consistently lost in 52 (91%) intestinal-type sinonasal adenocarcinomas, as compared with the normal epithelium. The expression of annexin A2 was more heterogeneous, and only 19 (33%) cases showed annexin A2 negative expression. Annexin A2 expression was correlated with the histopathological type, being lower in the mucinous type (P = .022). The loss of annexin A2 expression correlated with a reduced survival in univariate analysis (P = .004). However, the impact of annexin A2 expression on patient survival could be an indirect consequence of its association with the histopathological type, since annexin A2 expression was not found to be an independent predictor in multivariate analyses. These results suggest that annexin A1 expression is frequently and commonly lost in intestinal-type sinonasal adenocarcinoma development. Annexin A2 expression is also reduced in intestinal-type sinonasal adenocarcinoma, and this loss of expression is associated to the more aggressive histopathological types., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent studies have associated the dysregulated expression of Annexin-A1/Formyl peptide receptor 2 (FPR2/ALX) system with the development of autoimmune diseases. In this study we systematically scanned human leukocyte subsets for the presence of this pathway aiming to provide a roadmap that will help investigators to explore possible links between the development of immune related disorders and the expression of this system. Our results show that neutrophils, monocytes and NK cells express higher levels of both AnxA1 and FPR2/ALX compared to T or B cells. Further analysis of specific T cell subsets revealed higher levels in activated CD25(+) and memory CD45RO CD4 T cells compared to resting CD25(-) or naïve CD45RA CD4 T cells. Together the results expand our knowledge of the AnxA1-FPR2/ALX system in immune cells and provide new avenues for investigation into the functions of this signalling pathway in systems other than that classically described for neutrophils., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Inflammatory response is a kind of nonspecific immune response, with the central link of vascular response, which is mainly manifested by changes in neutrophils and vascular endothelial cells. In recent years, the in vivo and in vitro role of intravenous anesthetic propofol in inhibiting inflammatory response has been attracting more and more attention, but the anti-inflammatory mechanisms of propofol for mononuclear cells still remain undefined. In this study, proteomics analysis was applied to investigate protein expression profile changes in serum mononuclear cells following intervention of rats with endotoxemia using propofol. After two-dimensional electrophoresis and mass spectrometric identification, it has been found that the protein Annexin A1 was up-regulated in the propofol intervention group. Annexin A1 is a glucocorticoid-dependent anti-inflammatory protein. After detection using ELISA and Western blot assays, it has also been found that propofol can not only promote the expression of Annexin A1, but also inhibit the phosphorylation level of p38 and release of inflammatory factors (IL-1β, IL-6 and TNF-α) in rats with endotoxemia. In order to further determine the role of up-regulated expression of Annexin A1 in anti-inflammation of propofol, this gene was silenced in vitro in human THP-1 cells, to detect the phosphorylation status of p38 and release of inflammatory factors. The results show that Annexin A1 can negatively regulate phosphorylation of p38 and release of IL-1β, IL-6 and TNF-α in THP-1 cells following propofol intervention and lipopolysaccharide (LPS) stimulation. Our results clearly indicate that propofol can up-regulate Annexin A1 to inhibit the phosphorylation level of p38 and release of IL-1β, IL-6 and TNF-α, so as to inhibit inflammatory response. Therefore, it can be speculated that Annexin A1 might be the key signaling protein in the in vivo and in vitro anti-inflammatory mechanisms of propofol.

Metastasis is the major cause of carcinoma-induced death, but mechanisms involved are poorly understood. Metastasis crucially involves epithelial-to-mesenchymal transition (EMT), causing loss of epithelial polarity. Here we identify Annexin A1 (AnxA1), a protein with important functions in intracellular vesicle trafficking, as an efficient suppressor of EMT and metastasis in breast cancer. AnxA1 levels were strongly reduced in EMT of mammary epithelial cells, in metastatic murine and human cell lines and in metastatic mouse and human carcinomas. RNAi-mediated AnxA1 knockdown cooperated with oncogenic Ras to induce TGFβ-independent EMT and metastasis in non-metastatic cells. Strikingly, forced AnxA1 expression in metastatic mouse and human mammary carcinoma cells reversed EMT and abolished metastasis. AnxA1 knockdown stimulated multiple signalling pathways but only Tyk2/Stat3 and Erk1/2 signalling were essential for EMT.

Background and Objective: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium., Material and Methods: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry., Results: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium., Conclusion: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium., ((c) 2010 John Wiley & Sons A/S.)

Background: The aim of this study is to identify the differentially expressed proteins in circulating polymorphonuclear neutrophils (PMN) from scalded bacteremia rabbits infected with Staphylococcus aureus to provide a basis to reveal the pathogenesis of burns and sepsis., Methods: Rabbits were subjected to sham burn (A), A + bacterial challenge (B), 30% scald injury (C), or C + bacterial challenge (D). Bacterial challenge was inflicted as an injection of 2.0 x 10(8) cfu S. aureus 18 h after burn procedure. Animals were sacrificed 24 h after burn. PMN were isolated, and the differential proteins in the PMN from these animals were identified by two-dimensional electrophoresis coupled with MALDI-TOF-MS; two proteins were confirmed by Western blotting., Results: Twenty-one differential protein spots were found, and seven differential proteins were identified. Among the identified proteins, the expression levels of protein disulfide-isomerase and thiol-specific antioxidant protein were down-regulated in groups C and D, and two protein spots of annexin I were identified, one of which was down-regulated and another up-regulated in groups C and D., Conclusions: Preliminary proteome changes in PMN from rabbits experiencing scald injury and S. aureus sepsis were revealed, which possibly play an important role in the inflammation and pathogenesis of sepsis after scald injury.

Annexin A1 (ANXA1), a mediator of the anti-inflammatory action of glucocorticoids, is important in cancer development and progression, whereas NF-kappaB regulates multiple cellular phenomena, some of them associated with inflammation and cancer. We showed that glucocorticoids and chemopreventive modified nonsteroidal anti-inflammatory drugs, such as nitric oxide-donating aspirin (NO-ASA) and phospho-aspirin, induced ANXA1 in cultured human colon and pancreatic cancer cells. ANXA1 associated with NF-kappaB and suppressed its transcriptional activity by preventing NF-kappaB binding to DNA. The induction of ANXA1 by glucocorticoids was proportional to their anti-inflammatory potency, as was the suppression of NF-kappaB activity, which was accompanied by enhanced apoptosis and inhibition of cell growth mediated by changes in NF-kappaB-dependent cell signaling. The proposed novel mechanism was operational in the intestinal mucosa of mice treated with dexamethasone or NO-ASA. ANXA1-based oligopeptides displayed the same effects as ANXA1 on NF-kappaB. One such tripeptide (Gln-Ala-Trp) administered to nude mice inhibited the growth of SW480 human colon cancer xenografts by 58% compared with control (P < 0.01). Our findings reveal that ANXA1 is an inducible endogenous inhibitor of NF-kappaB in human cancer cells and mice, provide a novel molecular mechanism for the action of anti-inflammatory agents, and suggest the possibility of mechanism-driven drug development.

The systemic inflammatory activity in patients with stable coronary artery disease (CAD) is associated with a dysregulated cortisol response. Moreover, an aberrant activation status of neutrophils in CAD has been discussed; and the question of glucocorticoid resistance has been raised. The anti-inflammatory actions of glucocorticoids are mediated by annexin-1 (ANXA1). We investigated the expression of glucocorticoid receptors (GR) and ANXA1, as well as the exogenous effects of ANXA1 on neutrophils in CAD patients and related the data to diurnal salivary cortisol. Salivary cortisol levels were measured in the morning and evening during 3 consecutive days in 30 CAD patients and 30 healthy individuals. The neutrophil expression of GR and ANXA1 was determined by flow cytometry. The effect of exogenous ANXA1 was determined in a neutrophil stimulation assay. The patients showed a flattened diurnal cortisol pattern compared with healthy subjects, involving higher levels in the evening. The neutrophil expression of GR-total and GR-alpha was decreased, whereas the GR-beta expression did not differ compared with controls. The neutrophil expression of ANXA1 was significantly increased in patients. Ex vivo, ANXA1 impaired the leukotriene B(4)-induced neutrophil production of reactive oxygen species in patients but not in controls. Our findings indicate a persistent overactivation of the hypothalamic-pituitary-adrenal axis in CAD patients but do not give any evidence for glucocorticoid resistance, as assessed by the neutrophil expression of GR and ANXA1. The altered neutrophil phenotype in CAD may thus represent a long-term response to disease-related activation., ((c) 2010 Elsevier Inc. All rights reserved.)

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB96 cells. In this study, comparative proteomic analysis identified that Annexin A1 was one of the significantly down-regulated genes in the cancerous HB96 cells. To investigate Annexin A1 down-regulation and its potential usefulness as a molecular marker in OSCC, we further screened Annexin A1 expressions with a panel of OSCC lines, and clinical samples of cancerous and the paired adjacent normal tissues from primary OSCC patients. By Western blot analysis and real-time PCR, we showed that both Annexin A1 mRNA and protein expressions decreased in OSCC cell lines except in two cell lines for the mRNA levels. Immunohistochemistry and real-time PCR also showed that both Annexin A1 mRNA and protein expressions decreased in the cancerous tissues from OSCC patients compared with those in the paired adjacent non-malignant epithelia. More importantly, both Annexin A1 mRNA and protein expressions negatively correlated with the pathologic differentiation grades of cancerous tissues. The lower Annexin A1 mRNA or protein expressions correlated with the poorer pathologic differentiation grades. These results suggest that decreased expression of Annexin A1 contributes to the cancerous progression of OSCC, and Annexin A1 may be a potential biomarker for pathologic differentiation grade of OSCC.

Background: Annexin A1 (ANXA1) is a potential marker of development of breast cancer. However, previous studies of ANXA1 expression in primary breast carcinoma and lymph node metastasis have yielded conflicting results. Therefore, to accurately characterize the ANXA1 expression pattern, we used microarray analysis and matched patient samples to evaluate progressive alterations in ANXA1 protein expression during malignant transformation and metastasis., Design: We constructed a tissue microarray using 82 pairs of primary breast cancers and lymph node metastases from archival materials. We also identified 21 cases of breast carcinoma for which a single slide contained the entire progression from benign breast tissue, carcinoma in situ, to invasive carcinoma. Immunohistochemical staining for ANXA1 and various prognostic markers was performed., Result: Microarray analysis revealed that ANXA1 expression was lost in 79% of breast carcinomas, and there was no difference in ANXA1 expression between primary breast carcinoma and lymph node metastasis. Most ANXA1-negative tumors were positive for estrogen and progesterone receptors but negative for HER2/neu and epidermal growth factor receptor. In contrast, most ANXA1-positive tumors were negative for estrogen, progesterone, and HER2/neu. In the whole tissue sections, ANXA1 is heterogeneously expressed in benign epithelium and is lost in both in situ carcinoma and invasive carcinoma., Conclusions: The lack of ANXA1 expression in the majority of breast carcinomas and the early loss of ANXA1 expression in in situ carcinoma, which is maintained in both invasive and metastatic tumors, suggests a possible role for ANXA1 in the early events of malignant transformation.

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation -0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation -0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3'-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-8 attract neutrophils into inflammatory sites. During emigration from the blood neutrophils interact with extracellular matrix proteins such as fibronectin. Fibronectin provides beta2-integrin co-stimulation, allowing GM-CSF and IL-8 to activate nuclear factor (NF)-kappaB, an effect that does not occur in suspension. We tested the hypothesis that exposure of mice to fever-like temperatures abrogates neutrophil recruitment and NF-kappaB activation in a mouse model of skin inflammation. Mice that were exposed to 40 degrees C for 1 hour showed strongly reduced GM-CSF- and IL-8-induced neutrophilic skin inflammation. In vitro heat exposure did not interfere with neutrophil adhesion or spreading on fibronectin but strongly inhibited migration toward both cytokines. Using specific inhibitors, we found that PI3-K/Akt was pivotal for neutrophil migration and that heat down-regulated this pathway. Furthermore, neutrophils on fibronectin showed abrogated NF-kappaB activation in response to GM-CSF and IL-8 after heat. In vivo heat exposure of mice followed by ex vivo stimulation of isolated bone marrow neutrophils confirmed these results. Finally, less NF-kappaB activation was seen in the inflammatory lesions of mice exposed to fever-like temperatures as demonstrated by in situ hybridization for IkappaBalpha mRNA. These new findings suggest that heat may have anti-inflammatory effects in neutrophil-dependent inflammation.

We recently demonstrated that arsenic trioxide (ATO) induced apoptosis in human neutrophils and increased de novo protein synthesis. Here, we identified one of these newly synthesized proteins as annexin-1 (AnxA1), a protein recently found to be proapoptotic in neutrophils when added exogenously. AnxA1 was detected at the cell membrane of ATO-induced neutrophils as well as in the supernatants. Using neutrophils harvested from AnxA1 knockout mice, we found that the proapoptotic activity of ATO was similar in neutrophils, regardless of AnxA1 levels. A second protein was identified as heat shock protein (Hsp) 89alpha. Because ATO is known to induce a HS-like response in a variety of cells, we investigated its ability to induce gene expression of Hsp in neutrophils and found that ATO increases HSP90AA1, HSPA1 and HSPB1 mRNA in these cells. We conclude that ATO-induced neutrophil apoptosis by an AnxA1-independent mechanism. Our data provide the first evidence that ATO induces a stress response in human neutrophils and that de novo synthesis of AnxA1 is related to this event rather than to the proapoptotic activity of ATO.

Background: Nerve growth factor (NGF) is a potent mediator, which regulates characteristics of mast cells, but its biological function is not well characterized. This study aimed to screen proteins associated with the maturation of human mast cells-1 (HMC-1) or mouse bone marrow-derived mast cells (BMMCs) cultured with NGF, and to examine the functions of proteins involved., Methods: NGF (10 ng/ml) was added to cell culture medium every other day for 10 days for HMC-1 or twice a week for 5 weeks for BMMCs. Granule formation was determined by electron microscopy or May-Grunwald-Giemsa staining, TNF-alpha by ELISA, expressions of various proteins by two-dimensional gel electrophoresis (2-DE), siRNA transfection by Lipofectamine 2000, and the expressions of pyruvate kinase and annexin I by immunoblotting., Results: After NGF treatment, granule formation and total amounts of granular mediator, TNF-alpha increased in both mast cells. This TNF-alpha was released by calcium ionophore or by antigen/antibody reaction. Expressions of pyruvate kinase and annexin I obtained by 2-DE were confirmed by immunoblotting and siRNA-transfected HMC-1 cells. Expressions of proteins, granule formation and TNF-alpha content were blocked by both the TrkA inhibitor, K252a, and the ERK inhibitor, PD98059, but not by the PI3 kinase inhibitors, LY294002 and wortmannin., Conclusion: These data suggest that pyruvate kinase and annexin I expressed by NGF contribute to granule formation containing TNF-alpha as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway., (Copyright 2008 S. Karger AG, Basel.)

Prolonged cell proliferation in response to irritation by bladder calculi can evoke malignant transformation of the urothelium. However, the molecular mechanisms responsible for calculi-associated bladder carcinogenesis are unknown. We compared the protein expression pattern of rat bladder transitional cell carcinomas (TCCs) induced by terephthalic acid with that of normal bladder tissues using 2-DE. Comparative analysis of the respective spot patterns on 2-DE showed 146 spots that were markedly changed in TCC samples. Subsequently, 56 of the variant protein spots were identified by MALDI-TOF MS. Among them, overexpression of annexin a1 (ANNA1) in rat TCCs was confirmed by Western blotting and real-time RT-PCR analysis. Immunohistochemical staining revealed that ANNA1, usually a cytoplasmic protein in normal urothelium, was translocated to the nucleus in rat bladder cancer cells. In contrast to the animal studies, examination of human clinical specimens showed that ANNA1 expression was reduced in TCC compared to normal urothelium. The expression of ANNA1 was inversely related to the level of differentiation of TCC. Our data suggest that overexpression of ANNA1 is involved in bladder carcinogenesis induced by bladder calculi and that translocation of the protein may be partly responsible for the effect. ANNA1 may serve as a new marker of differentiation for the histopathological grading of human TCC.

We have recently shown that Zap70 is important in retinoid receptor-dependent transactivation in T lymphocytes. We report that Zap70 signaling is also essential in dexamethasone-inducible glucocorticoid receptor (GR)-mediated transactivation in T lymphocytes. Zap70-negative Jurkat T cells and cells reconstituted with inactive Zap70 exhibited attenuated GR-mediated activation as compared with Zap70 reconstituted and wild-type cells. Lck-lacking Jurkat cells were also found to show markedly reduced GR activation, and reconstitution with Lck restored the activation. Gene array and protein analysis showed that the level of annexin 1 (ANXA1), an anti-inflammatory protein known to be induced and released by the glucocorticoid action, was significantly reduced in Zap70-negative and Zap70-inactive Jurkat cells as compared with wild-type cells. Lck-lacking cells were also found to have markedly reduced ANXA1 levels and reconstitution with Lck restored the ANXA1 expression. RNA interference-induced knockdown of Zap70 or Lck in Jurkat cells and peripheral blood T lymphocytes also resulted in the loss of ANXA1 expression. Transcriptional analysis revealed that dexamethasone-inducible GR-mediated activation of ANXA1 promoter was compromised in both Zap70 knocked down peripheral blood T cells and Zap70 or Lck-deficient/Lck-inactive Jurkat cells, indicating an essential role of these kinases in GR-mediated ANXA1 promoter activation in T lymphocytes. To summarize, our data demonstrate an important role for Zap70 signaling in GR-mediated transactivation in T lymphocytes and also point out a crucial role of this kinase in maintaining normal ANXA1 levels in these cells.

The chimeric fusion protein AML1-ETO, created by the t(8;21) translocation, recruits histone deacetylase (HDAC) to AML1-dependent promoters, resulting in transcriptional repression of the target genes. We analyzed the transcriptional changes in t(8;21) Kasumi-1 AML cells in response to the HDAC inhibitors, depsipeptide (FK228) and suberoylanilide hydroxamic acid (SAHA), which induced marked growth inhibition and apoptosis. Using cDNA array, annexin A1 (ANXA1) was identified as one of the FK228-induced genes. Induction of ANXA1 mRNA was associated with histone acetylation in ANXA1 promoter and reversal of the HDAC-dependent suppression of C/EBPalpha by AML1-ETO with direct recruitment of C/EBPalpha to ANXA1 promoter. This led to increase in the N-terminal cleaved isoform of ANXA1 protein and accumulation of ANXA1 on cell membrane. Neutralization with anti-ANXA1 antibody or gene silencing with ANXA1 siRNA inhibited FK228-induced apoptosis, suggesting that the upregulation of endogenous ANXA1 promotes cell death. FK228-induced ANXA1 expression was associated with massive increase in cell attachment and engulfment of Kasumi-1 cells by human THP-1-derived macrophages, which was completely abrogated with ANXA1 knockdown via siRNA transfection or ANXA1 neutralization. These findings identify a novel mechanism of action of HDAC inhibitors, which induce the expression and externalization of ANXA1 in leukemic cells, which in turn mediates the phagocytic clearance of apoptotic cells by macrophages.

We tested whether glucocorticoids modulated osteoblast expression of the annexin 1 system, including the ligand and two G-coupled receptors termed formyl-peptide receptor (FPR) and FPR-like-1 (FPRL-1). In Saos-2 cells, rapid up-regulation of FPR mRNA upon cell incubation with dexamethasone (0.01-1 microM) was observed, with significant changes as early as 2h and a more marked response at 24h; annexin 1 and FPRL-1 mRNA changes were more subtle. At the protein level, dexamethasone provoked a rapid externalization of annexin 1 (maximal at 2h) followed by delayed time-dependent changes in the cell cytosol. Saos-2 cell surface expression of FPR or FPRL-1 could not be detected, even when dexamethasone was added with the bone modelling cytokines interleukin-6 or interleukin-1. The uneven modulation of the annexin 1 system (mediator and its putative receptors) in osteoblasts might lead to a better understanding of how these complex biochemical pathways become operative in bone.