Annexin A1 Levels Research Articles

ANXA1Ac2–26 peptide, a possible therapeutic approach in inflammatory ocular diseases

The eye is immunologically privileged when inflammatory responses are suppressed. One component responsible for the suppression of inflammatory responses is the blood retinal barrier, which comprises the retinal pigment epithelium. The destruction of this barrier initiates inflammation, which can affect any part of the eye. Therefore, inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1) protein acts as a modulator of inflammation. In this study we aimed to improve the knowledge of this area by investigating how a peptide of the ANXA1 protein (ANXA1Ac2–26) modulates the morphology, proliferation, migration and expression of genes and proteins in human retinal pigment epithelium cells (ARPE-19). Determining how signaling pathways (NF-κB and UBC) are modulated by the ANXA1Ac2–26 peptide could be important for understanding the inflammatory process. ARPE-19 cells were activated by bacterial lipopolysaccharide endotoxin (LPS) and treated with ANXA1Ac2–26 peptide, in a concentration of 1.7μM and 33.8μM. We observed that the LPS activation diminished the levels of endogenous ANXA1 after 2h and 24h and ANXA1Ac2–26 peptide decreased the proliferation and re-establishes the migration of ARPE-19 cells. After using a hybridization approach, 80 differentially expressed genes were found. Five of these genes were selected (LRAT, CTGF, MAP1B, ALDH1A3 and SETD7) and all were down-regulated after treatment with the peptide. The genes CTGF and LRAT would be considered as potential molecular markers of ophthalmologic inflammation. The expression of pro-inflammatory cytokines was also decreased after the treatment, indicating the efficiency of the anti-inflammatory peptide at high concentrations, since the reduction in the levels of these mediators were observed after the treatment with ANXA1Ac2–26 peptide at 33.8μM. Our results suggest that the retinal pigment epithelial cells are a potential target of the ANXA1 protein and point to possible applications of the ANXA1Ac2–26 peptide as an innovative therapy for the treatment of ocular inflammation.

Effect of Annexin A1 gene on the proliferation and invasion of esophageal squamous cell carcinoma cells and its regulatory mechanisms

The aim of this study was to examine the effect of Annexin A1 (ANXA1) on the proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC) cells and its possible mechanisms of action. After constructing the ANXA1 overexpression plasmid, we transfected this plasmid and/or microRNA (miRNA)-196a mimic into ESCC cells (Eca109 cell line). Methyl thiazolyl tetrazolium (MTT) assay and Transwell chamber assay were performed to determine cell proliferation, migration and invasion, respectively. Western blot analysis was used to examine the protein expression levels of ANXA1, Snail and E-cadherin. RT-PCR was used to detect the expression of miRNA-196a. Our results revealed that ANXA1 expression was upregulated in the cells transfected with the ANXA1 overexpression plasmid, and cell proliferation, migration and invasion were significantly increased (p=0.004, p<0.001 and p=0.011, respectively). In the cells transfected with the miRNA-196a mimic, miRNA-196a expression was significantly upregulated (p<0.001). However, miRNA-196a expression was downregulated in the cells transfected with the ANXA1 overexpression plasmid. In addition, in the cells transfected with the miRNA-196a mimic, cell proliferation, migration and invasion were significantly decreased (p=0.027, p=0.009 and p=0.021, respectively). In the cells transfected with the ANXA1 overexpression plasmid, the expression of Snail was upregulated and that of E-cadherin was downregulated. However, the opposite was observed in the cells transfected with the miRNA-196a mimic. Our findings thus demonstrate that ANXA1 promotes the proliferation of Eca109 cells, and increases the expression of Snail, whereas it inhibits that of E-cadherin, thus enhancing the migration and invasion of ESCC cells. miRNA-196a negatively regulates the expression of ANXA1, thereby inhibiting the proliferation, invasion and metastasis of ESCC cells.

Caffeine prevents kidney stone formation by translocation of apical surface annexin A1 crystal-binding protein into cytoplasm: In vitro evidence.

Recent large 3 cohorts have shown that caffeinated beverage consumption was associated with lower risk of kidney stone disease. However, its protective mechanisms remained unknown and had not been previously investigated. We thus evaluated protective effects of caffeine (1 μM–10 mM) on calcium oxalate monohydrate (COM) kidney stone formation, using crystallization, crystal growth, cell-crystal adhesion, Western blotting, and immunofluorescence assays. The results showed that caffeine reduced crystal number but, on the other hand, increased crystal size, resulting in unchanged crystal mass, consistent with crystal growth that was not affected by caffeine. However, caffeine significantly decreased crystal-binding capacity of MDCK renal tubular cells in a dose-dependent manner. Western blotting and immunofluorescence study of COM crystal-binding proteins revealed significantly decreased level of annexin A1 on apical surface and its translocation into cytoplasm of the caffeine-treated cells, but no significant changes in other COM crystal-binding proteins (annexin A2, α-enolase, HSP70, and HSP90) were observed. Moreover, caffeine decreased intracellular [Ca2+] but increased [Ca2+] secretory index. Taken together, our findings showed an in vitro evidence of the protective mechanism of caffeine against kidney stone formation via translocation of annexin A1 from apical surface into cytoplasm to reduce the crystal-binding capacity of renal tubular epithelial cells.

Serum Annexin A2 Level Is Associated With Diagnosis and Prognosis in Patients With Oral Squamous Cell Carcinoma

In several human cancer types, serum annexin A2 levels are increased, but little is known regarding oral squamous cell carcinoma (OSCC). This study aimed to measure serum annexin A2 levels in OSCC patients and assess the association with diagnosis and prognosis. This case-control study compared serum annexin A2 concentrations in a group of OSCC patients and a control group. The predictor variable was the presence or absence of OSCC, and the outcome variable was the level of serum annexin A2. Annexin A2 concentrations were measured with an enzyme-linked immunosorbent assay, and correlations with clinicopathologic characteristics of OSCC were further evaluated. Receiver operating characteristic (ROC) curves, Kaplan-Meier curves, log-rank analyses, and a Cox proportional hazards model were used to evaluate the diagnostic and prognostic value of annexin A2. Serum samples were taken from 399 individuals: 126 patients with OSCC (aged 62.7 ± 10.6years, 79 men and 47 women); 115 patients with benign oral disease (aged 63.9 ± 10.8years, 73 men and 42 women); and 158 healthy controls (aged 65.4 ± 12.8years, 92 men and 66 women). The annexin A2 level was significantly higher in OSCC patients than in patients with benign disease and controls (27.1 ± 9.81ng/mL vs 15.9 ± 6.97ng/mL and 15.0 ± 6.69ng/mL, respectively). To distinguish OSCC patients from the other 2 groups, ROC curve-area under the ROC curve (AUC) analysis for serum annexin A2 levels provided an AUC of 0.80 (sensitivity, 0.62; specificity, 0.87) and an AUC of 0.77 (sensitivity, 0.57; specificity, 0.89). Furthermore, OSCC patients with high annexin A2 levels had poorer overall survival. This study suggested that an elevated serum annexin A2 level might be a novel diagnostic and prognostic biomarker for OSCC patients.

Inflammation in Sickle Cell Disease: Differential and Down-Expressed Plasma Levels of Annexin A1 Protein.

Sickle cell disease (SCD) is an inherited hemolytic anemia whose pathophysiology is driven by polymerization of the hemoglobin S (Hb S), leading to hemolysis and vaso-occlusive events. Inflammation is a fundamental component in these processes and a continuous inflammatory stimulus can lead to tissue damages. Thus, pro-resolving pathways emerge in order to restore the homeostasis. For example there is the annexin A1 (ANXA1), an endogenous anti-inflammatory protein involved in reducing neutrophil-endothelial interactions, accelerating neutrophil apoptosis and stimulating macrophage efferocytosis. We investigated the expression of ANXA1 in plasma of SCD patients and its relation with anemic, hemolytic and inflammatory parameters of the disease. Three SCD genotypes were considered: the homozygous inheritance for Hb S (Hb SS) and the association between Hb S and the hemoglobin variants D-Punjab (Hb SD) and C (Hb SC). ANXA1 and proinflammatory cytokines were quantified by ELISA in plasma of SCD patients and control individuals without hemoglobinopathies. Hematological and biochemical parameters were analyzed by flow cytometry and spectrophotometer. The plasma levels of ANXA1 were about three-fold lesser in SCD patients compared to the control group, and within the SCD genotypes the most elevated levels were found in Hb SS individuals (approximately three-fold higher). Proinflammatory cytokines were higher in SCD groups than in the control individuals. Anemic and hemolytic markers were higher in Hb SS and Hb SD genotypes compared to Hb SC patients. White blood cells and platelets count were higher in Hb SS genotype and were positively correlated to ANXA1 levels. We found that ANXA1 is down-regulated and differentially expressed within the SCD genotypes. Its expression seems to depend on the inflammatory, hemolytic and vaso-occlusive characteristics of the diseased. These data may lead to new biological targets for therapeutic intervention in SCD.

Evaluation of CRRT effects on pyemic secondary AKI by serum cartilage glycoprotein 39 and Annexin A1.

The aim of the present study was to examine the effects of continuous renal replacement therapy (CRRT) on pyemic secondary acute kidney injury (AKI) by serum cartilage glycoprotein 39 (YKL-40) and Annexin A1. From October, 2013 to October, 2015, 45 pyemic secondary AKI cases and 40 pyemic non-secondary AKI cases were selected for the present study. There were also 35 cases of physical examination volunteers. The serum YKL-40 and Annexin A1 levels were compared. CRRT was applied to pyemic secondary AKI patients and based on the obtained results the patients were divided into the success and failure groups. YKL-40, Annexin A1, hs-CRP, creatinine and urea nitrogen levels after 1, 6, 12, 24, 48 and 72 h of AKI were measured. The YKL-40 and Annexin A1 levels in the pyemic secondary AKI group were significantly higher than those in other two groups and differences were statistically significant (P<0.05). There was no statistically significant difference regarding time period for applying CRRT in the success and failure groups (P>0.05). The peak level of YKL-40 and Annexin A1 in the success group decreased more rapidly compared to the failure group and the difference was statistically significant (P<0.05). When the differences in creatinine and urea nitrogen levels at different time points were compared between the success and failure groups, no statistical significance was observed (P>0.05). However, the success group showed a significantly lower level compared to the failure group at 72 h. Comparisons for other time periods showed no statistical significance (P>0.05). Thus, the serum cartilage glycoprotein 39 and Annexin A1 level were able to predict the clinical effects of CRRT on pyemic secondary AKI.

Concomitant measurements of serum annexin A5 levels and hematological indices as markers in recent and old myocardial infarction with low ejection fraction: A preliminary study

BackgroundSerum annexin A5 (anxA5) level is significantly increased in patients with acute coronary syndrome. Hematological indices are significantly increased in patients with ischemic heart disease. This study aimed to demonstrate the changes in the distribution of blood cells and the levels of anxA5 in patients presented with significant low ejection fraction ST-elevation acute myocardial infarction (STEMI) in comparison with corresponding patients with ischemic heart disease. MethodsPatients with low ejection fraction presenting at Hospital of Diyala University of Iraq were enrolled. Electrocardiograph (ECG), echocardiograph, hematological indices, serum annexin V (anxV) levels and the determinants of the cardio-metabolic risk factors were performed. Based on clinical examination, ECG findings and laboratory tests, patients were divided into healthy subjects (n=20); patients with acute MI (n=40) and with chronic MI (n=12). ResultsAcute MI has significant high levels of serum triglyceride, uric acid and high mean value of red cell distribution width (RDW) compared with healthy subjects and chronic MI. Platelet distribution width (PDW) is significantly reduced in patients of acute MI and chronic MI compared with healthy subjects, whereas the plateletcrit (PCT) is significantly higher in acute MI compared with healthy subjects. There is an insignificant difference between the means of serum anxA5 levels of acute MI (35.6±7.2ng/ml) and chronic MI (32.4±8.9ng/ml), but significantly higher than the cutoff level of the healthy subjects (5ng/ml). ConclusionsMeasurement of serum annexin level is a useful diagnostic marker of acute or chronic MI with low ejection fraction.

High Podocalyxin levels promote cell viability partially through up-regulation of Annexin A2

Podocalyxin (PODXL) is a highly glycosylated and sialylated transmembrane protein that is up-regulated in various types of tumors and whose expression levels positively correlate with tumor grade. We previously found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells (NSPs). Here we investigated the effects of elevated Podxl levels in these cells. NSPs overexpressing Podxl did not form brain tumors upon intracranial transplantations, indicating that high levels of this gene alone are not sufficient for tumor initiation. However, Podxl overexpression had a positive effect on cell number, sphere formation and cell viability, indicating that it might in this way contribute to the development and/or maintenance of tumors. Proteome analyses of Podxl-overexpressing and control NSPs revealed increased levels of Annexin A2 (ANXA2). We also found increased transcript levels, indicating that PODXL stimulates expression of the Anxa2 gene. Lack of Anxa2 in Podxl-overexpressing NSPs resulted in reduced viability of these cells, suggesting that PODXL-mediated pro-survival effects can at least in part be explained by increased ANXA2 levels. Finally, our data indicate that Podxl overexpression activates the MAP kinase (MAPK) pathway which in turn up-regulates Anxa2 expression. Our data indicate a novel molecular connection between PODXL and ANXA2: both exert pro-survival effects in NSPs, and PODXL positively regulates ANXA2 expression through the MAPK pathway.

Annexin A5 promotes macrophage activation and contributes to pulmonary fibrosis induced by silica particles.

To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis. Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 μg/ml for 6 or 12 h, the expression levels of transforming growth factor β1 (TGF-β1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-β1 secretion into the cell culture medium was measured by ELISA assays or Western blots. Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-β1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-β1, IL-1α, and FasL downstream target molecules. These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis.

Functional characterization of annexin A5 gene promoter allelic variants

BackgroundM1 and M2 haplotypes are defined by 4 consecutive allelic variants in regulatory regions of the annexin A5 gene and have been found to reduce promoter activity. To date, no research has been carried out to investigate differential and individual impact each of the allelic variants has on promoter activity. In the current study, we functionally characterized the M1 and M2 haplotype allelic variants (c.-467G>A, c.-448A>C, c.-422T>C, c.-373G>A). We also characterized two other allelic variants located in the same regulatory region (c.-628C>T, c.-302T>G). Materials and methodsTheir impact on the ANXA5 promoter activity was examined using a luciferase reporter assay in BeWo cells. Electrophoretic mobility shift assay with probes centered around each polymorphism was used to examine the binding ability of the allelic variants to nuclear proteins from BeWo cells. ResultsOnly the c.-467G>A and c.-628C>T allelic variants influenced the activity of the ANXA5 promoter, as measured by luciferase activity. Differential specific interactions with nuclear proteins were obtained for all allelic variants, except for the c.-302T>G, indicating that these polymorphisms could have an impact on the ANXA5 expression. ConclusionsWe have functionally characterized allelic variants in the ANXA5 promoter, both alone and in combinations, and the results suggest that combinations of several individual variants contribute to modulate the ANXA5 transcriptional activity, most likely through binding of nuclear factors. These results provide new knowledge and insight into the mechanisms underlying the regulation of annexin A5 levels in healthy controls.

Annexin A2 and S100A10 are independent predictors of serous ovarian cancer outcome

Annexin A2, a calcium phospholipid binding protein, has been shown to play an important role in ovarian cancer metastasis. This study examined whether annexin A2 and S100A10 can be used as prognostic markers in serous ovarian cancer. ANXA2 and S100A10 gene expressions were assessed in publicly available ovarian cancer data sets and annexin A2 and S100A10 protein expressions were assessed by immunohistochemistry in a uniform cohort of stage III serous ovarian cancers (n = 109). Kaplan-Meier and Cox regression analyses were performed to assess the relationship between annexin A2 or S100A10 messenger RNA (mRNA) and protein expressions with clinical outcome. High ANXA2 mRNA levels in stage III serous ovarian cancers were associated with reduced progression-free survival (PFS; P = 0.023) and overall survival (OS; P = 0.0038), whereas high S100A10 mRNA levels predicted reduced OS (P = 0.0019). Using The Cancer Genome Atlas data sets, ANXA2 but not S100A10 expression was associated with higher clinical stage (P = 0.005), whereas both ANXA2 and S100A10 expressions were associated with the mesenchymal molecular subtype (P < 0.0001). Kaplan-Meier and Cox regression analyses showed that high stromal annexin A2 immunostaining was significantly associated with reduced PFS (P = 0.013) and OS (P = 0.044). Moreover, high cytoplasmic S100A10 staining was significantly associated with reduced OS (P = 0.027). Multivariate Cox regression analysis showed stromal annexin A2 (P = 0.009) and cytoplasmic S100A10 (P = 0.016) levels to be independent predictors of OS. Patients with high stromal annexin A2 and high cytoplasmic S100A10 expressions had a 3.4-fold increased risk of progression (P = 0.02) and 7.9-fold risk of ovarian cancer death (P = 0.04). Our findings indicate that together annexin A2 and S100A10 expressions are powerful predictors of serous ovarian cancer outcome.

Changes in the expression of three markers in T lymphocytes of peripheral blood and immunoregulatory mechanisms of burned mice with sepsis at early stage

Changes in the expression of three markers in T lymphocytes of peripheral blood and immunoregulatory mechanisms of burned mice with sepsis at early stage

Assessment of the Parameters of Myeloma Bone Disease Signaling to the Extent of Skeletal Involvement, Bone Mineral Turnover and Activity of Multiple Myeloma

Background: Myeloma bone disease (MBD) is present in up to 80% patients with newly diagnosed multiple myeloma (MM). It is known to be activated by three separate signaling pathways - receptor activator of nuclear factor κB/osteoprotegerin pathway (RANK/OPG), macrophage inflammatory factor pathway (MIP) and Wnt pathway. Except of these, several other factors have been described in relationship to MBD development. The aim of our study was to assess the activity of selected parameters of MBD signaling with respect to macroscopic skeletal changes detectable using imaging techniques, and in comparison with markers of bone turnover and the activity of MM.Patients and methods: We assessed 92 patients with monoclonal gammopathies: 56 patients with active multiple myeloma, 12 with smoldering myeloma and 24 individuals with monoclonal gammopathy of undetermined significance (MGUS). Following parameters of MBD signaling were measured in the patients´serum: hepatocyte growth factor (HGF, Human HGF Quantine ELISA), macrophage inflammatory factor alpha (MIP-1α, Human CCL3/MIP-1α Quantikine ELISA), syndecan-1 (Human Syndecan-1 (CD138) ELISA), osteoprotegerin (OPG, Human Osteoprotegerin ELISA), Activin A (Human Activin A Quantikine ELISA), Dickkopf-related protein-1 (DKK1, Human Dkk-1 Quantikine ELISA), Annexin A2 (Human Annexin A2 ELISA kit), nuclear factor kappa B (NF-κB, Human Nuclear factor-kappa B). The extent of skeletal involvement was evaluated using whole body magnetic resonance and low-dose computed tomography. The levels of the selected parameters of MBD signaling were compared to serum levels of Ca, P, PINP, ICTP, CTx, parathormone (PTH), calcitonine (CLC), 25-hydroxyvitamin D2 (D2), 1,25dihydroxyvitamin D3 (D3), bone isoenzyme of alkaline phosphatase (bALP) and sclerostin (SCL). The parameters were measured at the time of diagnosis and then after 4 months. In patients with MM we measured selected parameters of MBD in comparison with the stage of the disease according to Durie-Salmon (DS) and according to International Staging System (ISS). In MGUS we assessed the relationship of the parameters to the risk of transformation to MM based on the Mayo clinic stratification system. For statistical estimation we used Kruskal-Wallis test and Mann-Whitney U post hoc test with Bonferroni correction at p < 0,05.Results: Statistically significant difference between patients with low and high skeletal involvement was found in serum levels of MIP-1α, syndecan-1, DKK-1 and Annexin A2. None of the parameters correlated with the presence of extramedullary disease. With respect to markers of bone turnover we found following significant relationships: HGF to PINP, ICTP, CTx and inverse relationship to D3; MIP-1α to PINP, ICTP, CTx and inverse relationship to D3; Syndecan-1 to ICTP, CTx, and inversely to D2 and D3; OPG to ICTP and SCL; Activin A to PINP, ICTP, CTx and SCL; NF-κB to ICTP and CTx. We found significant differences between MGUS and MM in serum levels of HGF, MIP-1α, syndecan-1, DKK-1 and NF-κB. There was no significant relationship of any of the parameters to DS, however, we found significant relationship of HGF, syndecan-1, OPG and Activin A to ISS. MIP-1α only had a significant relationship to the risk stratification of MGUS. There was no significant change in the parameters in MM patients after 4 months of treatment with respect to treatment response.Conclusions: We confirmed the relationship of MBD signaling to the extent of skeletal changes using imaging techniques. All three signalling pathways were involved in the development of MBD in MM in comparison with MGUS. Signaling involved in osteoclast activation was more activated than the parameters preventing osteoblast differentiation. There was, however, no significant change in any of the parameters in MM patients with respect to treatment response after 4 months, suggesting that bone reparation processes are delayed after hematological response. Significant relationship to the markers of bone turnover and to the activity of MM suggests possible use of the parameters of MBD signaling as tools for early detection of bone marrow changes and timely indication for anti-resorption therapy.Supported by the grant NT 14393. DisclosuresNo relevant conflicts of interest to declare.

Abstract A09: Effects of Podocalyxin on neural stem/progenitor cells

Abstract Podocalyxin (PODXL) is a highly glycosylated and sialylated protein found mainly on podocytes and up-regulated in various types of tumors. Known to be a marker of hematopoietic progenitors and pluripotent stem cells, PODXL is also increasingly considered a marker of malignancy in several aggressive tumor types. Though the exact role of PODXL in tumors is not yet established, it has increasingly been shown in vitro to be involved in cell migration and invasion. We found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells. To investigate effects of elevated Podxl levels in neural stem/progenitor cells (NSPs), Podxl or an empty control construct were over-expressed in these cells. Podxl-overexpressing NSPs did not form brain tumors upon intracranial transplantations, indicating that high levels of this gene alone are not sufficient for tumorigenesis. In vitro studies however revealed how Podxl might contribute to the development and /or maintenance of tumors. Podxl overexpression had a positive effect on cell number, sphere formation and cell viability. To find out how Podxl exerts its pro-survival effects, the proteome of Podxl-overexpressing and empty control NSPs was analysed by mass spectrometry. These experiments revealed an up-regulation of Annexin A2, a calcium-regulated phospholipid binding protein interacting with the cytoskeleton and extracellular matrix. In addition to elevated Annexin A2 protein levels, we also found increased transcript levels indicating that PODXL stimulates expression of the Annexin A2 gene. NSPs lacking Annexin A2 and overexpressing Podxl are less viable than wild type Podxl-overexpressing NSPs, suggesting that PODXL-mediated pro-survival effects can at least in part be explained by increased Annexin A2 levels. We are now investigating how Podxl and Annexin A2 mechanistically interact to enhance cell viability Citation Format: Isabelle V. Leefa Chong San, Gaelle Prost, Ulrike Nuber. Effects of Podocalyxin on neural stem/progenitor cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A09.

Atorvastatin protects endothelial colony‑forming cells against H2O2‑induced oxidative damage by regulating the expression of annexin A2.

Endothelial dysfunction and injury are central events in the pathogenesis of ischemic vascular disorders. Endothelial progenitor cells (EPCs) are mobilized from the bone marrow into the peripheral circulation, where they locate to sites of injured endothelium and are involved in endothelial repair and vascular regeneration. During these processes, EPCs are exposed to oxidative stress, a crucial pathological condition, which occurs during vascular injury and limits the efficacy of EPCs in the repair of injured endothelium. Statins are effective inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, and are commonly used to manage and prevent ischemic vascular disease by reducing plasma cholesterol levels. In addition to lowering cholesterol, statins have also been reported to exert pleiotropic actions, including anti-inflammatory and anti-oxidative activities. The present study aimed to investigate the ability of atorvastatin to protect endothelial colony-forming cells (ECFCs), a homogeneous subtype of EPCs, from hydrogen peroxide (H2O2)-induced oxidative damage, and to determine the mechanism underlying this protective action. MTT assay, acridine orange/ethidium bromide staining, reactive oxygen species assay, western blot analysis and tube formation assay were employed. The results demonstrated that H2O2 induced cell death and decreased the tube-forming ability of the ECFCs, in a concentration-dependent manner; however, these effects were partially attenuated following administration of atorvastatin. The reversion of the quantitative and qualitative impairment of the H2O2-treated ECFCs appeared to be mediated by the regulation of annexin A2, as the expression levels of annexin A2 were decreased following treatment with H2O2 and increased following treatment with atorvastatin. These results indicated that annexin A2 may be involved in the H2O2-induced damage of ECFCs, and in the protective activities of atorvastatin in response to oxidative stress.

Lack of TNFRI signaling enhances annexin A1 biological activity in intestinal inflammation

We evaluated whether the lack of TNF-α signaling increases mucosal levels of annexin A1 (AnxA1); the hypothesis stems from previous findings showing that TNF-α neutralization in Crohn’s disease patients up-regulates systemic AnxA1 expression. Biopsies from healthy volunteers and patients under anti-TNF-α therapy with remittent ulcerative colitis (UC) showed higher AnxA1 expression than those with active disease. We also evaluated dextran sulfate sodium (DSS)-acute colitis in TNF-α receptor 1 KO (TNFR1−/−) strain with impaired TNF-α signaling and C57BL/6 (WT) mice. Although both strains developed colitis, TNFR1−/− mice showed early clinical recovery, lower myeloperoxidase (MPO) activity and milder histopathological alterations. Colonic epithelium from control and DSS-treated TNFR1−/− mice showed intense AnxA1 expression and AnxA1+ CD4+ and CD8+ T cells were more frequent in TNFR1−/− animals, suggesting an extra supply of AnxA1. The pan antagonist of AnxA1 receptors exacerbated the colitis outcome in TNFR1−/− mice, supporting the pivotal role of AnxA1 in the early recovery. Our findings demonstrate that the TNF-α signaling reduction favors the expression and biological activity of AnxA1 in inflamed intestinal mucosa.

Abstract 3906: Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling

Abstract Introduction: Annexin A1 is an anti-inflammatory protein reported to play a role in cell proliferation, apoptosis and to be deregulated in breast cancer. The role and regulation of annexin A1 in the biology and prognosis of breast cancer remain unclear because most of the studies did not consider specific differences between the different subtypes of breast cancer. Thus, in this study, we hypothesize that annexin A1 plays subtype-specific role in basal like breast cancer progression and metastasis. Methods: By mining The Cancer Genome Atlas-Breast Cancer Dataset (TCGA-BRCA) and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. Results: Our in-silico analysis of TCGA-BRCA dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer tumors than luminal and Her2/neu-positive breast cancer tumors. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway. Specifically, annexin A1 knockdown inhibited phosphorylation of mTOR and S6 (18-73% inhibition in pmTOR and 50-60% inhibition in pS6). But annexin A1 knockdown had no impact on the MAPK, c-Met, and EGFR pathways. In cell migration assays, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells which we found to be through suppression of MMP9. Conclusions: Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be due in part to the role of annexin A1 in activating mTOR-pS6 pathway. Citation Format: Kazunoshin Tachibana, Anjana Bhardwaj, Nivetha Ganesan, Kimal Rajapakshe, Constance T. Albarracin, Preethi H. Gunaratn, Cristian Coarfa, Isabelle Bedrosian. Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3906. doi:10.1158/1538-7445.AM2015-3906

Evaluation of annexin A2 and as potential biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) ranks as the fifth most common malignancy worldwide. Early detection of HCC is difficult due to the lack of reliable markers. We aimed to assess the diagnostic role of annexin A2 (ANXA2) and follistatin as serum markers for HCC patients. This study included 50 patients with confirmed diagnosis of HCC, 30 patients with chronic liver disease, and 20 normal persons. Subjects performed thorough assessment and laboratory investigations. Serum levels of alpha fetoprotein (AFP), annexin A2, and follistatin were measured using ELISA technique. Annexin A2 significantly increased in the sera of HCC patients (median, 69.6ng/ml) compared to chronic liver disease patients (median, 16.8ng/ml) and control group (median, 9.5ng/ml) (p < 0.001). Follistatin was higher in sera of HCC patients (median, 24.4ng/ml) compared to the control group (median, 4.2ng/ml) (p = 0.002) while no such significant difference was achieved between HCC and chronic liver disease patients. At a cutoff level 29.3ng/ml, area under the receiver-operating characteristic curve for ANXA2 was 0.910 (95% confidence interval (CI) 0.84-0.97). For follistatin, it was 0.631 (95% confidence interval 0.52-0.74) at cutoff level 15.7ng/ml. Combining both annexin A2 and AFP increased the diagnostic efficiency (98% specificity, LR + 41 and 97.6% PPV). Follistatin combined with AFP provided 92% specificity while lower sensitivity (50%) was observed. Serum ANXA2 is a promising biomarker for HCC, certainly when measured with AFP. Follistatin could not differentiate between HCC and chronic liver disease, but its combination with AFP improved the specificity for HCC diagnosis.

Brain microvascular endothelium induced-annexin A1 secretion contributes to small cell lung cancer brain metastasis

Small cell lung cancer is the most aggressive histologic subtype of lung cancer, with a strong predilection for metastasizing to brain early. However, the cellular and molecular basis is poorly known. Here, we provided evidence to reveal the role of annexin A1 in small cell lung cancer metastasis to brain. Firstly, the elevated annexin A1 serum levels in small cell lung cancer patients were associated with brain metastasis. The levels of annexin A1 were also upregulated in NCI-H446 cells, a small cell lung cancer cell line, upon migration into the mice brain. More interestingly, annexin A1 was secreted by NCI-H446 cells in a time-dependent manner when co-culturing with human brain microvascular endothelial cells, which was identified with the detections of annexin A1 in the co-cultured cellular supernatants by ELISA and western blot. Further results showed that blockage of annexin A1 in the co-cultured cellular supernatants using a neutralized antibody significantly inhibited NCI-H446 cells adhesion to brain endothelium and its transendothelial migration. Conversely, the addition of Ac2-26, an annexin A1 mimic peptide, enhanced these effects. Furthermore, knockdown of annexin A1 in NCI-H446 cells prevented its transendothelial migration in vitro and metastasis to mice brain in vivo. Our data showed that small cell lung cancer cell in brain microvasculature microenvironment could express much more annexin A1 and release it outside, which facilitated small cell lung cancer cell to gain malignant properties of entry into brain. These findings provided a potential target for the management of SCLC brain metastasis.

Role of RNA-interference-induced zinc finger protein 139 suppression in gastric cancer cell sensitivity to chemotherapeutic agents.

Zinc finger proteins (ZNFs) are a class of proteins widely distributed in the human genome, which have been found to play a role in the regulation of gene transcription and the occurrence and development of gastric cancer (GC). ZNF139 was found to be associated with GC in our previous experiments. The present study aimed to analyse the differences in ZNF139 protein expression in SGC7901 GC cells and in situ grafted GC tumors in nude mice prior to and following RNA interference inhibition, and to investigate the mechanisms underlying ZNF139 involvement in the occurrence, development and chemosensitivity of GC. A ZNF139-targeted small interfering (si)RNA plasmid was constructed and transfected into the cancer cells and in situ grafted tumors. The MTT assay was used to investigate the alterations in chemosensitivity prior to and following transfection of siRNA-ZNF139. The two-dimensional difference gel electrophoresis and liquid chromatography-mass spectrometry techniques were used to identify the different protein points prior to and following siRNA-ZNF139 transfection. Western blot analysis was performed to confirm the identified proteins. In the siRNA-ZNF139 group, the growth of the cancer cells and in situ grafted tumors significantly decreased. However, the post-interference chemosensitivity to 5-fluorouracil, cisplatin and mitomycin C significantly increased. In the in vivo and in vitro experiments, the expression of pyridoxal kinase (PDXK) was upregulated, whereas the expression levels of annexin A2 (ANXA2) and fascin were downregulated following transfection. Western blot analysis confirmed the results for PDXK, ANXA2 and fascin by proteomics. Therefore, ZNF139 may participate in the occurrence, development and chemosensitivity of GC by promoting the expression of ANXA2 and fascin, while inhibiting the expression of PDXK.