Neutrophil Gelatinase-associated Lipocalin Overexpression Research Articles

NGAL Mediates Anaplastic Thyroid Carcinoma Cells Survival Through FAS/CD95 Inhibition.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

Sex-dependent expression of neutrophil gelatinase-associated lipocalin in aortic stenosis

BackgroundAccumulating evidence suggest the existence of sex-related differences in the pathogenesis of aortic stenosis (AS) with inflammation, oxidative stress, fibrosis and calcification being over-represented in men. Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in a myriad of tissues and cell types, and it is associated with acute and chronic pathological processes comprising inflammation, fibrosis or calcification. Sex-dependent signatures have been evidenced for NGAL which expression has been associated predominantly in males to metabolic and cardiovascular disorders. We aimed to analyse sex-related differences of NGAL in AS and its role in the inflammatory and fibrocalcific progression of AS.Methods and results220 (60.45% men) patients with severe AS elective for surgical aortic valve (AV) replacement were recruited. Immunohistochemistry revealed higher expression of NGAL in calcific areas of AVs and that was validated by qPCR in in 65 (60% men) donors. Valve interstitial cells (VICs) were a source of NGAL in these samples. Proteome profiler analyses evidenced higher expression of NGAL in men compared to women, and that was further validated by ELISA. NGAL expression in the AV was correlated with inflammation, oxidative stress, and osteogenic markers, as well as calcium score. The expression of NGAL, both intracellular and secreted (sNGAL), was significantly deregulated only in calcifying male-derived VICs. Depletion of intracellular NGAL in calcifying male-derived VICs was associated with pro-inflammatory profiles, dysbalanced matrix remodelling and pro-osteogenic profiles. Conversely, exogenous NGAL mediated inflammatory and dysbalanced matrix remodelling in calcifying VICs, and all that was prevented by the pharmacological blockade of NGAL.ConclusionsOwing to the over-expression of NGAL, the AV from men may be endowed with higher expression of inflammatory, oxidative stress, matrix remodelling and osteogenic markers supporting the progression of calcific AS phenotypes. The expression of NGAL in the VIC emerges as a potential therapeutic checkpoint, with its effects being potentially reverted by the pharmacological blockade of extracellular NGAL.

Preliminary study on 24p3 / neutrophil gelatinase-associated lipocalin (NGAL) ferroptosis inhibition in renal tubular epithelial cells

ABSTRACT The 24p3/neutrophil gelatinase-associated lipocalin (NGAL) protein plays an important protective role in acute kidney injury (AKI), but the exact mechanism remains unclear. Therefore, we have made a preliminary exploration of its mechanism. The experimental group was formed by constructing and transfecting 24P3 overexpressed plasmid into renal tubular epithelial cells. Western Bolt was used to detect NGAL expression. Cell proliferation was detected by CCK8 kit, cell death was detected by Hoechst 33342 and PI kit, mitochondrial morphology was observed under light microscope, reactive oxygen species (ROS) content was detected by fluorescence probe, and iron level and glutathione peroxidase 4 (GPX4) activity were detected by kit. Furthermore, the mechanism of NGAL action was further demonstrated by adding ferrostein-1 (Fer-1), an ferroptosis inhibitor, and erastin (containing DMSO),an ferroptosis inductor. We found that ferroptosis-related indicators were lower in the NGAL overexpression group than in the control group. At the same time, we found that NGAL alleviated ferroptosis induced by erastin and coordinated with Fer-1 to alleviate ferroptosis. In conclusion, NGAL inhibits ferroptosis in renal tubular epithelial cells, which may be associated with the progression of AKI and may provide a new therapeutic target for the transition from acute kidney injury to chronic kidney injury.

The factors involved in the induction of neutrophil gelatinase-associated lipocalin overexpression in renal tubular epithelial cells under endoplasmic reticulum stress.

Our previous work found that neutrophil gelatinase-associated lipocalin (NGAL) expression increases when endoplasmic reticulum stress (ERS) occurs in human kidney-2 (HK-2) tubular epithelial cells. However, the reason for this is not yet known. This study investigated the factors involved when inducing NGAL overexpression in HK-2 cells during ERS. The cells were divided into six groups: the control group (normal HK-2 cells), the ERS group (HK-2 cells cultured in complete medium with thapsigargin (TG)), the transfection group (HK-2 cells transfected with activating transcription factor 4 small interfering ribonucleic acid (ATF4 siRNA), the ERS after transfection group (HK-2 cells transfected with ATF4 siRNA, then cultured in complete medium with TG), the negative control group (HK-2 cells transfected with siRNA-negative contrast), and the dimethyl sulfoxide (DMSO) group (HK-2 cells cultured in complete medium with DMSO). Western blot and a real-time polymerase chain reaction were used to measure the expression of protein and messenger ribonucleic acid (mRNA). As a result NGAL, ATF4, C/EBP homologous protein, glucose-regulated protein 78 kDa, ATF4 mRNA, and NGAL mRNA were clearly overexpressed in the ERS group compared with the control group (p < 0.05). The expression of NGAL and ATF4 were similar in the control group, the negative control group, and the DMSO group (p > 0.05). Meanwhile, ATF4, NGAL, ATF4 mRNA, and NGAL mRNA in the ERS after transfection group were significantly lower compared with the ERS group (p < 0.05), which showed that NGAL was affected by ATF4. There was a close correlation between NGAL and ATF4; when the expression of ATF4 was inhibited, NGAL was significantly lower. Therefore, ATF4 may be one of the upstream regulators of NGAL.

Relation of Neutrophil Gelatinase-Associated Lipocalin Overexpression to the Resistance to Apoptosis of Tumor B Cells in Chronic Lymphocytic Leukemia.

The resistance to apoptosis of chronic lymphocytic leukemia (CLL) cells partly results from the deregulated production of survival signals from leukemic cells. Despite the development of new therapies in CLL, drug resistance and disease relapse still occur. Recently, neutrophil gelatinase-associated lipocalin (NGAL), a secreted glycoprotein, has been suggested to have a critical role in the biology of tumors. Thus, we investigated the relevance of NGAL in CLL pathogenesis, analyzed the expression of its cellular receptor (NGAL-R) on malignant B cells and tested whether CLL cells are resistant to apoptosis through an autocrine process involving NGAL and NGAL-R. We observed that NGAL concentrations were elevated in the serum of CLL patients at diagnosis. After treatment (and regardless of the therapeutic regimen), serum NGAL levels normalized in CLL patients in remission but not in relapsed patients. In parallel, NGAL and NGAL-R were upregulated in leukemic cells from untreated CLL patients when compared to normal peripheral blood mononuclear cells (PBMCs), and returned to basal levels in PBMCs from patients in remission. Cultured CLL cells released endogenous NGAL. Anti-NGAL-R antibodies enhanced NGAL-R+ leukemia cell death. Conversely, recombinant NGAL protected NGAL-R+ CLL cells against apoptosis by activating a STAT3/Mcl-1 signaling pathway. Our results suggest that NGAL and NGAL-R, overexpressed in untreated CLL, participate in the deregulation of the apoptotic machinery in CLL cells, and may be potential therapeutic clues for CLL treatment.

NGAL protects in nasopharyngeal carcinoma by inducing apoptosis and blocking epithelial-mesenchymal transition.

In recent years, neutrophil gelatinase-associated lipocalin (NGAL) has been considered to be a key molecule in different cancer types and its carcinogenesis may be related to the NGAL/MMP-9 complex. However, its expression pattern and role in nasopharyngeal carcinoma (NPC) has rarely been reported. In the current study, 158 tumor tissues from NPC patients were collected and immunohistochemistry was performed to determine the NGAL protein expression, to investigate the correlation between its expression and clinical and pathological parameters using Chi square analysis. Furthermore, by over-expressing NGAL in NPC cell lines, biological alteration of NPC cells with respect to cell proliferation, migration and invasion was analyzed. Results suggested that high expression of NGAL predicts better prognosis and longer survival. Overexpression of NGAL significantly reduced the proliferation and migration of NPC cells, and induced the apoptosis by activating caspase 3, 8 and 9, and blocking epithelial-mesenchymal transition by inhibiting mothers against decapentaplegic homolog 2/3 phosphorylation.

Dendritic cells are crucial for cardiovascular remodeling and modulate neutrophil gelatinase-associated lipocalin expression upon mineralocorticoid receptor activation.

Adaptive immunity is crucial in cardiovascular and renal inflammation/fibrosis upon hyperactivation of mineralocorticoid receptor. We have previously demonstrated that dendritic cells can respond to mineralocorticoid receptor activation, and the neutrophil gelatinase-associated lipocalin (NGAL) in dendritic cells is highly increased during aldosterone (Aldo)/mineralocorticoid receptor-dependent cardiovascular damage. However, the interrelationship among dendritic cells, target organs inflammation/fibrosis induced by mineralocorticoid receptor, and NGAL-dependence remains unknown. We studied the role of dendritic cells in mineralocorticoid receptor-dependent tissue remodeling and whether NGAL can modulate the inflammatory response of dendritic cells after mineralocorticoid receptor activation. Cardiovascular and renal remodeling induced by Aldo and high-salt diet [nephrectomy-Aldo-salt (NAS) model] were analyzed in CD11c.DOG mice, a model which allows dendritic cells ablation by using diphtheria toxin. In addition, in-vitro studies in NGAL-knock out dendritic cells were performed to determine the immunomodulatory role of NGAL upon Aldo treatment. The ablation of dendritic cells prevented the development of cardiac hypertrophy, perivascular fibrosis, and the overexpression of NGAL, brain natriuretic peptide, and two profibrotic factors induced by NAS: collagen 1A1 and connective tissue growth factor. We determined that dendritic cells were not required to prevent renal hypertrophy/fibrosis induced by NAS. Between different immune cells analyzed, we observed that NGAL abundance was higher in antigen-presenting cells, while in-vitro studies showed that mineralocorticoid receptor stimulation in dendritic cells favored NGAL and IL-23 expression (p19 and p40 subunits), which are involved in the development of fibrosis and the Th17-driven response, respectively. NGAL produced by dendritic cells may play a pivotal role in the activation of adaptive immunity that leads to cardiovascular fibrosis during mineralocorticoids excess.

Ulcer-associated cell lineage expresses genes involved in regeneration and is hallmarked by high neutrophil gelatinase-associated lipocalin (NGAL) levels.

Neutrophil gelatinase‐associated lipocalin (NGAL), also known as Lipocalin 2, is an antimicrobial protein, encoded by the gene LCN2, strongly upregulated in inflammatory bowel disease (IBD) and a promising biomarker for IBD. Here we demonstrate that NGAL is highly expressed in all parts of pyloric metaplasia, also known as the ulcer‐associated cell lineage (UACL), a metaplastic cell lineage suggested to play a role in wound healing in Crohn's disease (CD). We further show NGAL expression in regenerative intestinal crypts and in undifferentiated patient‐derived colonoids. This indicates that NGAL is important in the tissue regeneration process. The remarkable overexpression of NGAL in UACL led us to explore the pathobiology of these cells by transcriptome‐wide RNA sequencing. This study is, to our knowledge, the first to characterize the UACL at this level. Biopsies with UACL and inflamed non‐UACL epithelium from the terminal ileum of CD patients and epithelium from healthy controls were laser capture microdissected for RNA sequencing. Among the 180 genes differentially expressed between UACL and control epithelium, the ten most‐upregulated genes specific for UACL were MUC5AC, PGC, MUC6, MUC5B, LCN2, POU2AF1, MUC1, SDC3, IGFBP5, and SLC7A5. PDX1 was among the most upregulated in both UACL and inflamed non‐UACL epithelium. Immunohistochemistry and iDisco 3D visualization was used to characterize UACL histo‐morphologically, and to validate protein expression of 11 selected differentially expressed genes. Among these genes, LCN2, NOTCH2, PHLDA1, IGFBP5, SDC3, BPIFB1, and RCN1 have previously not been linked to UACL. Gene expression results were analyzed for functional implications using MetaCore, showing that differentially expressed genes are enriched for genes involved in cell migration and motility, and for biomarkers of gastrointestinal neoplasia. These results support a role for UACL as part of the reepithelialization process during and after destructive intestinal inflammation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Shortest path analyses in the protein-protein interaction network of NGAL (neutrophil gelatinase-associated lipocalin) overexpression in esophageal squamous cell carcinoma.

NGAL (neutrophil gelatinase-associated lipocalin) is a novel cancer-related protein involves multiple functions in many cancers and other diseases. We previously overexpressed NGAL to analyze its role in esophageal squamous cell carcinoma (ESCC). In this study, a protein-protein interaction (PPI) was constructed and the shortest paths from NGAL to transcription factors in the network were analyzed. We found 28 shortest paths from NGAL to RELA, most of them obeying the principle of extracellular to cytoplasm, then nucleus. These shortest paths were also prioritized according to their normalized intensity from the microarray by the order of interaction cascades. A systems approach was developed in this study by linking differentially expressed genes with publicly available PPI data, Gene Ontology and subcellular localizaton for the integrated analyses. These shortest paths from NGAL to DEG transcription factors or other transcription factors in the PPI network provide important clues for future experimental identification of new pathways.

Functional analysis of the mRNA profile of neutrophil gelatinase‑associated lipocalin overexpression in esophageal squamous cell carcinoma using multiple bioinformatic tools.

Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily; dysregulated expression of NGAL has been observed in several benign and malignant diseases. In the present study, differentially expressed genes, in comparison with those of control cells, in the mRNA expression profile of EC109 esophageal squamous cell carcinoma (ESCC) cells following NGAL overexpression were analyzed by multiple bioinformatic tools for a comprehensive understanding. A total of 29 gene ontology (GO) terms associated with immune function, chromatin structure and gene transcription were identified among the differentially expressed genes (DEGs) in NGAL overexpressing cells. In addition to the detected GO categories, the results from the functional annotation chart revealed that the differentially expressed genes were also associated with 101 functional annotation category terms. A total of 59 subpathways associated locally with the differentially expressed genes were identified by subpathway analysis, a markedly greater total that detected by traditional pathway enrichment analysis only. Promoter analysis indicated that the potential transcription factors Snail, deltaEF1, Mycn, Arnt, MNB1A, PBF, E74A, Ubx, SPI1 and GATA2 were unique to the downregulated DEG promoters, while bZIP910, ZNF42 and SOX9 were unique for the upregulated DEG promoters. In conclusion, the understanding of the role of NGAL overexpression in ESCC has been improved through the present bioinformatic analysis.

Targeting of Receptor for Advanced Glycation End Products Suppresses Cyst Growth in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a myriad of research groups have attempted to identify a new therapeutic target for ADPKD, no drug has worked well in clinical trials. Our research group has focused on the receptor for advanced glycation end products (RAGE) gene as a novel target for ADPKD. This gene is involved in inflammation and cell proliferation. We have already confirmed that blocking RAGE function attenuates cyst growth in vitro. Based on this previous investigation, our group examined the effect of RAGE on cyst enlargement in vivo. PC2R mice, a severe ADPKD mouse model that we generated, were utilized. An adenovirus containing anti-RAGE shRNA was injected intravenously into this model. We observed that RAGE gene knockdown resulted in loss of kidney weight and volume. Additionally, the cystic area that originated from different nephron segments decreased in size because of down-regulation of the RAGE gene. Blood urea nitrogen and creatinine values tended to be lower after inhibiting RAGE. Based on these results, we confirmed that the RAGE gene could be an effective target for ADPKD treatment.

Clinical significance of neutrophil gelatinase-associated lipocalin (NGAL) in colorectal cancer: a meta-analysis

Growing evidence has implicated that neutrophil gelatinase-associated lipocalin (NGAL) plays a role in a spectrum of human cancers. Several observational studies from different parts of the world have been devoted to elucidate the clinical relationship between NGAL and colorectal cancer. This meta-analysis aimed to explore the overall accuracy of NGAL detection for the diagnosis and prognosis of colorectal cancer. Based on comprehensive literature screening on Pubmed, Ovid, and CNKI databases, our screening covered all published papers until March 2013. The relevant papers were selected according to some stringent inclusion criteria. Essential data were extracted from the recruited papers and further processed by systematic meta-analysis. The meta-analysis included 5 studies for diagnosis of colorectal cancer. Overall, the pooled sensitivity and specificity of all studies were 73% (95%CI=0.69-0.76) and 89% (95%CI=0.85-0.93). The pooled positive likelihood ratio and negative likelihood ratio were 5.41 (95%CI=3.85- 7.59) and 0.37 (95%CI=0.22-0.62). The pooled diagnostic odds ratios was 18.05 (95%CI=11.77-27.69). The area under the summary receiver operating characteristic curve for the diagnosis of colorectal cancer was 0.87. Meanwhile, 3 studies were included to evaluate the prognostic significance of NGAL overexpression in colorectal cancer patients. The pooled hazard ratio was 2.12 (95%CI=1.35-3.33). High level of NGAL predicted poor disease-free survival. Thus, NGAL is a potential biomarker for the diagnosis and prognosis of colorectal cancer.

NGAL and NGALR overexpression in human hepatocellular carcinoma toward a molecular prognostic classification

Aim: Neutrophil gelatinase-associated lipocalin (NGAL) and its cell surface receptor, NGALR, have been implicated in tumorigenesis and tumor progression of various human malignant neoplasms. In particularly, it has been demonstrated that NGAL is overexpressed in hepatocellular carcinoma (HCC) tissues and closely associated with the proliferation and invasion of HCC cells. The aim of this study was to investigate the clinical significance of NGAL and NGALR in HCC. Methods: Expression of NGAL and NGALR was evaluated by immunohistochemistry in tumor tissues from 138 patients who underwent curative resection of HCC. The association of NGAL or NGALR expression with the clinicopathologic features was analyzed. Univariate and multivariate analyses were performed to evaluate the prognostic value of NGAL and/or NGALR expression for HCC patients. Results: The expression levels of NGAL and NGALR were both up-regulated in HCC tissues, and to be associated with vascular invasion (both P=0.03), TNM stage (both P=0.004), and tumor recurrence (both P<0.001). A positive correlation between expression of the two markers was also observed (r=0.89; P<0.001). Additionally, survival analysis showed that high expression of NGAL or NGALR was significantly associated with poor prognosis for patients with HCC (both P=0.003). Patients with high expression of both NGAL and NGALR had a shorter overall survival (P<0.001) than those with low expression of both. Furthermore, multivariate analysis showed both NGAL and NGALR were independent predictors of overall survival. Conclusion: Our data demonstrate for the first time that the up-regulations of NGAL and NGALR expression in HCC were both significantly correlated with unfavorable clinicopathologic features and independent poor prognostic factor for overall survival in patients. These findings suggest that NGAL and NGALR expression might be served as novel prognostic factors and potential therapeutic targets in HCC.

Neutrophil gelatinase-associated lipocalin (NGAL) in inflammatory bowel disease: association with pathophysiology of inflammation, established markers, and disease activity

Neutrophil gelatinase-associated lipocalin (NGAL) is a multi-potent 25-kDa protein mainly secreted by neutrophils. In inflammatory bowel disease (IBD), overexpression of NGAL in colon epithelium has been previously shown. This is the first study analyzing serum and urinary NGAL levels in IBD patients, with regard to specific characteristics of patients and disease. Serum and urinary NGAL levels were determined in 181 patients with IBD, 93 with ulcerative colitis (UC), and 88 with Crohn's disease (CD), 82 healthy controls (HC), and 41 patients with irritable bowel syndrome (IBS). Serum NGAL levels were elevated in IBD patients (88.19 ± 40.75 ng/mL) compared with either HC (60.06 ± 24.18 ng/mL) or IBS patients (60.80 ± 20.30 ng/mL), P < 0.0001. No significant difference was shown between UC (86.62 ± 35.40 ng/mL) and CD (89.92 ± 46.05 ng/mL). Significantly higher levels of serum NGAL were observed in patients with active (120.1 ± 38.46) versus inactive IBD (61.58 ± 15.98), P < 0.0001. Serum NGAL displayed a strong ability to distinguish active IBD from inactive disease, healthy controls, or IBS patients with a sensitivity of 100, 95, and 95% and a specificity of 68, 83, and 79%, respectively, performing better than erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in the assessment of disease activity in both UC and CD. Urinary NGAL levels showed neither significant difference among patients and controls nor correlation with disease activity. Serum NGAL is elevated particularly in active IBD and correlates with established markers of inflammation and disease activity, implicating its role in the pathophysiology of IBD.

NGAL Expression Is Elevated in Both Colorectal Adenoma–Carcinoma Sequence and Cancer Progression and Enhances Tumorigenesis in Xenograft Mouse Models

There is growing evidence implicating that neutrophil gelatinase-associated lipocalin (NGAL) plays a role in the development and progression of cancers. However, the effect of NGAL in colorectal carcinoma (CRC) has not been clearly elucidated. In this study, we investigated the role of NGAL in the tumorigenesis and progression of CRC and evaluated the clinical value of NGAL expression. We examined NGAL expression in 526 colorectal tissue samples, including 53 sets of matched specimens (histologically normal mucosa, adenomas, and carcinomas) using immunohistochemical analysis. In CRCs, correlations between NGAL expression and clinicopathologic parameters were analyzed, and survival analysis was conducted. The role of NGAL was further tested using mouse xenograft models. NGAL expression was elevated during the colorectal adenoma-carcinoma sequence both among the 526 cases (r(s) = 0.66, P < 0.001) and in the 53 sets of matched specimens (r(s) = 0.60, P < 0.001). In CRCs, NGAL expression was associated with cancer stage (P = 0.041) and tumor recurrence in stage II patients (P = 0.037). Survival analysis revealed that NGAL expression was an independent prognostic factor for overall survival (HR = 1.84, P = 0.004) and for disease-free survival of stage II patients (HR = 5.88, P = 0.021). In mouse models, the xenografts in cecum and spleen were heavier and more numerous in the group injected with NGAL-overexpressing CRC cells (P < 0.05). NGAL overexpression may promote the tumorigenesis and progression of CRC. Detecting NGAL expression in tumor tissues may be useful for evaluating prognosis of patients with CRC.

Abstract 5008: Epidermal growth factor downregulates expression of neutrophil gelatinase-associated lipocalin (NGAL) through E-cadherin in pancreatic cancer cells

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, is a secreted protein that is upregulated in multiple cancer types. Our group previously reported that NGAL overexpression significantly blocked invasion and angiogenesis in pancreatic ductal adenocarcinoma (PDAC). We and others have also demonstrated loss NGAL expression in advanced stages of PDAC. However, little is known about mechanisms that regulate NGAL expression in PDAC. As EGF-EGFR axis is significantly upregulated in PDAC, we examined EGF-mediated NGAL regulation in these cells. Methods: NGAL-positive AsPC-1 and BxPC-3 cells were used as model system. Quantitative RT-PCR, western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or shRNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay (EMSA). Results: NGAL expression was positively associated with tumor differentiation and was significantly downregulated after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin downregulation was partly through the EGF receptor (EGFR)-dependent MEK1/2-ERK1/2 signaling pathway, which was demonstrated using pharmacologic inhibitors of EGFR (AG1478) and MEK1/2 (U0126 and PD98059). In addition, E-cadherin downregulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued inhibition of NGAL expression by EGF. Furthermore, EGF in part through E-cadherin reduced NGAL promoter activity by blocking NF-κB activation. Conclusion: We demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect is partly mediated through activation of the EGFR-MEK1/2-ERK1/2 signaling pathway, which in turn downregulated E-cadherin with a subsequent reduction in NF-κB activation. Our findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2011-5008

NGAL and NGALR are frequently overexpressed in human gliomas and are associated with clinical prognosis

Recently, neutrophil gelatinase-associated lipocalin (NGAL) and its cell surface receptor, NGALR, have been shown to have critical roles in the biology of various tumors. Therefore, we investigated the expression of NGAL and NGALR in tumor sections obtained from patients with gliomas, and compared these results with the clinical characteristics of the patients. Using immunohistochemical assays, the expression levels of NGAL and NGALR were found to be up-regulated in tumor tissues, and to be related to tumor grade (p < 0.001). A positive correlation between expression of the two markers was also observed in these assays (r = 0.849; p < 0.001). Overexpression of NGAL and NGALR in glioma tissues was also confirmed in western blot analysis and real-time quantitative RT-PCR assays. Furthermore, overexpression of NGAL and NGALR was found to be significantly associated with poor prognosis (p < 0.001 in each case). Multivariate analysis identified patient age, tumor grade, and expression levels of NGAL and NGALR to be independent prognostic factors. In particular, NGAL(2+)/NGALR(2+) tissues were associated with lower rates of survival (risk ratio, 1.378; 95% CI, 1.102-1.724; p = 0.005). These findings suggest that NGAL and NGALR expression are frequently up-regulated in gliomas, and are closely associated with poor clinical outcome.

Abstract 3074: Neutrophil gelatinase-associated lipocalin inhibits proliferation of human esophageal adenocarcinoma cells both in vitro and in vivo

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL), a member of lipocalin family, is a 25-kDa secreted glycoprotein. NGAL modulates multiple biological functions including cell proliferation, invasion, and metastasis. NGAL is upregulated in multiple gastrointestinal cancers including colorectal, pancreatic, and esophageal adenocarcinoma (EAC). Our group recently showed that NGAL over-expression significantly blocked metastasis of pancreatic adenocarcinoma cells [Cancer Research 68(15): 6100-6108, 2008]. However, the biological role of NGAL in EAC is not known. Aim: To examine the biological role of NGAL in human EAC cells both in vitro and in vivo. Methods and Results: We used OE33 (well-differentiated) and Flo-1 (poorly differentiated) EAC cell lines as our model system. We observed that OE33 cells have significantly higher levels of NGAL expression than Flo-1 cells both by qPCR and western blot analyses. To further study the biological role of NGAL in EAC, we created stable NGAL over-expression clones in Flo-1 (Flo1-NGAL) and NGAL under-expression clones in OE33 (OE33-shNGAL) using lentiviral system. We observed that under-expression of NGAL in OE33 significantly increased cell proliferation as determined by clonogenic assays. In contrast, NGAL over-expression in Flo-1 significantly suppressed cell proliferation. We also observed that NGAL under-expression increased cyclin D1 expression, a known cell cycle regulator. This, in part, explained increased EAC cell proliferation secondary to NGAL under-expression. In addition, NGAL over-expression potently suppressed and conversely, NGAL under-expression significantly increased invasion of EAC clones through MatrigelTM in vitro. Furthermore, to confirm our in vitro findings, we observed that OE33-shNGAL clones strikingly increased tumor volume in a subcutaneouos heterotopic xenograft model in nude mice. Conclusion: We demonstrated for the first time that NGAL under-expression increased cell proliferation and invasion in vitro, which could be in part secondary to regulation of cyclin D1 expression. NGAL under-expression also enhanced tumor growth in vivo. Thus, modulation of NGAL can be a novel therapeutic target in progression of EAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3074.

Overexpression of neutrophil gelatinase-associated lipocalin and its receptor in colorectal carcinoma: Significant correlation with cell differentiation and tumour invasion

Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, is related to imflammation and tumour. Recently, a specific cell-surface receptor (24p3R/NGALR) for lipocalin 24p3 was reported. However, the characteristics of NGALR expression in colorectal carcinoma (CRC) are not known. The objectives of this study were to investigate the expression of NGAL and NGALR in CRC specimens, and determine any relationship between the expression of these proteins and tumour progression. In the present study, CRC specimens of 102 patients were obtained, and the expression of NGAL, NGALR, ferritin and Ki67 was analyzed in paraffin sections by immunohistochemistry. Statistical analyses of the data collected were performed with SPSS software. We found that the cytoplasmic staining of NGAL, NGALR and ferritin, as well as the nuclear staining of Ki67 were significantly up-regulated in CRC tissues compared with normal colorectal tissues. Expression of NGAL was related to the deeper invasion of CRC (P=0.026), while NGALR was significantly associated with a deeper invasion (P=0.018) and a high degree of Tumor, Node and Metastasis stages (P=0.042) in CRC. The NGAL/NGALR co-expression was associated with poor cellular differentiation (P=0.004). Positive correlations between NGAL and NGALR (r=0.432, P<0.01), NGAL and ferritin (r=0.374, P<0.001), NGALR and Ki67 (r=0.228, P<0.05), NGAL/NGALR co-expression and ferritin (r=0.349, P<0.001), as well as NGAL/NGALR co-expression and Ki67 (r=0.205, P<0.05) were observed. However, the expression of NGAL or NGALR was not significantly associated with patient survival. These findings detected an elevated expression of NGAL and NGALR resulting in poor cellular differentiation and a deeper invasion of CRC. Thus, NGALR may be a novel target for the treatment of CRC.

NGAL decreases E-cadherin-mediated cell–cell adhesion and increases cell motility and invasion through Rac1 in colon carcinoma cells

Expression of neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin2, a recently recognized iron regulatory protein that binds to matrix metalloproteinase-9 (MMP9), is increased in a spectrum of cancers, including those of the colorectum. Using colon carcinoma cell lines stably transfected with NGAL or antisense NGAL, we showed that NGAL overexpression altered subcellular localization of E-cadherin and catenins, decreased E-cadherin-mediated cell–cell adhesion, enhanced cell–matrix attachment, and increased cell motility and in vitro invasion. Conversely, a decrease in NGAL enhanced more aggregated growth pattern and decreased in vitro invasion. We further showed that NGAL exerted these effects through the alteration of the subcellular localization of Rac1 in an extracellular matrix-dependent, but MMP9-independent, manner. Furthermore, we observed that the NGAL-overexpressing cells tolerated increased iron levels in the culture environment, whereas the NGAL-underexpressing cells showed significant cell death after prolonged incubation in high-iron condition. Thus, overexpressing NGAL in colon carcinomas is an important regulatory molecule that integrates extracellular environment cues, iron metabolism, and intracellular small GTPase signaling in cancer migration and invasion. NGAL may therefore be a new target for therapeutic intervention in colorectal carcinoma.