Novas formulações de Anexina A1 como estratégia terapêutica para a colite experimental / New formulations of Annexin A1 as a therapeutic strategy to experimental colitis

A Anexina A1 (AnxA1) é uma proteína de 37 kDa que controla o desenvolvimento da reação inflamatória inata, e favorece a eferocitose e o reparo tecidual. Em doenças inflamatórias intestinais (DIIs), tanto a AnxA1 endógena, como a sintética e o peptídeo sintético mimético ao N-terminal da proteína (Ac2-26) inibem o desenvolvimento de doença e induzem a cicatrização. O presente projeto teve o objetivo de obter novas formulações para carrear a AnxA1 recombinante (rAnxA1) ou o Ac2-26 e testar suas eficácias no modelo de colite experimental induzida pelo dextram sulfato de sódio (DSS, 0-6 dias) em camundongos C57Bl/6 machos. A rAnxA1 foi funcionalizada em nanocápsulas de núcleo lipídico de parede múltipla (MLNC) pela ligação Zn2+, com alta eficência de incorporação (92%) e adminsitrada pelas vias oral, intravenosa ou intraperitoneal durante a fase latente da doença (6º-9º dia). Somente o tratamento intraperitoneal com MLNC-AnxA1 (12,5 µg/mL) reduziu significativamente os sinais clínicos da doença, restaurou a integridade da estrutura colônica e a proliferação celular, bem como aumentou expressão de junções celulares da barreira intestinal. Ainda, MLNC-AnxA1 induziu a polarização de macrófagos para o fenótipo M2 in vivo no tecido inflamado e in vitro após estímulo com lipopolissacarídeos (LPS) bacteriano. Na tentativa de obter uma formulação terapêutica com atividade por vial oral, o peptídeo Ac2-26 foi incorporado em sílica mesoporosa ordenada SBA-15 e revestidos com Eudragit® L30-D55. A incorporação do peptídeo foi efetiva (88%) e a administração oral de Eudragit-SBA15-Ac2-26 (6º-9º dia; 200 µg/camundongo; 8 mg/kg) reduziu significativamente os sintomas clínicos e inflamação. De fato, ensaios de PET-SCAN mostraram que o SBA-15 permaneceu no intestino por até 16 horas após a administração e promoveu a liberação do peptídeo no intestino inflamado. Em cultura celular de epitélio (Caco-2), Eudragit-SBA15-Ac2-26 favoreceu a internalização de Ac2-26. Em conjunto, as duas estratégias expermentais de entrega do rAnxA1 ou Ac2-26 foram eficientes e os resultados obtidos sugerem que mais estudos devem ser realizados para a confirmação das estratégias de tratamento. Com o intuito de buscar ferramentas para ampliar estes estudos, durante estágio BEPE foram realizados estudos em cultura de células epiteliais baseado em células-tronco adultas diferenciadas in vitro. Os resultados mostraram que rAnxA1 ou Ac2-26 protegeram a integridade epitelial após desafio com LPS, pela regulação positiva da expressão das junções oclusivas e aderentes e redução da expressão de claudina-2, responsável pelo aumento da permeabilidade intercelular; pela modulação negativa decitocinas pró-inflamatórias CXCL-1 e MCP-1, e positiva de citocina antiinflamatória IL-10. Desta forma, padronizamos um novo modelo de cultura celular ainda não testada para a AnxA1 ou Ac2-26, que poderá ser empregada para desvendar os mecanismos da MLNC-AnxA1 e do Eudragit-SBA15-Ac2-26

Annexin Al (AnxA1) is a 37 kDa protein that controls the development of the innate inflammatory reaction, and favors efferocytosis and tissue repair. In inflammatory bowel diseases (IBDs), both endogenous and synthetic AnxA1 and the synthetic peptide mimetic to the N-terminal of the protein (Ac2-26) inhibit the development of disease and induce healing. The present project aimed to obtain new formulations to carry recombinant AnxA1 (rAnxA1) or Ac2-26 and test their efficacy in the experimental colitis model induced by dextram sodium sulfate (DSS, 0-6 days) in C57Bl/6 mice. rAnxA1 was functionalized into multiwall lipid core nanocapsules (MLNC) by Zn2+ binding, with high incorporation efficiency (92%) and administered orally, intravenously or intraperitoneally during the latent phase of the disease (6º-9º day). Only intraperitoneal treatment with MLNC-AnxA1 (12.5 µg/mL) significantly reduced the clinical signs of the disease, restored the integrity of the colonic structure and cell proliferation, as well as increased the expression of intestinal barrier cell junctions. Furthermore, MLNC-AnxA1 induced macrophage polarization to the M2 phenotype in vivo in inflamed tissue and in vitro after stimulation with bacterial lipopolysaccharides (LPS). In an attempt to obtain a therapeutic formulation with oral activity, the Ac2-26 peptide was incorporated into ordered mesoporous silica SBA-15 and coated with Eudragit® L30-D55. Peptide incorporation was effective (88%) and oral administration of Eudragit-SBA15-Ac2-26 (6º-9º day; 200 µg/mice; 8 mg/kg) significantly reduced clinical symptoms and inflammation. PET-SCAN assays showed SBA-15 remained in the intestine for up to 16 hours after administration and promoted the release of the peptide in the inflamed intestine. In epithelial cell culture (Caco-2), SBA15-Ac2-26 favored the internalization of Ac2-26. Taken together, the two experimental delivery strategies for rAnxA1 or Ac2-26 were efficient and the results obtained suggest that more studies should be carried out to confirm the treatment strategies. In order to seek tools to expand these studies, during the BEPE internship, studies were carried out in epithelial cell cultures based on adult stem cells differentiated in vitro. The results showed rAnxA1 or Ac2-26 protected epithelial integrity after challenge with LPS, by upregulating the expression of tight and adherens junctions and reducing the expression of claudin-2, responsible for increasing intercellular permeability; by negative modulation of pro-inflammatory cytokines CXCL-1 and MCP-1, and positive modulation of anti-inflammatory cytokine IL-10. In this way, we standardized a new cell culture model that has not yet been tested for AnxA1 or Ac2-26, which could be used to unravel the mechanisms of MLNC-AnxA1 and Eudragit-SBA15-Ac2-26

Salivary annexin A1: A candidate biomarker for periodontitis.

AIM: To compare the salivary proteomic profile of periodontitis-affected (PA) parents and their offspring to periodontally healthy (PH) dyads in the pursuit of possible biomarkers for early diagnosis of this disease. MATERIALS AND METHODS: Unstimulated saliva samples collected from 17 pairs of PA or PH individuals and their children were submitted to mass spectrometric analyses followed by proteomic analyses. Primary PA fibroblasts were triggered towards having an inflammatory response, and an immunoenzymatic assay of its supernatant was performed to validate the obtained data. RESULTS: ANXA1, KRT4, GSTP1, HPX, A2M and KRT13 were lower in PA parents and their children, and IGHG1, CSTB, KRT9, SMR3B, IGHG4 and SERPINA1 were higher. ANXA1 presented the highest fold change, 7.1 times less produced in children of PA parents, and was selected as a potential biomarker for periodontitis. The in vitro assay also showed lower ANXA1 production by cells of PA patients. CONCLUSION: Before any clinical sign of periodontal loss, descendants of PA patients have an altered proteomic profile compared to PH individuals, presenting a lower abundance of ANXA1. This protein is suggested as a potential biomarker for periodontitis.

Effects of hyperosmolarity on annexin A1 on ocular surface epithelium in vitro.

Osmotic stress is an important challenge to cell function. Dry eye pathology is characterized by elevated tear film osmolarity as consequence of decreased tear secretion and/or increased evaporation. Dry eye pathogenesis is not completely clarified. However, it is known that tear hyperosmolarity induces NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain-cointaining 3) inflammasome activation and inflammatory mediators release that leads to ocular surface damage. Annexin A1 is a protein involved in anti-inflammatory or pro-resolution actions in different tissues while its presence and biological role on ocular surface has been scarcely examined. In this study, potential changes in annexin A1 protein expression and secretion on the ocular surface after exposure to hyperosmolar conditions were evaluated. In addition, considering the significant role of inflammation in dry eye pathology, the potential anti-inflammatory activity of Ac2-26, an annexin A1 peptide mimicking its N-terminus, was assessed. Cytosolic and membrane staining was detected for annexin A1 in corneal and conjunctival epithelial cells. A native form of annexin A1 together with a truncated form were detected by western blot analysis. Under hyperosmotic conditions increased protein levels of intracellular and secreted annexin A1 as well as higher expression of its receptor Fpr2 (formyl peptide receptor type 2) were found. Treatment with mimetic peptide Ac2-26 ameliorated NLRP3 activation and interleukin 1ß (IL-1ß) release triggered by elevated osmolarity in corneal and conjunctival epithelial cells. These findings suggest a potential role of annexin A1 and its mimetic peptide modulating key inflammatory events associated to dry eye.

Calculus-related functional protein expression in ureteral calculus-adhered polyp: A preliminary study.

ABSTRACT: To explore the expressions of calculus-related functional proteins in the ureteral calculus-adhered polyp tissues and investigate the role of these proteins in the formation of adhesions between the calculus and polyp.Patients with ureteral calculi and polyps who underwent ureteroscopic lithotripsy for the excision of polyps between January 2019 and June 2019 were enrolled. Polyps obtained from each patient were divided into 2 groups using a matched pairs design: observation group (polyps adhered to calculus) and control group (polyps not adhered to calculus). Histopathological examination of polyps was performed using hematoxylin and eosin staining. Polyp tissues were immunohistochemically stained to assess the expressions of calculus-related functional proteins, that is, annexin A1, calcium-binding protein S100A9 (S100A9), uromodulin, and osteopontin. Furthermore, quantitative analysis was performed using the H-score of tissue staining; Pearson correlation analysis was performed for proteins with high expression.Overall, 40 polyp specimens were collected from 20 patients with ureteral calculi combined with polyps (observation group, 20 specimens; control group, 20 specimens). Hematoxylin and eosin staining revealed obvious epithelial cell proliferation in polyps of both groups; crystals were observed in the epithelial cells of the polyp tissue in the observation group. The expression levels of annexin A1 and S100A9 in the observation group were significantly greater than those in the control group (P < .05). However, no obvious expression of osteopontin or uromodulin was observed in the polyp tissues of both groups. There was a strong correlation between the increased expressions of annexin A1 and S100A9 in the observation group (R = 0.741, P = .022).We documented increased expressions of annexin A1 and S100A9 in the ureteral calculus-adhered polyp tissues. Annexin A1 and S100A9 may play an essential role in the adhesion of calculus and polyp and the growth of calculi.

Proteomics of exhaled breath condensate in lung cancer and controls using data-independent acquisition (DIA): a pilot study.

Lung cancer, the leading cause of cancer mortality worldwide has a poor prognosis. To develop a non-invasive method for early lung cancer detection, exhaled breath condensate (EBC) was explored in this study. EBC samples were collected from lung cancer patients (n= 10) and healthy controls (n= 10), and a proteomic study was performed to identify potential biomarkers. Data-dependent acquisition was used to build the spectral library, and a data-independent acquisition (DIA) approach was applied for quantification of EBC proteomics. A total of 1151 proteins were identified, and several proteins were significantly upregulated in the lung cancer group compared to the control group. The Gene Ontology analysis revealed that most of the proteins were located within several organelles in the cells and were involved in binding and catalytic activity, and the Kyoto Encyclopedia Genes and Genomes results revealed that the proteins were mainly related to organismal systems and human disease. And S100A11, ANXA1, ENO1, and FABP5 might play a vital role in the EBC proteome. In summary, we demonstrated that the DIA-based quantification method was efficient in performing proteomic analysis in individual EBC samples, and some of the proteins might be novel biomarkers for lung cancer.

Analysis of macrophage subtypes and annexin A1 expression in lesions of patients with cutaneous leishmaniasis.

INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.

Assessment of annexin A1 release during immunogenic cell death.

The protein annexin A1 (ANXA1) belongs to the danger-associated molecular patterns (DAMPs) that alert the innate immune system about tissue perturbations. In the context of immunogenic cell death (ICD), ANXA1 is released from the cytoplasm of dying cells and, once extracellular, acts on formyl peptide receptor 1 (FPR1) expressed on dendritic cells to favor long-term interactions between dying and dendritic cells. As a result, the accumulation of extracellular ANXA1 constitutes one of the hallmarks of ICD. In the past, the detection of ANXA1 was based on semiquantitative immunoblots. More recently, a commercial enzyme-linked immunosorbent assay (ELISA) has been developed to measure ANXA1 in an accurate fashion. Here, we detail the protocol to measure the concentration of ANXA1 in the supernatants of cancer cells treated with chemotherapy.

Analysis of macrophage subtypes and annexin A1 expression in lesions of patients with cutaneous leishmaniasis

Abstract INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.

Annexin A1 in plasma from patients with bronchial asthma: its association with lung function.

BACKGROUND: Annexin-A1 (ANXA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The anti-inflammatory protein ANXA1 and its N-formyl peptide receptor 2 (FPR2) have protective effects on organ fibrosis. However, the exact role of ANXA1 in asthma remains to be determined. The aim of this study was to identify the role of ANXA1 in bronchial asthma. METHODS: In mice sensitized and challenged with ovalbumin (OVA-OVA mice) and mice sensitized with saline and challenged with air (control mice), we investigated the potential links between ANXA1 levels and bronchial asthma using ELISA, immunoblotting, and immunohistochemical staining. Moreover, we also determined ANXA1 levels in blood from 50 asthmatic patients (stable and exacerbated states). RESULTS: ANXA1 protein levels in lung tissue and bronchoalveolar lavage fluid were significantly higher in OVA-OVA mice compared with control mice. FPR2 protein levels in lung tissue were significantly higher in OVA-OVA mice compared with control mice. Plasma ANXA1 levels were increased in asthmatic patients compared with healthy controls. Plasma ANXA1 levels were significantly lower in exacerbated patients compared with stable patients with bronchial asthma (p < 0.05). The plasma ANXA1 levels in controlled asthmatic patients were correlated with forced expiratory volume in 1 s (FEV1) (r = - 0.191, p = 0.033) and FEV1/forced vital capacity (FVC) (r = -0.202, p = 0.024). CONCLUSION: These results suggest that ANXA1 may be a potential marker and therapeutic target for asthma.

Annexin A1 influences in breast cancer: Controversies on contributions to tumour, host and immunoediting processes.

Annexin A1 is a multifunctional protein characterised by its actions in modulating the innate and adaptive immune response. Accumulating evidence of altered annexin A1 expression in many human tumours raises interest in its functional role in cancer biology. In breast cancer, altered annexin A1 expression levels suggest a potential influence on tumorigenic and metastatic processes. However, reports of conflicting results reveal a relationship that is much more complex than first conceptualised. In this review, we explore the diverse actions of annexin A1 on breast tumour cells and various host cell types, including stromal immune and structural cells, particularly in the context of cancer immunoediting.

Proteomics-based analysis of lung injury-induced proteins in a mouse model of common bile duct ligation.

BACKGROUND: Lung injury is a life-threatening complication in patients with liver dysfunction. We recently provided an experimental lung injury model in mouse with common bile duct ligation. In this study, we aimed to characterize the pathologic and biochemical features of lung tissues in common bile duct ligation mice using a proteomic approach. METHODS: Common bile ducts of BALB/c mice, 8 weeks of age, were ligated operatively. CD31-expressing pulmonary cells were sorted with immunomagnetic microbeads, and protein profiles were examined by 2-dimensional gel electrophoresis. Based on the results of protein identification, immunohistochemistry and quantitative reverse transcription polymerase chain reaction were carried out in pulmonary and hepatic tissues. RESULTS: Two-dimensional gel electrophoresis revealed 3 major inflammation-associated proteins exhibiting considerable increases in the number of CD31-positive pulmonary cells after common bile duct ligation. Mass spectrometry analysis identified these proteins as SerpinB1a (48 kDa), ANXA1 (46 kDa), and S100A9 (16 kDa). Furthermore, the 3 proteins were more highly expressed in dilated pulmonary blood vessels of common bile duct ligation mice, in which neutrophils and monocytes were prominent, as shown by immunohistochemistry. More importantly, SerpinB1a mRNA and protein were significantly upregulated in the liver, whereas S100A9 and ANXA1 mRNA and protein were upregulated in the lungs, as shown by quantitative reverse transcription polymerase chain reaction and Western blotting. CONCLUSION: We identified 3 proteins that were highly expressed in the lung after common bile duct ligation using a proteomics-based approach.

Proteomic characterization of the acid-insoluble fraction of whole saliva from preterm human newborns.

The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC­ESI­MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100 A6, S100 A7, S100 A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and α-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100 A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain. SIGNIFICANCE: Differential protein expression analysis of the acid insoluble fraction of saliva from preterm, at-term newborns and adults has been performed in this study by coupling 2-DE analysis and high-resolution tandem mass spectrometry in order to complete the information previously obtained by top-down LC­MS only on the acid-soluble proteome. Several proteins identified in the acid insoluble fraction of both preterm newborn and adult saliva are not of glandular origin, being only prolactin-inducible protein, salivary cystatins, α-amylase and polymeric immunoglobulin receptor exclusive of salivary glands. Three proteins resulted increased in at-term newborns with respect to preterm newborns and adults: BPI fold-containing family A member 1, two proteoforms of annexin A1 and keratin type 1 cytoskeletal 13, while several proteins were significantly increased in adults.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features.

INTRODUCTION: The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects. METHODS: Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence. RESULTS: All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions. CONCLUSIONS: Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features

ABSTRACTINTRODUCTION:The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.METHODS:Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.RESULTS:All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.CONCLUSIONS:Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.

Overexpression of ANXA1 confers independent negative prognostic impact in rectal cancers receiving concurrent chemoradiotherapy.

Neoadjuvant concurrent chemoradiation therapy (CCRT) is an increasingly common therapeutic strategy for rectal cancer. Clinically, it remains a major challenge to predict therapeutic response and patient outcomes after CCRT. Annexin I (ANXA1), encoded by ANXA1, is a Ca(2+)/phospholipid-binding protein that mediates actin dynamics and cellular proliferation, as well as suggesting tumor aggressiveness and predicting therapeutic response in certain malignancies. However, expression of ANXA1 has never been reported in rectal cancer receiving CCRT. This study examined the predictive and prognostic impact of ANXA1 expression in patients with rectal cancer following neoadjuvant CCRT. We identified ANXA1 as associated with resistance to CCRT through data mining from a published transcriptomic dataset. Its immunoexpression was retrospectively assessed using H scores on pre-treatment biopsies from 172 rectal cancer patients treated with neoadjuvant CCRT followed by curative surgery. Results were correlated with clinicopathological features, therapeutic response, tumor regression grade (TRG), and metastasis-free survival (MeFS), as well as local recurrent-free survival (LRFS) and disease-specific survival (DSS). High expression of ANXA1 was associated with advanced pre-treatment tumor status (T3, T4, p = 0.022), advanced pre-treatment nodal status (N1, N2, p = 0.004), advanced post-treatment tumor status (T3, T4, p < 0.001), advanced post-treatment nodal status (N1, N2, p = 0.001) and inferior TRG (p = 0.009). In addition, high expression of ANXA1 emerged as an adverse prognosticator for DSS (p < 0.0001), LRFS (p = 0.0001) and MeFS (p = 0.0004). Moreover, high expression of ANXA1 also remained independently prognostic of worse DSS (hazard ratio [HR] = 3.998; p = 0.007), LRFS (HR = 3.206; p = 0.028) and MeFS (HR = 3.075; p = 0.017). This study concludes that high expression of ANXA1 is associated with poor therapeutic response and adverse outcomes in rectal cancer patients treated with neoadjuvant CCRT.

Association of nuclear annexin A1 with prognosis of patients with esophageal squamous cell carcinoma.

Although recent progress has been made in the diagnosis and treatment of cancer, the prognosis of esophageal squamous cell carcinoma (ESCC) remains poor. The identification of biomarkers for ESCC prognosis is important for treatment decisions. The aim of this study was to evaluate the relationship between the expressions of Annexin A1 (ANXA1), three prime repair exonuclease 1 (TREX1) and apurinic/apyrimidinic endonuclease-1 (APE1) and clinical outcome of patients with ESCC. The expressions of ANXA1, TREX1 and APE1 in 93 pairs of ESCC and paracancerous tissues were tested using immunohistochemistry. ANX1, TREX1 and APE1 were dysregulated in ESCC. Nuclear expressions of ANXA1 and APE1 were significantly associated with pathologic type (P = 0.004 and 0.040, respectively). Patients with low expression of nuclear ANXA1 had a better prognosis than those with high expression of nuclear ANXA1 (HR = 0. 448, 95% CI 0.236-0.849, P = 0.014), especially for those with histologic grade 1 and 2 (HR = 0.303, 95% CI: 0.155-0.593, P < 0.001). In conclusion, nuclear ANXA1 may be potentially used as a prognostic biomarker for ESCC.

Identification of Annexin A1 protein expression in human gastric adenocarcinoma using proteomics and tissue microarray.

AIM: To study the differential expression of Annexin A1 (ANXA1) protein in human gastric adenocarcinoma. This study was also designed to analyze the relationship between ANXA1 expression and the clinicopathological parameters of gastric carcinoma. METHODS: Purified gastric adenocarcinoma cells (GAC) and normal gastric epithelial cells (NGEC) were obtained from 15 patients with gastric cancer by laser capture microdissection. All of the peptide specimens were labeled as ¹8O/¹6O after trypsin digestion. Differential protein expressions were quantitatively identified between GAC and NGEC by nanoliter-reverse-phase liquid chromatography-mass/mass spectrometry (nano-RPLC-MS/MS). The expressions of ANXA1 in GAC and NGEC were verified by western blot analysis. The tissue microarray containing the expressed ANXA1 in 75 pairs of gastric carcinoma and paracarcinoma specimens was detected by immunohistochemistry (IHC). The relationship between ANXA1 expression and clinicopathological parametes of gastric carcinoma was analyzed. RESULTS: A total of 78 differential proteins were identified. Western blotting revealed that ANXA1 expression was significantly upregulated in GAC (2.17/1, P < 0.01). IHC results showed the correlations between ANXA1 protein expression and the clinicopathological parameters, including invasive depth (T stage), lymph node metastasis (N stage), distant metastasis (M stage) and tumour-lymph node metastasis stage (P < 0.01). However, the correlations between ANXA1 protein expression and the remaining clinicopathological parameters, including sex, age, histological differentiation and the size of tumour were not found (P > 0.05). CONCLUSION: The upregulated ANXA1 expression may be associated with carcinogenesis, progression, invasion and metastasis of GAC. This protein could be considered as a biomarker of clinical prognostic prediction and targeted therapy of GAC.

Low Annexin A1 expression predicts benefit from induction chemotherapy in oral cancer patients with moderate or poor pathologic differentiation grade.

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.

Annexin A1: a new biomarker for predicting nasopharyngeal carcinoma response to radiotherapy.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance remains a serious obstacle to successful treatment in many cases. Therefore, the biomarkers for predicting NPC response to radiotherapy are very important for targeted therapy and individualized radiotherapy of NPC. Accumulating evidences have shown that Annexin A1 was correlated with NPC radioresistance. First, Annexin A1 is a potential tumor suppressor gene, and can regulate tumor cell proliferation and apoptosis, thus abnormal expression of Annexin A1 in NPC affects apoptosis of tumor cells induced by ionizing radiation and radiotherapeutic efficacy. Second, Annexin A1 is one of the proteins that are involved in p53-mediated radioresponse in NPC, and it might be related to NPC radioresistance. Third, the expression level of Annexin A1 is down-regulated in NPC, and is correlated with metastasis, recurrence and poor prognosis of NPC, thus Annexin A1 downregulation may increase NPC radioresistance, leading to poor prognosis. Last but not the least, Annexin A1 is closely related with tumor chemoresistance, whereas radioresistance is similar to chemoresistance in many aspects, thus Annexin A1 may also be involved in NPC radioresistance. Based on the above mentions, we hypothesize that Annexin A1 is closely correlated with NPC radioresistance and is an important new biomarker for predicting NPC response to radiotherapy.

Annexin A1 on the surface of early apoptotic cells suppresses CD8+ T cell immunity.

Prevention of an immune response against self-antigens derived from apoptotic cells is essential to preclude autoimmune and chronic inflammatory diseases. Here, we describe apoptosis induced externalization of endogenous cytosolic annexin 1 initiating an anti-inflammatory effector mechanism that suppresses the immune response against antigens of apoptotic cells. Cytosolic annexin 1 rapidly translocated to the apoptotic cell surface and inhibited dendritic cell (DC) activation induced by Toll like receptors (TLR). Annexin 1-inhibited DC showed strongly reduced secretion of pro-inflammatory cytokines (e.g. TNF and IL-12) and costimulatory surface molecules (e.g. CD40 and CD86), while anti-inflammatory mediators like PD-L1 remained unchanged. T cells stimulated by such DC lacked secretion of interferon-γ (IFN-γ) and TNF but retained IL-10 secretion. In mice, annexin 1 prevented the development of inflammatory DC and suppressed the cellular immune response against the model antigen ovalbumin (OVA) expressed in apoptotic cells. Furthermore, annexin 1 on apoptotic cells compromised OVA-specific tumor vaccination and impaired rejection of an OVA-expressing tumor. Thus, our results provide a molecular mechanism for the suppressive activity of apoptotic cells on the immune response towards apoptotic cell-derived self-antigens. This process may play an important role in prevention of autoimmune diseases and of the immune response against cancer.