Effects of ceramide C2 application on human laryngeal carcinoma cells: a cell culture study.

OBJECTIVE: In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma HEp-2 (ATCC® CCL-23™) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 µM were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp-2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent. RESULTS: MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 µM). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells' migration capability and proliferation for 24 hours. CONCLUSIONS: Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.

Macrophage migration inhibitory factor promotes glucocorticoid resistance of neutrophilic inflammation in a murine model of severe asthma.

BACKGROUND: Severe neutrophilic asthma is resistant to treatment with glucocorticoids. The immunomodulatory protein macrophage migration inhibitory factor (MIF) promotes neutrophil recruitment to the lung and antagonises responses to glucocorticoids. We hypothesised that MIF promotes glucocorticoid resistance of neutrophilic inflammation in severe asthma. METHODS: We examined whether sputum MIF protein correlated with clinical and molecular characteristics of severe neutrophilic asthma in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. We also investigated whether MIF regulates neutrophilic inflammation and glucocorticoid responsiveness in a murine model of severe asthma in vivo. RESULTS: MIF protein levels positively correlated with the number of exacerbations in the previous year, sputum neutrophils and oral corticosteroid use across all U-BIOPRED subjects. Further analysis of MIF protein expression according to U-BIOPRED-defined transcriptomic-associated clusters (TACs) revealed increased MIF protein and a corresponding decrease in annexin-A1 protein in TAC2, which is most closely associated with airway neutrophilia and NLRP3 inflammasome activation. In a murine model of severe asthma, treatment with the MIF antagonist ISO-1 significantly inhibited neutrophilic inflammation and increased glucocorticoid responsiveness. Coimmunoprecipitation studies using lung tissue lysates demonstrated that MIF directly interacts with and cleaves annexin-A1, potentially reducing its biological activity. CONCLUSION: Our data suggest that MIF promotes glucocorticoid-resistance of neutrophilic inflammation by reducing the biological activity of annexin-A1, a potent glucocorticoid-regulated protein that inhibits neutrophil accumulation at sites of inflammation. This represents a previously unrecognised role for MIF in the regulation of inflammation and points to MIF as a potential therapeutic target for the management of severe neutrophilic asthma.

Implications and Efficacy of Aromatase Inhibitors in Combination and Monotherapy for the Treatment of Lung Cancer.

BACKGROUND: Lung tumors express high levels of aromatase enzyme compared to surrounding normal tissue. Inhibition of aromatase has emerged as a recent therapeutic approach for the treatment of breast cancer. However, the role of aromatase inhibition in lung cancer treatment requires further investigation. METHODS: The anti-proliferative effects of aromatase inhibitors were evaluated by MTT assay. Cell migration was assessed using a wound healing assay. The mechanism of cell death was determined using the annexin VFITC/ propidium iodide staining flow cytometry method. The soft agar colony formation assay evaluated cells' capability to form colonies. RESULT: Exemestane and curcumin significantly inhibited the growth of lung cancer cell lines in a dose- and timedependent manner. The IC50 values after 48 hours of treatment with exemestane were 176, 180, and 120 µM in A549, H661, and H1299, respectively. Curcumin IC50 values after 48 hours were 80, 43, and 68 µM in A549, H661, and H1299, respectively. The combined treatment of exemestane or curcumin with cisplatin, raloxifene, and celecoxib resulted in a synergistic effect in the A549 lung cell line with a combination index of less than 1, suggesting synergism. Exemestane resulted in approximately 96% inhibition of wound closure at 100 µM, while curcumin resulted in approximately 63% inhibition of wound closure at 50 µM. Exemestane and curcumin inhibited the formation of cell colonies by reducing the number and size of formed colonies of A549, H661, and H1299 cell lines in a concentration dependent manner. Exemestane and curcumin had significantly induced apoptosis in A549 cells compared to control of untreated cells. CONCLUSION: Aromatase inhibition by exemestane or curcumin had significantly inhibited the growth of lung cancer cell lines, synergized with cisplatin, raloxifene, and celecoxib, suppressed lung cancer cell migratory potential, induced apoptosis, and reduced colony formation of lung cancer cells.

Exogenous Annexin 1 inhibits Th17 cell differentiation induced by anti-TNF treatment via activating FPR2 in DSS-induced colitis.

BACKGROUND: Anti-TNF treatment has played a vital role in treating Inflammatory Bowel Disease (IBD). However, T helper 17 (Th17) cells, which facilitate the development of IBD, are not reduced and even increased during anti-TNF clinical studies. Therefore, inhibition of Th17 cells is of great significance for improving anti-TNF treatment. METHOD: DSS-induced colitis mice were treated with the anti-TNF nanobody V7 and ANX1, and the variation in intestinal Th17 cells and DCs was estimated by flow cytometry. RAW264.7 cells were used to study the differentiation mechanism of Th17 cells from colon and mesenteric lymphocyte nodes (mLN). RESULTS: Intestinal Th17 cells increased after DSS-induced colitis was well treated with V7 (10 mg/kg). Th17 cell differentiation induced by V7 was accounted for by the accumulation of the V7-TNF complex, which was phagocytized by lamina propria (LP) DCs and induced the upregulation of MHCII. V7-TNF complex phagocytized RAW264.7 (CPR) was constructed and directly induced Th17 cell differentiation in colon LPLs and mLN in vitro. After knocking down CIITA in RAW264.7 cells, the induction was inhibited. Furthermore, Annexin 1 (ANX1) was upregulated and activated the FPR2-STAT3 pathway to inhibit the differentiation of Th17 cells. Then, animal assay demonstrated that ANX1 (500 µg/kg) enhanced the therapeutic effect of V7 (10 mg/kg) on DSS-induced colitis accompanied by a decrease in Th17 cells in mLN and colon. CONCLUSION: The differentiation of Th17 cells induced by V7 was mediated by phagocytosis of V7-TNF complexes by DCs and regulated by exogenous ANX1 via activation of the FPR2-STAT3 pathway. The combination of V7 and ANX1 presented a better therapeutic effect than monotherapy on DSS-induced colitis.

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

BACKGROUND & AIMS: Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. METHODS: Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. RESULTS: Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. CONCLUSION: Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. LAY SUMMARY: We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.

Suppression of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mice by transduced Tat-Annexin protein.

We examined that the protective effects of ANX1 on 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced skin inflammation in animal models using a Tat-ANX1 protein. Topical application of the Tat-ANX1 protein markedly inhibited TPAinduced ear edema and expression levels of cyclooxygenase-2 (COX-2) as well as pro-inflammatory cytokines such as interleukin- 1 beta (IL-1 ß), IL-6, and tumor necrosis factor-alpha (TNF-α). Also, application of Tat-ANX1 protein significantly inhibited nuclear translocation of nuclear factor-kappa B (NF-κ B) and phosphorylation of p38 and extracellular signalregulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TPA-treated mice ears. The results indicate that Tat-ANX1 protein inhibits the inflammatory response by blocking NF-κ B and MAPK activation in TPA-induced mice ears. Therefore, the Tat-ANX1 protein may be useful as a therapeutic agent against inflammatory skin diseases.

Anti-inflammatory effects of Tat-Annexin protein on ovalbumin-induced airway inflammation in a mouse model of asthma.

Chronic airway inflammation is a key feature of bronchial asthma. Annexin-1 (ANX1) is an anti-inflammatory protein that is an important modulator and plays a key role in inflammation. Although the precise action of ANX1 remains unclear, it has emerged as a potential drug target for inflammatory diseases such as asthma. To examine the protective effects of ANX1 protein on ovalbumin (OVA)-induced asthma in animal models, we used a cell-permeable Tat-ANX1 protein. Mice sensitized and challenged with OVA antigen had an increased amount of cytokines and eosinophils in their bronchoalveolar lavage (BAL) fluid. However, administration of Tat-ANX1 protein before OVA challenge significantly decreased the levels of cytokines (interleukin (IL)-4, IL-5, and IL-13) and BAL fluid in lung tissues. Furthermore, OVA significantly increased the activation of mitogen-activated protein kinase (MAPK) in lung tissues, whereas Tat-ANX1 protein markedly reduced phosphorylation of MAPKs such as extracellular signal-regulated protein kinase, p38, and stress-activated protein kinase/c-Jun N-terminal kinase. These results suggest that transduced Tat-ANX1 protein may be a potential protein therapeutic agent for the treatment of lung disorders including asthma.

Inflammation 2005 - Seventh World Congress. Respiratory inflammation.

In this conference, unlike previous conferences in this series, relatively little time was given to new drug therapy approaches for the treatment of inflammation. Instead, the emphasis was on basic mechanisms with a strong bias toward Australian research. The etiology of chronic obstructive pulmonary disease (COPD) and asthma was discussed in detail with an intensive consideration of mechanisms involved in the development of corticosteroid resistance, particularly the role of histone deacetylase. Various rodent model systems for testing potential inhibitors of COPD and asthma were also presented.

Construction, expression, and characterization of a recombinant annexin B1-low molecular weight urokinase chimera in Escherichia coli.

To produce a thrombi-targeting plasminogen activator, low molecular weight single-chain urokinase gene (scuPA32k) was spliced with the full-length cDNA of annexin B1 gene (anxB1) by overlap extension method. The fused gene anxB1scuPA was ligated into pET28a vector, transformed into E. coli BL21-RIL, and then induced to express under the control of T7 promoter. The AnxB1ScuPA protein expressed amounted to 22% of the total bacterial proteins. The product was refolded, and then purified by using DEAE Sepharose fast flow ion-exchange column and Superdex S-200 gel-filtration column. HPLC analysis revealed that the final purity is about 95%. The specific activity of AnxB1ScuPA, measured as amidolytic activity, reached 100,000 IU/mg. It had a similar S2444 catalytic efficiency (kcat/Km) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chimera fully retained the components of enzymatic and membrane-binding activities of the parent molecules. In vivo test revealed that, the dogs administered with AnxB1ScuPA had less reperfusion time, higher reperfusion ratio, and less bleeding effects than those with urokinase. These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.