Selective induction of cyclooxygenase-2 plays a role in lysophosphatidic acid regulated Fas ligand cell surface presentation

Previous studies found that lysophosphatidic acid (LPA) upregulated Fas ligand (FasL) presentation on the ovarian cancer cell surface and lead to apoptosis of activated lymphocytes. In this report, we investigated the role of selective induction of cyclooxygenase-2 (Cox-2) in FasL cell surface presentation stimulated by LPA. Ovarian cancer cells pretreated with general aspirin derivative acetylsalicylic acid and specific Cox-2 inhibitor (NS-398) before stimulation with LPA, FasL cell surface presentation was significantly blocked, so was the apoptosis of activated lymphocytes mediated by increasing FasL on the ovarian cancer cell surface. Using the specific inhibitors PD98059, AG1478 or dominant-negative epidermal-growth-factor receptor (EGFR-DN) plasmid, we found that the activation of ERK1/2 played a role in Cox-2 induction, and the transactivation of EGFR worked as an upstream signaling pathway in ERK1/2 phosphorylation. This study first revealed the selective induction of Cox-2 by LPA led to FasL presentation on ovarian cancer cell surface and provide cancer cell immune privilege, and might provide important information of Cox-2 in cancer progression and Cox-2 inhibitors' application in cancer chemoprevention.     

CSE1L,a Novel Microvesicle Membrane Protein,Mediates Ras-Triggered Microvesicle Generation and Metastasis of Tumor Cells

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1–like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal–regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras–induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody–conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the “early” and “late” metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.     

Apoptosis-inducing membrane vesicles. A novel agent with unique properties

The CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novo protein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL.     

Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle

Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid.     

Identification of a conserved glycan signature for microvesicles

Microvesicles (exosomes) are important mediators of intercellular communication, playing a role in immune regulation, cancer progression, and the spread of infectious agents. The biological functions of these small vesicles are dependent on their composition, which is regulated by mechanisms that are not well understood. Although numerous proteomic studies of these particles exist, little is known about their glycosylation. Carbohydrates are involved in protein trafficking and cellular recognition. Glycomic analysis may thus provide valuable insights into microvesicle biology. In this study, we analyzed glycosylation patterns of microvesicles derived from a variety of biological sources using lectin microarray technology. Comparison of the microvesicle glycomes with their parent cell membranes revealed both enrichment and depletion of specific glycan epitopes in these particles. These include enrichment in high mannose, polylactosamine, α-2,6 sialic acid, and complex N-linked glycans and exclusion of terminal blood group A and B antigens. The polylactosamine signature derives from distinct glycoprotein cohorts in microvesicles of different origins. Taken together, our data point to the emergence of microvesicles from a specific membrane microdomain, implying a role for glycosylation in microvesicle protein sorting.     

Expression of the functional soluble form of human fas ligand in activated lymphocytes.

Fas is a type I membrane protein which mediates apoptosis. Fas ligand (FasL) is a 40 kDa type II membrane protein expressed in cytotoxic T cells upon activation that belongs to the tumor necrosis factor (TNF) family. Here, we found abundant cytotoxic activity against Fas-expressing cells in the supernatant of COS cells transfected with human FasL cDNA but not with murine FasL cDNA. Using a specific polyclonal antibody against a peptide in the extracellular region of human FasL, a protein of 26 kDa was detected in the supernatant of the COS cells. The signal sequence of granulocyte colony-stimulating factor was attached to the extracellular region of human FasL. COS cells transfected with the cDNA coding for the chimeric protein efficiently secreted the active soluble form of human FasL (sFasL). Chemical crosslinking and gel filtration analysis suggested that human sFasL exists as a trimer. Human peripheral T cells activated with phorbol myristic acetate and ionomycin also produced functional sFasL, suggesting that human sFasL works as a pathological agent in systemic tissue injury.     

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca(2+)](i)) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.     

Activation of AMP-activated protein kinase is essential for lysophosphatidic acid-induced cell migration in ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-β3 (PLC-β3) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-β3, or (CaMKKβ) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.     

In vitro activity of immunoconjugates between cisplatin and an anti-CA125 monoclonal antibody on ovarian cancer cell lines

Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker. The coupling efficiencies of the different preparations synthesized, varied between 1.10±0.42 and 2.65±1.60 mol of CDDP per mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3. The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR3 and SKOV 3, applying a kinetic crystal violet microassay. Despite the promising results obtained with the specific immuno-staining of the target cells, no significant antiproliferative activity of our immunoconjugates against the cell lines tested was observed. One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune complexes.     

Effects of bile salts on human erythrocytes. Plasma membrane vesiculation,phospholipid solubilization and their possible relationships to bile secretion

Glycocholate removed approximately 25% of the membrane acetylcholinesterase and 10% of the membrane phospholipid from intact human erythrocytes prior to the onset of cell lysis. At low concentrations (up to 6 mM), glycocholate caused human erythrocytes to become echinocytic and to pinch off microvesicles, whereas at higher concentrations glycocholate also specifically released components from the outer leaflet of the plasma membrane in a ‘soluble’ form (as defined by their presence in a 150 000 × g/60 min supernatant) and caused the cells to become stomatocytic. Whilst the phospholipid profile of the ‘soluble’ material differed from that of the whole membrane, the profile of the microvesicle fraction was similar. The microvesicles were depleted in several membrane proteins with respect to phospholipids. These observations are discussed in relation to the possible role of bile salts in the origins of biliary phospholipid and protein.     

Ca2+-Mg2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca2+ +Mg2+ dependent ATPase activities (about 9 mumol/h per mg protein). The Na+ +K+ +Mg2+ dependent ATPase activity was 3.2 mumol/h per mg (+/- 1.0, S.D., n = 3) when microvesicles were prepared according to (1) and 1.5 mumol/h per mg (+/- 1.0, S.D., n = 3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca2+ transport in mitochondria and erythrocyte membranes had no effect on Ca2+ +Mg2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca2+ +Mg2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na+ +K+ +Mg2+ ATPase was decreased. Na+ +K+ +Mg2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na+ +K+ +Mg2+ dependent ATPase activity in the eluted coated microvesicle fraction. It was concluded that Ca2+ +Mg2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-β-bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.     

Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).

The goals of this study were to determine whether the Fas-dependent apoptosis pathway is active in the lungs of patients with the acute respiratory distress syndrome (ARDS), and whether this pathway can contribute to lung epithelial injury. We found that soluble Fas ligand (sFasL) is present in bronchoalveolar lavage (BAL) fluid of patients before and after the onset of ARDS. The BAL concentration of sFasL at the onset of ARDS was significantly higher in patients who died. BAL from patients with ARDS induced apoptosis of distal lung epithelial cells, which express Fas, and this effect was inhibited by blocking the Fas/FasL system using three different strategies: anti-FasL mAb, anti-Fas mAb, and a Fas-Ig fusion protein. In contrast, BAL from patients at risk for ARDS had no effect on distal lung epithelial cell apoptosis. These data indicate that sFasL is released in the airspaces of patients with acute lung injury and suggest that activation of the Fas/FasL system contributes to the severe epithelial damage that occurs in ARDS. These data provide the first evidence that FasL can be released as a biologically active, death-inducing mediator capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury.     

Microvesicles shed by oligodendroglioma cells and rheumatoid synovial fibroblasts contain aggrecanase activity

Membrane microvesicle shedding is an active process and occurs in viable cells with no signs of apoptosis or necrosis. We report here that microvesicles shed by oligodendroglioma cells contain an 'aggrecanase' activity, cleaving aggrecan at sites previously identified as targets for adamalysin metalloproteinases with disintegrin and thrombospondin domains (ADAMTSs). Degradation was inhibited by EDTA, the metalloproteinase inhibitor GM6001 and by tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. This inhibitor profile indicates that the shed microvesicles contain aggrecanolytic ADAMTS(s) or related TIMP-3-sensitive metalloproteinase(s). The oligodendroglioma cells were shown to express the three most active aggrecanases, namely Adamts1, Adamts4 and Adamts5, suggesting that one or more of these enzymes may be responsible for the microvesicle activity. Microvesicles shed by rheumatoid synovial fibroblasts similarly degraded aggrecan in a TIMP-3-sensitive manner. Our findings raise the novel possibility that microvesicles may assist oligodendroglioma and rheumatoid synovial fibroblasts to invade through aggrecan-rich extracellular matrices.     

Role for Mycobacterium tuberculosis Membrane Vesicles in Iron Acquisition

Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host.     

Mechanisms of nonspecific cytotoxic cell regulation of apoptosis: cytokine-like activity of Fas ligand

The role of FasL/FasR pathways of immunoregulation of programmed cell death in teleost cytotoxic innate immunity has not been previously examined. In the present study, constitutive cytosolic soluble FasL (sFasL) was detected in anterior kidney (AK), peripheral blood (PBL) and liver NCC obtained from tilapia. Ligation of NCC by tumour cells caused the release of sFasL that was associated with lysis of HL-60 targets in 14 h killing assays. Evidence that sFasL mediated this activity was that anti-(human) FasL inhibited tilapia and catfish (cf.) NCC lysis of FasR+ HL-60 tumour cells. Inhibition was concentration dependent. Lysis of IM-9 targets (12% positive for FasR) by (cf.) anterior kidney and PBL NCC was only partially inhibited by anti-FasL mab. Activated NCC from both species were negative for the expression of membrane FasL and FasR. These data confirmed that NCC lyse sensitive tumour cells by multiple effector pathways. Pretreatment of (FasR+) HL-60 cells with anti-FasR mab completely inhibited cf. cytotoxicity at low (100:1) E:T ratios. Anti-FasR mab did not inhibit the lysis of IM-9 targets by cf. NCC. This study demonstrated that for catfish and tilapia, initial target cell conjugate formation was required; however, the terminal killing mechanism depended on at least two different pathways of cytotoxicity. One pathway depended on the release of preformed soluble FasL by activated NCC in the presence of FasR positive target cells. A second pathway has yet to be determined.     

Serum soluble Fas ligand (sFasL) in patients with primary squamous cell carcinoma of the esophagus

Esophageal carcinomas have been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. Circulating soluble FasL (sFasL) has been suggested to provide protection from Fas-mediated apoptosis. The aim of this study was to assess serum sFasL levels in esophageal cancer. The pretreatment levels of sFasL in the serum of 100 patients with esophageal squamous cell cancer and 41 healthy volunteers were determined by ELISA. Probability of survival was calculated according to the method of Kaplan-Meier. The prognostic influence of high and low level of sFasL was analyzed with the log-rank test. The mean serum level of sFasL in patients with esophageal cancer was significantly higher than that in healthy donors (1.567+/-1.786 vs 0.261+/-0.435, p<0.0001). The levels of serum sFasL were significantly higher in advanced stages (II vs IV p<0.034; III vs IV p<0.041; except II vs III p=0.281), patients with lymph node (N0 vs N1 p<0.0389) or distant (M0 vs. M1 p<0.0388) metastases and significantly lower in patients with well differentiated tumors (G1 vs G2 p<0.0272). The serum levels of soluble FasL were not related to gender, age, tumor size, T-stage, tobacco smoking and history of chronic alcohol intake. The survival difference between pretreatment high and low level of sFasL in surgery and chemio- and/or radiotherapy group was not statistically significant (p=0.525; p=0.840). Our results indicate that elevated serum sFasL levels might be associated with a disease progression in patients with esophageal squamous cell carcinoma.