Development of a magnetic bead-based method for the collection of circulating extracellular vesicles.

Cells release different types of extracellular vesicles (EVs). These EVs contain biomolecules, including proteins and nucleic acids, from their parent cells, which can be useful for diagnostic applications. The aim of this study was to develop a convenient procedure to collect circulating EVs with detectable mRNA or other biomolecules. Magnetic beads coated with annexin A5 (ANX-beads), which bound to phosphatidylserine moieties on the surfaces of most EVs, were tested for their ability to capture induced apoptotic bodies in vitro and other phosphatidylserine-presenting vesicles in body fluids. Our results show that up to 60% of induced apoptotic bodies could be captured by the ANX-beads. The vesicles captured from cultured media or plasma contained amplifiable RNA. Suitable blood samples for EV collection included EDTA-plasma and serum but not heparin-plasma. In addition, EVs in plasma were labile to freeze-and-thaw cycles. In rodents xenografted with human cancer cells, tumor-derived mRNA could be detected in EVs captured from serum samples. Active proteins could be detected in EVs captured from ascites but not from plasma. In conclusion, we have developed a magnetic bead-based procedure for the collection of EVs from body fluids and proved that captured EVs contain biomolecules from their parent cells, and therefore have great potential for disease diagnosis.

Detection of reactive oxygen species during the cell cycle under normal culture conditions using a modified fixed-sample staining method.

We developed an alternative method of simultaneously monitoring the generation of reactive oxygen species (ROS) and cellular oxidative responses using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF) in fixed samples. In this study, we evaluated the ability of this method to detect ROS generation during the cell cycle under normal culture conditions using flow cytometric analyses. Among the fixatives tested, only acetone and paraformaldehyde did not alter the endogenous oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which is a chloromethyl derivative of H2DCFDA. Only acetone fixation followed by staining with propidium iodide was able to detect ROS generation during the cell cycle without altering DCF oxidation. Further thymidine treatment led to cell cycle arrest at the G1 phase followed by the downregulation of total intracellular ROS. Paraformaldehyde-based fixation enabled the evaluation of ROS generation by immunostaining at a different phase of the cell cycle, whereas MPM2 co-staining enabled identification of the specific mitotic phase. This study demonstrates a modified fixed-sample method that can be used to measure intracellular ROS production during the cell cycle using standard immunostaining techniques.

Rosiglitazone regulates anti-inflammation and growth inhibition via PTEN.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonist has anti-inflammatory and anticancer properties. However, the mechanisms by which PPARγ agonist rosiglitazone interferes with inflammation and cancer via phosphatase and tensin homolog-(PTEN)-dependent pathway remain unclear. We found that lower doses (<25 µ M) of rosiglitazone significantly inhibited lipopolysaccharide-(LPS)-induced nitric oxide (NO) release (via inducible nitric oxide synthase, iNOS), prostaglandin E2 (PGE2) production (via cyclooxygenase-2, COX-2), and activation of Akt in RAW 264.7 murine macrophages. However, rosiglitazone did not inhibit the production of reactive oxygen species (ROS). In PTEN knockdown (shPTEN) cells exposed to LPS, rosiglitazone did not inhibit NO release, PGE2 production, and activation of Akt. These cells had elevated basal levels of iNOS, COX-2, and ROS. However, higher doses (25-100 µ M) of rosiglitazone, without LPS stimulation, did not block NO release and PGE2 productions, but they inhibited p38 MAPK phosphorylation and blocked ROS generation in shPTEN cells. In addition, rosiglitazone caused G1 arrest and reduced the number of cells in S + G2/M phase, leading to growth inhibition. These results indicate that the anti-inflammatory property of rosiglitazone is related to regulation of PTEN independent of inhibition on ROS production. However, rosiglitazone affected the dependence of PTEN-deficient cell growth on ROS.

Blockade of reactive oxygen species and Akt activation is critical for anti-inflammation and growth inhibition of metformin in phosphatase and tensin homolog-deficient RAW264.7 cells.

CONTEXT: Metformin is widely used for treatment of type 2 diabetes and has a potential application on the treatment of inflammation and cancer. Phosphatase and tensin homolog (PTEN) plays a critical role in cancer cell growth and inflammation; however, precise mechanisms remain unclear. OBJECTIVE: We aimed to investigate the possible mechanisms of how PTEN regulates metformin against cell growth and inflammation. MATERIALS AND METHODS: We established PTEN knockdown in RAW264.7 murine macrophages (shPTEN cells) to detect inflammatory mediators using commercial kits, production of reactive oxygen species (ROS) by flow cytometry, cell growth by MTT assay and phosphorylated levels of signal molecules by western blot. RESULTS: The shPTEN cells had a significant large amount of inflammatory mediators, such as inducible nitric oxide synthase (iNOS)/nitric oxide (NO) and cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)); and also elevated the production of ROS and increased cell proliferation. These effects were accompanied with the activation of Akt and p38 mitogen-activated protein kinase (MAPK), and the inactivation of an AMP-activated protein kinase (AMPK) activator and extracellular signal-regulated kinase 1/2. Pretreatment with metformin not only blocked these inflammatory mediators, but also caused growth inhibition induced by significant apoptosis. Furthermore, inactivation of Akt, blockade of ROS generation and independence of activations of AMPK and MAPK by metformin were also observed. CONCLUSION: Macrophages with PTEN deficiency developed a continuous inflammatory microenvironment, which further aggravated tumor cell growth. Moreover, metformin affected PTEN-deficient cells dependent of inhibition of ROS production and Akt activation against enlarged inflammatory mediators and/or cell growth in shPTEN cells.

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses.

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H(2)O(2))-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), a chloromethyl derivative of H(2)DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H(2)O(2)-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H(2)O(2)-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.

Glycogen synthase kinase-3ß regulates anti-inflammatory property of fluoxetine.

A selective serotonin reuptake inhibitor fluoxetine not only is widely used in the treatment of depression but also has an anti-inflammatory property. Glycogen synthase kinase-3beta (GSK-3ß) is a vital factor in the inflammation process. How fluoxetine interferes with inflammation via a GSK-3ß-dependent pathway remains unclear. The aim of this study is to investigate the effects of fluoxetine on lipopolysaccharide (LPS)-induced inflammation. Results showed that fluoxetine decreased mortality rate of the mice. It also inhibited LPS-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) in serum and RAW264.7 murine macrophages and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Upon LPS stimulation, fluoxetine caused a delay but increased in the phosphorylated levels of GSK-3ß (ser9), whereas it did not affect LPS-induced activation of mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS). Fluoxetine in combination with phosphatidylinositol 3-kinases/Akt inhibitors (LY294002 and Wortmannin) did not have a synergistic inhibition on LPS-induced NO release and PGE2 production. In addition, peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9622 showed no reverse effects of this inhibition of fluoxetine. GSK-3ß knockdown blocked the inhibitory effects of fluoxetine on LPS-induced iNOS/NO release and COX-2/PGE2 production. These results indicated that GSK-3ß regulated anti-inflammatory property of fluoxetine. However, Akt activation, ROS generation, and altered PPARγ activity were not involved in this inhibition of fluoxetine.

Glycogen synthase kinase-3ß is critical for interferon-α-induced serotonin uptake in human Jurkat T cells.

Dysregulation of glycogen synthase kinase (GSK)-3ß contributes to the pathophysiology of mood disorders. However, how its regulation is responsible for the functioning of serotonin (5-HT) requires further investigation. Although enhancement of T-cell function may present an alternative strategy to treat depression, the precise mechanisms have yet to be established. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin transporter (5-HTT) expression and induces 5-HT uptake in T cells. The present study is to examine GSK-3ß regulation on IFN-α-induced 5-HTT functions. GSK-3ß short hairpin RNAs (shRNAs) or GSK-3ß inhibitors decreased IFN-α-induced 5-HT uptake and 5-HTT expression. Src activation and calcium/calcium-activated calmodulin kinase II (CaMKII) were involved in IFN-α-induced phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) (Tyr402) and GSK-3ß (Tyr216), which regulated 5-HT uptake. GSK-3ß knockdown blocked the IFN-α-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and signal transducer and transactivator (STAT) 1. In addition to inhibiting ERK, a selective 5-HTT inhibitor fluoxetine blocked IFN-α-induced activations of Src, CaMKII-regulated Pyk2/GSK-3ß cascade, as well as STAT1 activation and translocation. These results indicated that calcium/CaMKII- and Src-regulated Pyk2 participated in IFN-α-induced GSK-3ß activation and GSK-3ß-regulated 5-HT uptake. GSK-3ß signaling facilitated IFN-α-activated STAT1 by regulating ERK1/2, which controlled 5-HT uptake. Fluoxetine interfered with the Pyk2/GSK-3ß cascade, thereby inhibiting IFN-α-induced 5-HT uptake.

The acid sphingomyelinase inhibitors block interferon-α-induced serotonin uptake via a COX-2/Akt/ERK/STAT-dependent pathway in T cells.

Sphingomyelinase (SMase) regulates an activation of the sphingomyelin cycle. Recent studies have shown that it is a novel modulator of monoamine receptor and transporter functions; however, its mechanisms are not fully understood. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin (5-HT) transporter expression and induces 5-HT uptake via an extracellular signal-regulated kinase (ERK)1/2-dependent pathway in T cells, which is blocked by a selective 5-HT transporter inhibitor fluoxetine. In the present study, we further investigated the roles of various SMase inhibitors in IFN-α-induced 5-HT uptake, including sphingolactone-24 (sph24) for neutral SMase or tricyclodecan-9-yl-xanthogenate (D609) for acid SMase. Pretreatments with Sph24 and D609 inhibited IFN-α-induced 5-HT uptake, and activation of ERK1/2 and signal transducer and transactivator (STAT) 1 and STAT3. The elevated protein levels of pro-inflammatory enzyme cycloxygenase (COX)-2 were observed upon IFN-α stimulation. The COX-2 inhibitor celecoxib blocked IFN-α-induced COX-2 expression, 5-HT uptake and activation of Akt, ERK and STAT. Moreover, a PI3K/Akt inhibitor Wortamannin blocked IFN-α-induced 5-HT uptake and activation of Akt and ERK. D609 also blocked IFN-α-induced COX-2 and Akt activation. Contrarily, sph24 did not result in these effects. Furthermore, fluoxetine as an acid SMase inhibitor lowered IFN-α-induced SMase activity as well as attenuated COX-2, Akt, ERK, and STAT activation. These results suggest that inhibiting SMase attenuates IFN-α-induced ERK and STAT activation to regulate 5-HT uptake. Moreover, inhibition of COX-2 induction and an Akt-dependent pathway are involved in IFN-α-induced 5-HT uptake by the blockade of acid SMase activity.

Dermatophagoides pteronyssinus 2 regulates nerve growth factor release to induce airway inflammation via a reactive oxygen species-dependent pathway.

Group 2 allergen of Dermatophagoides pteronyssinus 2 (Der p2) induces airway inflammation without protease activity, and elevated nerve growth factor (NGF) levels are also found in this inflammation. How the allergen Der p2 regulates NGF release via reactive oxygen species (ROS) to induce inflammation remains unclear. In the present study, intratracheal administration of Der p2 to mice led to inflammatory cell infiltration, mucus gland hyperplasia, and NGF upregulation in the bronchial epithelium, as well as elevated ROS and NGF production in bronchoalveolar lavage fluids. In addition, Der p2 caused fibrocyte accumulation and mild fibrosis. p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) inhibitors inhibited Der p2-induced NGF release in LA4 lung epithelial cells and MLg lung fibroblasts. Pretreatment with an antioxidant, tiron, reduced the Der p2-induced ROS production, NGF expression and release, p38 MAPK or JNK phosphorylation, and airway inflammation. These results suggest that Der p2 allergen-induced airway inflammation and elevated NGF release were through increasing ROS production and a MAPK-dependent pathway. The use of an antioxidant, tiron, may provide a new therapeutic modality for the treatment of allergic asthma.