Encapsulation of cinnamon oil in whey protein counteracts the disturbances in biochemical parameters, gene expression, and histological picture of the liver and pancreas of diabetic rats.

This study aimed to evaluate the protective role of encapsulated cinnamon oil emulsion (COE) in whey protein concentrate (WPC) against the disturbance in lipid profile, oxidative stress markers, and gene expression in streptozotocin (STZ)-treated rats. COE was analyzed using GC-MS, and the emulsion was prepared and characterized. In the in vivo study, six groups of male rats were treated orally for 4 weeks, including the control group, the group treated with STZ (D-rats), the groups received a low or high dose of COE (200 or 400 mg/kg B.w.), and the D-rats groups received COE at the low or high dose. Blood and tissue samples were collected after the end of the treatment period for biochemical, genetical, and histological analyses. The GC-MS results revealed that the major components of the oil were cinnamaldehyde, 1,8 cineole, acetic acid, 1,7,7-trimethylbicyclo[2.2.1]hept2yl ester, α-Pinene, and α-Terpineol. The size, zeta potential, and polydispersity index (PDI) of COE were 240 ± 1.03 nm, - 7.09 ± 0.42, and 0.36, respectively. The in vivo results revealed that COE at the two tested doses improved the levels of glucose, insulin, amylase, lipid profile, hepatic MDA, SOD, and GSH. COE also downregulated hepatic GLU2, FAS, SREBP-1c, and PEPCK gene expression and upregulated IGF-1 mRNA expression in diabetic rats in a dose-dependent manner. Moreover, COE improved and the histological picture of the liver and pancreas. It could be concluded that COE overcomes the disturbances in biochemical, cytological, and histopathological changes in D-rats via the enhancement of antioxidant capacity; reduces the oxidative stress; modulates the concerned gene expression; and may be promising to develop new drugs for diabetic treatment.

EphA2 targeted intratumoral therapy for non-small cell lung cancer using albumin mesospheres.

Lung cancer, primarily non-small cell lung cancer (NSCLC), is the leading cause of cancer mortality and the prognosis of patients with advanced or metastatic NSCLC is poor. Despite significant advances in diagnosis and treatment, little improvement has been seen in NSCLC mortality. Recently, Intratumoral Chemotherapy, a direct local delivery of chemotherapeutic drugs, has shown promise in clinical studies. However, toxicity and high dosage of chemotherapeutic agents used for treatment are a limitation. Moreover, these drugs damage indiscriminately, cancerous as well as normal tissues. Thus, a novel therapeutic strategy that targets only malignant tissue sparing normal tissue becomes an urgent issue. Ephrin receptor-A2 (EphA2), a new biomarker, is over-expressed in NSCLC, but not on normal epithelial cells. Receptor EphA2 is a cell surface protein, which upon binding to its ligand EphrinA1 undergo phosphorylation and degradation which attenuates NSCLC growth. Targeting the tumor, sparing the normal tissue and enhancing the therapeutic effects of ligand proteins are the goal of this project. Thus a novel method, intratumoral EphA2 targeted therapy, has been developed to target the oncogenic receptors on tumor tissue by using albumin mesosphere (AMS) conjugated ephrinA1 in mice bearing NSCLC tumors.

Heme Oxygenase-1 Deficiency Diminishes Methicillin-Resistant Staphylococcus aureus Clearance Due to Reduced TLR9 Expression in Pleural Mesothelial Cells.

Methicillin Resistant Staphylococcus aureus (MRSA) cause pneumonia and empyema thoraces. TLR9 activation provides protection against bacterial infections and Heme oxygenase-1 (HO-1) is known to enhance host innate immunity against bacterial infections. However, it is still unclear whether HO-1 regulates TLR-9 expression in the pleura and modulates the host innate defenses during MRSA empyema. In order to determine if HO-1 regulates host innate immune functions via modulating TLR expression, in MRSA empyema, HO-1+/+ and HO-1-/- mouse pleural mesothelial cells (PMCs) were infected with MRSA (1:10, MOI) in the presence or absence of Cobalt Protoporphyrin (CoPP) and Zinc Protoporphyrin (ZnPP) or CORM-2 (a Carbon monoxide donor) and the expression of mTLR9 and mBD14 was assessed by RT-PCR. In vivo, HO-1+/+ and HO-1-/- mice were inoculated with MRSA (5x106 CFU) intra-pleurally and host bacterial load was measured by CFU, and TLR9 expression in the pleura was determined by histochemical-immunostaining. We noticed MRSA inducing differential expression of TLR9 in HO-1+/+ and HO-1 -/- PMCs. In MRSA infected HO-1+/+ PMCs, TLR1, TLR4, and TLR9 expression was several fold higher than MRSA infected HO-1-/- PMCs. Particularly TLR9 expression was very low in MRSA infected HO-1-/- PMCs both in vivo and in vitro. Bacterial clearance was significantly higher in HO-1+/+ PMCs than compared to HO-1-/- PMCs in vitro, and blocking TLR9 activation diminished MRSA clearance significantly. In addition, HO-1-/- mice were unable to clear the MRSA bacterial load in vivo. MRSA induced TLR9 and mBD14 expression was significantly high in HO-1+/+ PMCs and it was dependent on HO-1 activity. Our findings suggest that HO-1 by modulating TLR9 expression in PMCs promotes pleural innate immunity in MRSA empyema.

MicroRNA-302b targets Mcl-1 and inhibits cell proliferation and induces apoptosis in malignant pleural mesothelioma cells.

MicroRNAs belonging to the miR-302 family are emerging as key players in the control of cell growth, and maintaining pluripotency during cell fate determination and differentiation in embryonic stem cells. However, the mechanisms whereby ephA2/ephirnA1 signaling regulates miR-302b expression and attenuates malignant pleural mesothelioma (MPM) cell growth are not known. Our study identified a novel mechanism of ephrin-A1 mediated anti-oncogenic signaling in MPM. Ephrin-A1 treatment up regulates miR-302b expression in MPM cells and attenuates cell proliferation and tumorsphere formation via repression of myeloid cell leukemia-1 (Mcl-1). The expression of miR-302b was analyzed by qPCR, the expression of Mcl-1 was analyzed by RT-PCR, immuno-blotting and Immunofluorescence staining. To confirm that ephrin-A1 regulates the expression of Mcl-1 mRNA through miR-302b up regulation, cells were transfected with and without miR-302b and miR-302b inhibitor prior to ephrinA1 treatment. The cell proliferation and tumorsphere formation was measured by WST-1 and matrigel assays respectively. In addition, to confirm the binding of miR-302b to the 3'UTR of Mcl-1 Luciferase assay was performed. Ephrin-A1 treatment induced several fold increases of miR-302b expression in MM cells. In ephrin-A1 treated MM cells, Mcl-1 expression was significantly down regulated when compared to control. Moreover, ephrin-A1 activation significantly inhibited MM cell proliferation and tumorsphere growth. Furthermore, ephrinA1 and miR-302b induced apoptosis in MM cells. The present data suggests that ephrin-A1 induces the expression of miR-302b in MM cells which targets Mcl-1 thereby inhibits MM tumorsphere growth by inducing apoptosis.

Silencing Receptor EphA2 Enhanced Sensitivity to Lipoplatin™ in Lung Tumor and MPM Cells.

Receptor EphA2 is overexpressed in lung cancer and malignant pleural mesothelioma (MPM) which promote tumorogenesis. Lipoplatin™, a new liposomal cisplatin formulation, is used against resistant tumors. Use of cisplatin-based drugs leads to unacceptable toxicities. To improve the effectiveness of Lipoplatin, enhancing the cellular sensitivity of lung tumor and MPM cells is critical. Therefore, we targeted receptor EphA2 by silencing interference RNA (siRNA) and treated tumor cells with Lipoplatin. The combined effects of siRNA-EphA2 and Lipoplatin were determined. We report that silencing EphA2 significantly enhanced the cellular sensitivity of lung tumor and MPM cells to Lipoplatin and maybe a potential therapy for lung cancer.

Nanoparticle-based targeted gene therapy for lung cancer.

Despite striking insights on lung cancer progression, and cutting-edge therapeutic approaches the survival of patients with lung cancer, remains poor. In recent years, targeted gene therapy with nanoparticles is one of the most rapidly evolving and extensive areas of research for lung cancer. The major goal of targeted gene therapy is to bring forward a safe and efficient treatment to cancer patients via specifically targeting and deterring cancer cells in the body. To achieve high therapeutic efficacy of gene delivery, various carriers have been engineered and developed to provide protection to the genetic materials and efficient delivery to targeted cancer cells. Nanoparticles play an important role in the area of drug delivery and have been widely applied in cancer treatments for the purposes of controlled release and cancer cell targeting. Nanoparticles composed of artificial polymers, proteins, polysaccharides and lipids have been developed for the delivery of therapeutic deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences to target cancer. In addition, the effectiveness of cancer targeting has been enhanced by surface modification or conjugation with biomolecules on the surface of nanoparticles. In this review article we provide an overview on the latest developments in nanoparticle-based targeted gene therapy for lung cancers. Firstly, we outline the conventional therapies and discuss strategies for targeted gene therapy using nanoparticles. Secondly, we provide the most representative and recent researches in lung cancers including malignant pleural mesothelioma, mainly focusing on the application of Polymeric, Lipid-based, and Metal-based nanoparticles. Finally, we discuss current achievements and future challenges.

Cisplatin loaded albumin mesospheres for lung cancer treatment.

The low solubility of cisplatin in aqueous solution limits the treatment effectiveness and the application of cisplatin in various kinds of drug-eluting devices. Although cisplatin has a high solubility in Dimethyl sulfoxide (DMSO), the toxicity of cisplatin can be greatly reduced while dissolved in DMSO. In this study, the solid powder of cisplatin-loaded albumin mesospheres (CDDP/DMSO-AMS), in a size range of 1 to 10 µm, were post-loaded with cisplatin and showed high cisplatin content (16% w/w) and effective cytotoxicity to lung cancer cells. Cisplatin were efficiently absorbed into the albumin mesospheres (AMS) in DMSO and, most importantly, the toxicity of cisplatin was remained at 100% after the loading process. This CDDP/DMSO-AMS was designed for the intratumoral injection through the bronchoscopic catheter or dry powder inhalation (DPI) due to its high stability in air or in solution. This CDDP/DMSO-AMS showed a fast cisplatin release within 24 hours. In the in vitro study, CDDP/DMSO-AMS showed high effectiveness on killing the lung cancer cells including the non-small cell lung cancer (NCL-H23 and A549), malignant mesothelioma (CRL-2081) and the mouse lung carcinoma (Lewis lung carcinoma) cell lines. The albumin based mesospheres provide an ideal loading matrix for cisplatin and other metal-based drugs due to the high swelling degree and fast uptake rate in the organic solvents with high polarity. In addition, to investigate the effects of polysaccharides, such as chitosan and chondroitin, on enhancing loading efficiency and lasting cytotoxicity of cisplatin, the polysaccharide-modified albumin mesospheres were synthesized and loaded with cisplatin in this study.

FBXO11 promotes ubiquitination of the Snail family of transcription factors in cancer progression and epidermal development.

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFß and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarily conserved and critically governs carcinoma progression and mammalian epidermal development.

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling.

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

Fluticasone furoate is more effective than mometasone furoate in restoring tobacco smoke inhibited SOCS-3 expression in airway epithelial cells.

Fluticasone furoate (FF) and mometasone furoate (MF) are potent glucocorticoids recommended for the treatment of allergic rhinitis and other inflammatory diseases. However, whether these drugs render any anti-inflammatory effects in Chronic Obstructive Pulmonary Disease (COPD) is unclear. Emerging data on suppressors of cytokine signaling-3 (SOCS-3) activation in the lungs during inflammation suggests that SOCS3 can be potential targets for regulating pulmonary inflammatory responses in COPD. In this study, we compared the effect of FF with MF on SOCS-3 expression in tobacco smoke (TS) exposed BAEpCs in vitro and in a mouse model of COPD in vivo. BAEpCs were exposed to TS or room air and later were treated with either FF (1nmol-100nmol) or MF (10-500nmol) inhibitors in the presence and absence of Jak1 and Stat-3 inhibitors. C57BL/6 mice were exposed to TS for 6 months, and treated with either FF, MF for 2 and 4 weeks. FF induced 7 fold increases in SOCS-3 expression in BAEpCs whereas MF induced a three fold increase when compared to control. Jak1 and Stat-3 inhibitors significantly inhibited the FF and MF induced SOCS-3 expression in BAEpCs. In addition, FF and MF restored TS inhibited SOCS-3 expression in the airway epithelium of COPD mice. FF and MF treatments significantly reduced leukocyte infiltration in airways and inhibited lung inflammation. Our study elucidates a novel mechanism for the anti-inflammatory action of FF in COPD. The superior efficacy of FF may be in part due to the increased expression of SOCS-3 in BAEpCs.

Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer.

MicroRNAs (miRs) are small noncoding RNA sequences that negatively regulate the expression of target genes by posttranscriptional repression. miRs are dysregulated in various diseases, including cancer. let-7a miR, an antioncogenic miR, is downregulated in lung cancers. Our earlier studies demonstrated that let-7a miR inhibits tumor growth in malignant pleural mesothelioma (MPM) and could be a potential therapeutic against lung cancer. EphA2 (ephrin type-A receptor 2) tyrosine kinase is overexpressed in most cancer cells, including MPM and non-small-cell lung cancer (NSCLC) cells. Ephrin-A1, a specific ligand of the EphA2 receptor, inhibits cell proliferation and migration. In this study, to enhance the delivery of miR, the miRs were encapsulated in the DOTAP (N-[1-(2.3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium)/Cholesterol/DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000])-PEG (polyethylene glycol)-cyanur liposomal nanoparticles (LNP) and ephrin-A1 was conjugated on the surface of LNP to target receptor EphA2 on lung cancer cells. The LNP with an average diameter of 100 nm showed high stability, low cytotoxicity, and high loading efficiency of precursor let-7a miR and ephrin-A1. The ephrin-A1 conjugated LNP (ephrin-A1-LNP) and let-7a miR encapsulated LNP (miR-LNP) showed improved transfection efficiency against MPM and NSCLC. The effectiveness of targeted delivery of let-7a miR encapsulated ephrin-A1 conjugated LNP (miR-ephrin-A1-LNP) was determined on MPM and NSCLC tumor growth in vitro. miR-ephrin-A1-LNP significantly increased the delivery of let-7a miR in lung cancer cells when compared with free let-7a miR. In addition, the expression of target gene Ras was significantly repressed following miR-ephrin-A1-LNP treatment. Furthermore, the miR-ephrin-A1-LNP complex significantly inhibited MPM and NSCLC proliferation, migration, and tumor growth. Our results demonstrate that the engineered miR-ephrin-A1-LNP complex is an effective carrier for the targeted delivery of small RNA molecules to lung cancer cells. This could be a potential therapeutic approach against tumors overexpressing the EphA2 receptor.

Arginine-conjugated albumin microspheres inhibits proliferation and migration in lung cancer cells.

Arginine is one of the essential amino acid involved in numerous biosynthetic pathways that significantly influence tumor growth. It has been demonstrated that arginine is effective to inhibit proliferation of cancer cells when an appropriate dose is applied. Generally, induction of cell death requires high concentration of arginine while low concentration of arginine facilitates cell proliferation. In addition to the apoptosis induced by metabolism of arginine, it has also been reported that in an ideal solution environment, arginine may assemble into arginine clusters to kill cancer cells. Therefore, to make the arginine an effective anticancer agent, arginine/albumin microspheres were designed and synthesized to provide a localized high concentration of arginine on tumor sites. In addition, the arginine/albumin mesospheres (AAMS) are also expected to provide an arginine-rich surface on microspheres, which is similar to the arginine cluster, to effectively inhibit tumor growth. In this study, the AAMS were synthesized through a water/organic solvent emulsion system and the surface properties were characterized. The in vitro effects of AAMS on A549, CRL-2081, MAK9 lung cancer cells (LCC) proliferation, migration, and tumor growth were determined. The expression of oncogenic protein EphA2 and transcription factor slug was also determined. AAMS significantly inhibited the cell proliferation, cell migration and tumor growth in all the three LCC, while same concentration of free arginine promoted the LCC tumor growth and migration. Our studies indicate that the synthesized AAMS has a more effective inhibiting effect on proliferation, migration and tumor growth of LCC than freely released arginine. The expression of EphA2 receptor mRNA was significantly decreased when compared to control cells. In addition the mRNA expression of transcription factor slug was also inhibited by AAMS suggesting that AAMS affects the expression of EphA2 and slug and may regulate LCC proliferation and migration. These data suggests that the AAMS can be an ideal delivery vehicle for therapeutic interventions against LCCs.

Ephrin-A1 inhibits NSCLC tumor growth via induction of Cdx-2 a tumor suppressor gene.

BACKGROUND: Tumor formation is a complex process which involves constitutive activation of oncogenes and suppression of tumor suppressor genes. Receptor EphA2 and its ligand ephrin-A1 form an important cell communication system with its functional role in cell-cell interaction and tumor growth. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential. Claudins, the integrated tight junction (TJ) cell-cell adhesion proteins located on the apico-lateral portion of epithelial cells, functions in maintaining cell polarity. There is extensive evidence implicating Eph receptors and ephrins in malignancy, but the mechanisms how these molecular players affect TJ proteins and regulate tumor growth are not clear. In the present study we hypothesized that EphA2 signaling modulates claudin-2 gene expression via induction of cdx-2, a tumor suppressor gene in NSCLC cells. METHODS: The expression of EphA2, claudin-2 was determined in various NSCLC cell lines by using real-time quantitative polymerase chain reaction and Western blot analysis. The claudin-2 expression was also analyzed by immunofluorescence analysis. EphA2 and erk1/erk2 phosphorylation in ephrin-A1 activated cells was evaluated by Western blot analysis. The cell proliferation and tumor colony formation were determined by WST-1 and 3-D matrigel assays respectively. RESULTS: NSCLC cells over expressed receptor EphA2 and claudin-2. Ephrin-A1 treatment significantly down regulated the claudin-2 and EphA2 expression in NSCLC cells. The transient transfection of cells with vector containing ephrin-A1 construct (pcDNA-EFNA1) decreased the expression of claudin-2, EphA2 when compared to empty vector. In addition ephrin-A1 activation increased cdx-2 expression in A549 cells. In contrast over-expression of EphA2 with plasmid pcDNA-EphA2 up regulated claudin-2 mRNA expression and decreased cdx-2 expression. The transient transfection of cells with vector containing cdx-2 construct (pcMV-cdx-2) decreased the expression of claudin-2 in A549 cells. Moreover, silencing the expression of receptor EphA2 by siRNA significantly reduced claudin-2 expression and decreased cell proliferation and tumor formation. Furthermore, silencing cdx-2 gene expression before ephrin-A1 treatment increased claudin-2 expression along with increased cell proliferation and tumor growth in A549 cells. CONCLUSIONS: Our study suggests that EphA2 signaling up-regulates the expression of the TJ-protein claudin-2 that plays an important role in promoting cell proliferation and tumor growth in NSCLC cells. We conclude that receptor EphA2 activation by ephrin-A1 induces tumor suppressor gene cdx-2 expression which attenuates cell proliferation, tumor growth and thus may be a promising therapeutic target against NSCLC.

Advances in malignant pleural mesothelioma therapy: targeting EphA2 a novel approach.

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm with a poor prognosis. MPM grows from the mesothelial cells lining the surface of the lung and chest wall called Pleura. Exposure to asbestos is mainly linked to the development of MPM. Approximately 80% of the tumors are pleural in origin, and up to 3000 people are diagnosed with MPM in the United States annually. The incidence of MPM is expected to rise in the coming decades particularly in the developing countries. Although there is an increase in the awareness of danger associated with the use of asbestos, its use is still prevalent in Australia and Asia because of its durability and low cost. This further warns and adds to the mortality and morbidity of patients with MPM globally. The traditional treatment strategies have shown only modest improvement towards the disease. MPM is difficult to treat because of the fact that the time between the exposure to asbestos and the appearance of symptoms is extremely delayed, and also due to tumor involvement with the pleural surface and the adjoining tissues such as the chest wall, pericardium and sub-diaphragmatic organs. Despite advances in the diagnostic and treatment approaches the median survival rate for MPM is between 9 to 17 months. The standard care with double agent has shown modest improvement however, multimodality approach using novel targets may have potential to achieve the improvement in the survival rate. In this review we give an update on the conventional treatment modalities and discuss about various molecular targets including receptor EphA2, a novel target gene which may be considered as a biomarker for the diagnosis and treatment of MPM.

Fluticasone propionate and Salmeterol combination induces SOCS-3 expression in airway epithelial cells.

Fluticasone propionate (FP) and Salmeterol (SAL) are commonly used in combination therapy for patients with Chronic obstructive pulmonary disease (COPD). Clinical studies show that FP/SAL used in combination therapy was found to inhibit airway inflammation in COPD patients. However, the mechanisms associated with FP/SAL induced anti-inflammatory effects were not clear. We have evaluated the effect of FP/SAL and tobacco smoke (TS) on SOCS-3 and interleukine-6 expression in bronchial airway epithelial cells (BAEpCs). Human BAEpCs were exposed to TS and subsequently treated with FP or SAL alone or in combinations in the presence and absence of mitogen activated protein kinase (MAPK) inhibitors for either Erk1/Erk2, or p38 or PI3 kinase. In BAEpCs, TS induced IL-6 expression via ERK1/ERK2 MAPK pathway and FP/SAL inhibited TS mediated IL-6 expression. TS down regulated the SOCS-3 expression via activation of Erk1/Erk2, and p38 MAPK signaling. When TS exposed BAEpCs were treated with FP/SAL SOCS-3 expression was restored. FP/SAL combinations induced significantly higher expression of SOCS-3 in BAEpCs when compared to individual drug. Pretreatment with Ly294002 a PI3 MAPK inhibitor significantly attenuated FP/SAL induced SOCS-3 expression in BAEpCs. Furthermore, FP/SAL blunted TS induced phosphorylation of Erk1/Erk2 and p38 MAPK in BAEpCs. Our study suggests that TS inhibits SOCS-3, combination of FP/SAL has a profound synergistic effect on SOCS-3 induction in BAEpCs and it is dependent on PI3 kinase signaling pathway. SOCS-3 may represent a potential biomarker for understanding the efficacy and a novel anti-inflammatory mechanism of FP/SAL combination therapy in the treatment of COPD.

Silencing receptor EphA2 induces apoptosis and attenuates tumor growth in malignant mesothelioma.

Receptor EphA2 over-expression is associated with the aggressive nature of growth in malignant mesothelioma (MM) and silencing EphA2 with interference RNA suppressed MM proliferation. The mechanisms associated with targeting the EphA2 gene in MM were not clear. We sought to determine whether silencing EphA2 induces apoptosis in MM cells by either extrinsic or intrinsic pathways. The receptor EphA2 signaling pathway may provide attractive therapeutic strategy for MM. Apoptosis was determined by Cell Death ELISA in MM Cells transfected with siRNA-EphA2 and control siRNA. The gene expression profile of apoptosis pathways were analyzed by GEArray. Selected genes were further studied by quantitative PCR, Western analysis, and immunofluorescence. Caspases activities were measured by fluorescence spectrometer. Silencing EphA2 expression induced apoptosis in MMC. Apoptosis was characterized by FADD expression, activated caspase-8, caspase-3 and induction of Bax, Bak, and Bid as revealed by GEArray and protein fractionation assays. The expression of FADD, Bid, caspase-8, cytochrome-c and apaf-1 were significantly higher in the cytosolic fractions of EphA2-siRNA transfected cells. Furthermore, blocking the expression of caspase-8 by an inhibitor blunted FADD expression, indicating that caspase-8 is implicated in EphA2-siRNA induced apoptosis in MMC. Our data indicates that targeting the EphA2 gene by siRNA induced both extrinsic and intrinsic apoptotic pathways in MM Cells. Receptor EphA2 inhibition may be an effective approach for inhibiting MM growth and a promising direction for MM therapy.

Targeted lung cancer therapy using ephrinA1-loaded albumin microspheres.

OBJECTIVES: EphrinA1, the ligand of EphA2 receptor tyrosine kinase, has been proven to suppress the growth of tumours. The aim of this study was to conjugate ephrinA1 on the surface of albumin microspheres and investigate the non-small cell lung carcinoma growth and migration in vitro. METHODS: Bovine serum albumin microspheres were designed and synthesized using a natural polymer albumin by emulsification chemical cross-linking. EphrinA1 was then conjugated on the surface of microspheres by imine formation. The microspheres conjugated with ephrinA1 (ephrinA1-MS) were characterized for particle size, surface morphology, loading efficiency and stability in vitro. The ephrinA1-MS were labelled with fluorescein isothiocyanate to determine phagocytosis. In addition, the effects of ephrinA1-MS on A549 cell growth and migration were determined. KEY FINDINGS: Albumin microspheres exhibited low toxicity for A549 cells (above 90% cell viability). More than 80% of microspheres were phagocytosed within 2 h of incubation. EphrinA1-MS decreased the expression of focal adhesion kinase more effectively than recombinant ephrinA1 alone. Furthermore, ephrinA1-MS showed significant inhibition of non-small cell lung cancer migration when compared with resting cells. EphrinA1-MS attenuated the growth of tumour colonies in matrigels. CONCLUSIONS: The developed ephrinA1-MS may serve as potential carriers for targeted delivery of the tumour suppressive protein ephrinA1, with minimal cytotoxic effects and greater antitumour therapeutic efficacy against non-small cell lung cancer.

Targeted delivery of amikacin into granuloma.

RATIONALE: Nontuberculous mycobacterial (NTM) infection is a growing problem in the United States and remains underrecognized in the developing world. The management of NTM infections is further complicated by several factors, including the need to use high systemic doses of toxic agents, the length of therapy, and the development of drug resistance. OBJECTIVES: We have evaluated the use of monocyte-derived dendritic cells (DCs) as a delivery vehicle for a luminescent derivative of amikacin prepared by conjugation to fluorescein isothiocyanate (FITC) (amikacin-FITC) into granulomas formed in the tissues of mice infected with Mycobacterium avium. METHODS: Amikacin-FITC was prepared and quantitative fluorescence was used to track the intracellular uptake of this modified antibiotic. The antibiotic activity of amikacin-FITC was also determined to be comparable to unmodified amikacin against M. avium. Amikacin-FITC-loaded DCs were first primed with M. avium, and then the cells were injected into the tail vein of infected mice. After 24 hours, the mice were sacrificed and the tissues were analyzed under fluorescence microscope. MEASUREMENTS AND MAIN RESULTS: We found that we were able to deliver amikacin into granulomas in a mouse model of disseminated mycobacterial infection. No increase in levels of monocyte chemoattractant protein-1 and its CCR2 as markers of inflammation were found when DCs were treated with amikacin-FITC. CONCLUSIONS: DC-based drug delivery may be an adjunct and useful method of delivering high local concentrations of antibiotics into mycobacterial granulomas.

Alpha-defensins increase lung fibroblast proliferation and collagen synthesis via the beta-catenin signaling pathway.

Alpha-defensins are released from granules of leukocytes and are implicated in inflammatory and fibrotic lung diseases. In the present study, the effects of alpha-defensins on the proliferation and collagen synthesis of lung fibroblasts were examined. We found that alpha-defensin-1 and alpha-defensin-2 induced dose-dependent increases in the incorporation of 5-bromo-2'-deoxy-uridine into newly synthesized DNA in two lines of human lung fibroblasts (HFL-1 and LL-86), suggesting that alpha-defensin-1 and alpha-defensin-2 stimulate the proliferation of lung fibroblasts. alpha-defensin-1 and alpha-defensin-2 also increased collagen-I mRNA (COL1A1) levels and protein contents of collagen-I and active/dephosphorylated beta-catenin without changes in total beta-catenin protein content in lung fibroblasts (HFL-1 and LL-86). Inhibition of the beta-catenin signaling pathway using quercetin prevented increases in cell proliferation and the protein content of collagen-I and active/dephosphorylated beta-catenin in lung fibroblasts, and in COL1A1 mRNA levels and collagen release into culture medium induced by alpha-defensin-1 and alpha-defensin-2. Knocking-down beta-catenin using small interfering RNA technology also prevented alpha-defensin-induced increases in cell proliferation and the protein content of collagen-I and active/dephosphorylated beta-catenin in lung fibroblasts, and in COL1A1 mRNA levels. Moreover, increases in the phosphorylation of glycogen synthase kinase 3beta, accumulation/activation of beta-catenin, and collagen synthesis induced by alpha-defensin-1 and alpha-defensin-2 were prevented by p38 mitogen-activated protein kinase inhibitor SB203580 and phosphoinositide 3-kinase inhibitor LY294002. These results indicate that alpha-defensin-1 and alpha-defensin-2 stimulate proliferation and collagen synthesis of lung fibroblasts. The beta-catenin signaling pathway mediates alpha-defensin-induced increases in cell proliferation and collagen synthesis of lung fibroblasts. alpha-defensin-induced activation of beta-catenin in lung fibroblasts might be caused by phosphorylation/inactivation of glycogen synthase kinase 3beta as a result of the activation of the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase/Akt pathways.

Pleural mesothelial cell transformation into myofibroblasts and haptotactic migration in response to TGF-beta1 in vitro.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis noted in the pulmonary parenchyma. Pleural mesothelial cells (PMC) are metabolically dynamic cells that cover the lung and chest wall as a monolayer and are in intimate proximity to the underlying lung parenchyma. The precise role of PMC in the pathogenesis of pulmonary parenchymal fibrosis remains to be identified. Transforming growth factor (TGF)-beta1, a cytokine known for its capacity to induce proliferative and transformative changes in lung cells, is found in significantly higher quantities in the lungs of patients with IPF. High levels of TGF-beta1 in the subpleural milieu may play a key role in the transition of normal PMC to myofibroblasts. Here we demonstrate that PMC activated by TGF-beta1 undergo epithelial-mesenchymal transition (EMT) and respond with haptotactic migration to a gradient of TGF-beta1 and that the transition of PMC to myofibroblasts is dependent on smad-2 signaling. The EMT of PMC was marked by upregulation of alpha-smooth muscle actin (alpha-SMA), fibroblast specific protein-1 (FSP-1), and collagen type I expression. Cytokeratin-8 and E-cadherin expression decreased whereas vimentin remained unchanged over time in transforming PMC. Knockdown of smad-2 gene by silencing small interfering RNA significantly suppressed the transition of PMC to myofibroblasts and significantly inhibited the PMC haptotaxis. We conclude that PMC undergo EMT when exposed to TGF-beta1, involving smad-2 signaling, and PMC may be a possible source of myofibroblasts in IPF.