Downregulation Of Adhesion Molecules Research Articles

Fraxetin reduces endometriotic lesions through activation of ER stress, induction of mitochondria-mediated apoptosis, and generation of ROS

BackgroundFraxetin, a phytochemical obtained from Fraxinus rhynchophylla, is well known for its anti-inflammatory and anti-fibrotic properties. However, fraxetin regulates the progression of endometriosis, which is a benign reproductive disease that results in low quality of life and infertility. Hypothesis/purposeWe hypothesized that fraxetin may have therapeutic effects on endometriosis and aimed to elucidate the underlying mechanisms of mitochondrial function and tiRNA regulation. Study designEndometriotic animal models and cells (End1/E6E7 and VK2/E6E7) were used to identify the mode of action of fraxetin. MethodsAn auto-implanted endometriosis animal model was established and the effects of fraxetin on lesion size reduction were analyzed. Cell-based assays including proliferation, cell cycle, migration, apoptosis, mitochondrial function, calcium efflux, and reactive oxygen species (ROS) were performed. Moreover, fraxetin signal transduction was demonstrated by western blotting and qPCR analyses. ResultsFraxetin inhibited proliferation and migration by inactivating the P38/JNK/ERK mitogen-activated protein kinase (MAPK) and AKT/S6 pathways. Fraxetin dissipates mitochondrial membrane potential, downregulates oxidative phosphorylation (OXPHOS), and disrupts redox and calcium homeostasis. Moreover, it triggered endoplasmic reticulum stress and intrinsic apoptosis. Furthermore, we elucidated the functional role of tiRNAHisGTG in endometriosis by transfection with its inhibitor. Finally, we established an endometriosis mouse model and verified endometriotic lesion regression and downregulation of adhesion molecules with inflammation. ConclusionThis study suggests that fraxetin is a novel therapeutic agent that targets mitochondria and tiRNAs. This is the first study to demonstrate the mechanisms of tiRNAHisGTG with mitochondrial function and cell fates and can be applied as a non-hormonal method against the progression of endometriosis.

A Systematic Review of Aminaphtone from Pathophysiology to Clinical Applications: Focus on New Rheumatological Acquisitions.

Aminaphtone is a chemical drug that has been used for more than thirty years to treat a variety of vascular disorders, with good clinical results and a satisfying safety profile. In the last two decades, multiple clinical studies have reported the efficacy of the drug in different clinical scenarios of altered microvascular reactivity, describing the downregulation of adhesion molecules (i.e., VCAM, ICAM, Selectins), vasoconstrictor peptides (i.e., Endothelin-1), and pro-inflammatory cytokine expression (i.e., IL-6, IL-10, VEGF, TGF-beta) by Aminaphtone. In this review, we summarize the current knowledge concerning Aminaphtone, with particular attention to rheumatological conditions in which microvascular disfunction plays a pivotal role, such as Raynaud's phenomenon and systemic sclerosis. These latter conditions may represent a promising field of application for Aminaphtone, due to the growing pre-clinical, clinical, and instrumental reports of efficacy. However, randomized, double-blind, placebo-controlled clinical trials are lacking and are desirable.

PB1962: COMPARISON OF EXPRESSION OF NSD2, CYCLIN D1 AND C-MAF BY PLASMA CELLS IN BONE MARROW AND BONE PLASMACYTOMA IN PATIENTS WITH PLASMA CELL MYELOMA

Background: Bone plasmacytomas forms as a result of destruction of bone structures by tumor proliferate and its escape out of the medullar cavity. According published data, the incidence of bone plasmacytomas at the time of plasma cell myeloma (PCM) diagnosis ranges from 7 to 32,5%, whereas rates are higher in a relapse. Bone plasmacytomas in most cases are composed of mature plasma cells, while immature morphology is mainly characteristic for extramedullary plasmacytomas. It is shown, that plasmacytoma cells are the descendants of bone marrow tumor plasma cells. Due to acquisition of new qualities as a downregulation of adhesion molecules, secretion of growth factors, activation of oncogenes and antiapoptotic factors, new subclones of tumor cells can survive without support from bone marrow microenvironment. Aims: to compare morphological consistency and immunohistochemical characteristics of PCM substrate in bone marrow trephines and bone plasmacytomas biopsy specimens. Methods: In retrospective study were included 13 patients with PCM aged from 36 to 67 years (median 55 years). At the time of diagnosis all patients underwent biopsy of bone plasmacytoma. The diagnosis of PCM verified according to IMWG criteria (2014). All patients have been examined with routine laboratory tests, immunochemical analysis of serum proteins and urine, cytologic examination of bone marrow smears, histologic and immunohistochemical examinations of bone marrow trephines and bone plasmacytomas biopsy specimens, instrumental examinations (CT, MRI). Immunohistochemical staining of bone marrow trephines and bone plasmacytomas biopsy specimens made with using a stainer Leica Bond-Max, reactions with antibodies to CCND1 (clone SPA4, Cell Marque), NSD2 (clone 29D1, Abcam), c-MAF (clone ERP16484, Abcam). The cut-off scores for determining positivity for markers have been taken 5% for CCND1, 10% for NSD2 and c-MAF (by T. Murase et al., 2019). Microscopic examination of histologic and immunohistochemical slides have been done on light microscope Leica 3000. Results: PCM substrate by histologic examination in bone marrow trepnines and bone plasmacytomas biopsy specimens were presented by mature plasma cells. In two patients (1 and 8) in bone plasmacytoma have been seen proplasmocytes between mature plasma cells, but in bone marrow have been seen only clusters of mature plasma cells. According to the results of immunohistochemical examination the expression of proteins-products of oncogenes have been revealed in most patients (12 of 13) in plasma cells of bone marrow and bone plasmacytomas, wherein in half of the patients (patients 7-12) the expression of proteins-products of oncogenes coincides in bone marrow and bone plasmacytoma. We have revealed a discordant expression in another patients (tabl. 1). We’ve paid a special attention to the patients 1-3, in whom in bone marrow have been revealed an expression of 1/3 protein, but in bone plasmacytoma – 2/3 (patients 2 and 3) or all of 3 studied proteins (patient 1). This phenomenon is possible to consider as a heterogeneity of clonal composition of bone plasmacytoma as a consequence of tumor evolution. Image:Summary/Conclusion: Discordant expression of proteins-products of oncogenes NSD2, cyclin D1 and c-Maf in tumor cells in bone marrow and bone plasmacytoma could be evidence of different tumor clones activation in PCM.

The Intercellular Communication Between Mesenchymal Stromal Cells and Hematopoietic Stem Cells Critically Depends on NF-κB Signalling in the Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) regulate the fate of the hematopoietic stem cells (HSCs) through both cell-cell interactions and paracrine mechanisms involving multiple signalling pathways. We have previously shown that co-culturing of HSCs with CoCl2-treated MSCs expands functional HSCs. While performing these experiments, we had observed that the growth of CoCl2-treated MSCs was significantly stunted. Here, we show that CoCl2-treated MSCs possess activated NF-κB signalling pathway, and its pharmacological inhibition significantly relieves their growth arrest. Most interestingly, we found that pharmacological inhibition of NF-κB pathway in both control and CoCl2-treated MSCs completely blocks their intercellular communication with the co-cultured hematopoietic stem and progenitor cells (HSPCs), resulting in an extremely poor output of hematopoietic cells. Mechanistically, we show that this is due to the down-regulation of adhesion molecules and various HSC-supportive factors in the MSCs. This loss of physical interaction with HSPCs could be partially restored by treating the MSCs with calcium ionophore or calmodulin, suggesting that NF-κB regulates intracellular calcium flux in the MSCs. Importantly, the HSPCs co-cultured with NF-κB-inhibited-MSCs were in a quiescent state, which could be rescued by re-culturing them with untreated MSCs. Our data underscore a critical requirement of NF-κB signalling in the MSCs in intercellular communication between HSCs and MSCs for effective hematopoiesis to occur ex vivo. Our data raises a cautionary note against excessive use of anti-inflammatory drugs targeting NF-κB.

Cordycepin prevents and ameliorates experimental autoimmune encephalomyelitis by inhibiting leukocyte infiltration and reducing neuroinflammation

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease characterized by multifocal perivascular infiltration of immune cells in the central nervous system (CNS). Cordycepin (3′-deoxyadenosine), an adenosine analogue initially extracted from the fungus Cordyceps militarisa, is one of the candidates that has multiple actions. We investigated that cordycepin attenuated the activation of LPS-induced mouse bone marrow-derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells (MoDCs) through the inhibition of the AKT, ERK, NFκB, and ROS pathways and impaired the migration of BMDCs through the downregulation of adhesion molecules and chemokine receptors in vitro. In experimental autoimmune encephalomyelitis (EAE) model, preventive treatment with cordycepin decreased the expression of trafficking factors in the CNS, inhibited the secretion of inflammatory cytokines (IFN-γ, IL-6, TNF-α, and IL-17), and attenuated disease symptoms. A chemokine array indicated that cordycepin treatment reversed the high levels of CCL6, PARRES2, IL-16, CXCL10, and CCL12 in the brain and spinal cord of EAE mice, consistent with the RNA-seq data. Moreover, cordycepin suppressed the release of neuroinflammatory cytokines by activated microglial cells, macrophages, Th17 cells, Tc1 cells, and Th1 cells in vitro. Furthermore, cordycepin treatment exerted therapeutic effects on attenuating the disease severity in the early disease onset stage and late disease progression stage. Our study suggests that cordycepin treatment may not only prevent the occurrence of MS by inhibiting DC activation and migration but also potentially ameliorates the progression of MS by reducing neuroinflammation, which may provide insights into the development of new approaches for the treatment of MS.

Whole Transcriptome Analysis Identifies Distinct Gene Expression Profiles between SF3B1mut and SF3B1 wt Myelodysplastic Syndrome with Ring Sideroblasts

Background and aims - The 2016 revised WHO classification incorporated somatic mutation in SF3B1 spliceosome gene within the diagnostic criteria of myelodysplastic syndrome (MDS) with ring sideroblasts (RS). However, SF3B1wt MDS-RS display significantly different clinical features and outcome from those of SF3B1mut MDS-RS. Recently, the recognition of SF3B1-mutant MDS as a distinct nosologic entity has been proposed to overcome this limitation.Methods - To evaluate the biological relevance of this proposal, we studied a consecutive cohort of 132 MDS with RS >5% using a pangenomic approach (targeted-DNA sequencing, genome-wide copy number variation analysis and bulk RNA-sequencing of CD34+ bone marrow mononuclear cells). 16 age-matched healthy individuals and 43 MDS-SLD/MLD negative for both splicing mutation and RS were included in this study as controls.Results - Unsupervised clustering analysis based on mutation profiles identified two major clusters predicted by SF3B1 mutation (87 MDS-RS-SF3B1mut and 45 MDS-RS-SF3B1wt). The most recurrently mutated genes in MDS-RS-SF3B1wt were TP53(40%), SRSF2(38%), TET2 (33%), ASXL1 (21%) and DNMT3A (12%). SRSF2 and TP53 mutations were found to be mutually exclusive with SF3B1 (p-value <0.05), whereas no difference was found in TET2, DNMT3A and ASXL1 frequencies between MDS-RS-SF3B1 mutand MDS-RS-SF3B1wt subgroups. TP53-mutated MDS-RS exhibited shorter overall survival (median 1.3 years, log-rank p-value<0.0001) compared to SF3B1mut (median 7.6 years), SRSF2mut (median 3.4 years) and MDS-RS without the abovementioned aberrations (median 4.4 years). Notably, allelic imbalances analysis of oncogenic variants identified a significant enrichment of TP53 biallelic inactivation in MDS-RS-SF3B1wt (64% vs 0, p<0.01).Differential gene expression analysis results were incorporated into a specific expression signature highly predictive of MDS-RS-SF3B1 mutand MDS-RS-SF3B1wt subgroups (Figure 1). The resulting gene clusters were classified in RS-specific genes (cluster 1 and 2) and SF3B1-specific genes (cluster 3 and 4). RS-specific genes comprising heme and hypoxia genes were enriched (Figure 2AB) and correlated with RS percentage (p<0.01). Pathway analysis revealed a specific downregulation of adhesion molecules and an upregulation of G-protein coupled receptor signaling molecules in MDS-RS-SF3B1mut. Among SF3B1-specific genes (Figure 2CD), we confirmed ABCB7 downregulation and identified new molecular targets that may concur to the pathophysiology of SF3B1-driven myeloid neoplasms.Conclusions - This study contributes to unveil molecular features of SF3B1-mutant MDS and provides further evidence to support recognition of somatic SF3B1 mutation as a disease-defining genetic lesion. DisclosuresPapaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH.

Background: Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH). Methods: We performed in vivo targeted silencing of the RUNX1 gene in liver sinusoidal endothelial cells (LSECs) by using vegfr3 antibody tagged immunonano-lipocarriers encapsulated RUNX1 siRNA (RUNX1 siRNA) in murine models of methionine choline deficient (MCD) diet-induced NASH. MCD mice given nanolipocarriers-encapsulated negative siRNA were vehicle, and mice with standard diet were controls. Results: Liver RUNX1 expression was increased in the LSECs of MCD mice in comparison to controls. RUNX1 protein expression was decreased by 40% in CD31-positive LSECs of RUNX1 siRNA mice in comparison to vehicle, resulting in the downregulation of adhesion molecules, ICAM1 expression, and VCAM1 expression in LSECs. There was a marked decrease in infiltrated T cells and myeloid cells along with reduced inflammatory cytokines in the liver of RUNX1 siRNA mice as compared to that observed in the vehicle. Conclusions: In vivo LSEC-specific silencing of RUNX1 using immunonano-lipocarriers encapsulated siRNA effectively reduces its expression of adhesion molecules, infiltrate on of immune cells in liver, and inflammation in NASH.

Myosin light chain kinase mediates intestinal barrier dysfunction following simulated microgravity based on proteomic strategy

Microgravity induces injury of intestinal barrier. However, the underlying mechanism remains unclear. The present study aimed to investigate the pathological change of intestinal mucosa induced by long term simulated microgravity and to explore its etiological mechanism using a proteomic approach. The well accepted tail-suspended rat model was used to simulate microgravity. The damage of rat small intestine was evaluated via histological and molecular test, and a label-free comparative proteomic strategy was used to determine the molecular mechanism. Simulated microgravity for 21 days damaged intestine barrier with decreased numbers of the goblet cells, large intercellular space, and down-regulated adhesion molecules, accompanied by increased intestinal permeability. Proteomic analysis identified 416 differentially expressed proteins and showed simulated microgravity dramatically down-regulated the adhesion molecules and deteriorated several pathways for metabolism, focal adhesion, and regulation of actin cytoskeleton. Western-blot analysis confirmed that myosin regulatory light chain (MLC) 12B was significantly down-regulated, while rho-associated protein kinase, myosin light chain kinase (MLCK), and phosphorylated MLC were dramatically up-regulated. Taken together, these data reveal that down-regulation of adhesion molecules and MLCK dependent up-regulation MLC phosphorylation mediate intestinal barrier dysfunction during simulated microgravity injury. Our results also indicate that regulation of epithelial MLCK is a potential target for the therapeutic treatment of microgravity injury.

Broad and thematic remodeling of the surfaceome and glycoproteome on isogenic cells transformed with driving proliferative oncogenes

The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.

Abstract A070: A phase 1/2a, open-label, dose-escalation study of EC-18 in patients with metastatic breast cancer for the prevention of chemotherapy-induced neutropenia

Abstract Background/Aim: Chemotherapy-induced neutropenia (CIN) is the major dose-limiting toxicity of systemic cancer chemotherapy leading to dose reductions, treatment delays or treatment early termination, which may compromise treatment outcome. EC-18 is a synthetic, orally available and lipid-based small molecular compound (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol). EC-18 prevents CIN by attenuating neutrophil extravasation via down-regulation of adhesion molecules, pro-inflammatory cytokines &amp; chemokines (Cell &amp; Bioscience 2019;9:4). The aim of this study is to evaluate the safety, tolerability, pharmacokinetics (PK) and preliminary activity of EC-18 for CIN (NCT03104595). Methods: A standard 3+3 design was used. EC-18 (softgel capsule) was administered on Day 1 through Day 21 to patients with metastatic breast cancer receiving combined doxorubicin and cyclophosphamide (AC regimen) in four dose-escalation cohorts (500mg/day, 1,000mg/day, 1,500mg/day, 2,000mg/day, respectively) with 3 subjects per each cohort. The first cohort was treated at a starting dose (500mg/day) and the subsequent cohorts were sequentially treated at increasing doses levels. PK was assessed on Day 1 and Day 15 in the first cycle of chemotherapy. Results: EC-18 was shown to be well tolerated in all participants and none of the subjects experienced drug-related severe adverse events or adverse events leading to discontinuation of treatment. Dose-limiting toxicity (DLT) did not occur and maximum-tolerated dose (MTD) was not determined accordingly. The baseline-adjusted absolute neutrophil count (ANC) of the patients showed a tendency to improve the neutrophil decline with increasing doses of EC-18 during the course of the study. With increasing dosage amount of EC-18, the time period (day) for the recovery of neutropenia from nadir to ANC &amp;gt; 500/mm3 showed a tendency to become shorter proportionally; 4.0 days in 500mg/day, 3.7 days in 1,000mg/day, 3.0 days in 1,500mg/day and 2.3 days in 2,000mg/day, respectively. In the PK outcomes, the plasma exposure on 500-2000 mg was observed in a dose-dependent manner. No plasma accumulation by the repeated dosing was observed based on the PK results of Day 1 and 15. The PK analysis and simulation suggested that the higher dose than 2000 mg would reach a systemic steady-state. Conclusion: EC-18 is safe and well tolerated. Studies in patients clearly demonstrated pharmacokinetics. Preliminary results suggest EC-18 has the potential to prevent and treat chemotherapy-induced neutropenia. Further dose-expansion cohorts trial is planned to explore a maximum tolerated dose and determine an efficacious dose for phase 2 clinical trial. Citation Format: Sung-Bae Kim, Myung-Hwan Kim. A phase 1/2a, open-label, dose-escalation study of EC-18 in patients with metastatic breast cancer for the prevention of chemotherapy-induced neutropenia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A070. doi:10.1158/1535-7163.TARG-19-A070

Abstract C037: Anti-metastatic effect of PLAG via interference of neutrophil elastase/PAR2/EGFR signaling on A549 lung cancer orthotopic implantation model

Abstract Background: A recent study reported that tumor-infiltrating neutrophil (TIN) has potential on malignant tumor progression leading to metastasis. In our study, to control the effect of TIN, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) was used. PLAG has shown its efficacy in modulating the neutrophil counts in chemotherapy-induced neutropenia by attenuating neutrophil extravasation via down-regulation of adhesion molecules, inflammatory cytokines, and chemokines. In this study, we investigated whether PLAG has an anti-metastatic effect on A549 lung cancer orthotopic implantation model via modulating TIN. Methods: To investigate the anti-metastatic effect of PLAG on lung cancer metastasis, RFP-labeled A549 cells were implanted into the lungs of BALB/c nude mice via intratracheal injection and bred for 12 weeks. After 6 weeks from tumor implantation, PLAG with three concentrations (25/50/100 mpk) were daily administrated for another 6 weeks. Each image of the growth of the primary tumor and colonization of metastatic tumor to other sites were acquired using CT and IVIS. TIN was analyzed in the primary tumor using immunohistochemistry (IHC). To identify the mechanism of PLAG on preventing the neutrophil-associated metastasis of cancer cells, direct or non-direct co-culture methods were used. Results: In the orthotopic implantation model, metastatic tumors to GI-track and brain were detected by IVIS. It was confirmed that metastasis to GI-track was decreased by 73% in 100 mpk PLAG-treated group, while metastasis to the brain was decreased by about 92% in PLAG-diet compared with positive control. Primary lung tumor growth was retarded by PLAG in a dose-dependent manner. The size of the primary tumor was much smaller than the positive control and alveolar tissue was similar to that of normal mice in 100 mpk PLAG-diet. In contrast, massive neutrophil infiltrations in positive and delivery groups, but significantly reduced upon PLAG administration. Moreover, p-EGFR was concomitantly detected with a similar pattern of neutrophil infiltration. During the direct contact in vitro assay, the A549 lung cancer spheroid cells were scattered by neutrophils, but it was effectively hindered by PLAG treatment. Neutrophil-stimulated cancer in the non-direct contact method showed enhanced metastatic activity. However, such a phenomenon was significantly reduced with the PLAG-treated group. Moreover, neutrophil stimulated the expression of EMT markers, whereas it was effectively down-regulated by PLAG administration. Transactivation between tumor cells and neutrophil was mediated by neutrophil elastase (NE). NE binds to and activates protease-activated receptor 2 (PAR2) on cancer cells and subsequently activated EGFR for metastasis to occur by cleaving HB-EGF through the ARR2/clathrin complex. On the other hand, the suppression of metastasis by PLAG was mediated through the regulation of the NE-PAR2 pathway and acceleration of intracellular trafficking and degradation of PAR2. Conclusion: In this study, TIN was observed in the primary lung tumor and metastasis into GI-track and brain, but PLAG dramatically reduced infiltration of neutrophil. The collective results suggest that TIN educates tumor cells for enhancing metastasis by EGFR transactivation. However, PLAG effectively interfered the transactivation. Having not met the medical needs for treating metastasis, PLAG may be a promising candidate as an anti-metastatic drug by modulating the TIN. Citation Format: GUEN TAE KIM, SUN YOUNG YOON, KI-YOUNG SOHN, MYUNG-HWAN KIM, JAE WHA KIM. Anti-metastatic effect of PLAG via interference of neutrophil elastase/PAR2/EGFR signaling on A549 lung cancer orthotopic implantation model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C037. doi:10.1158/1535-7163.TARG-19-C037

Inhibitory effects of vasostatin-1 against atherogenesis.

Vasostatin-1, a chromogranin A (CgA)-derived peptide (76 amino acids), is known to suppress vasoconstriction and angiogenesis. A recent study has shown that vasostatin-1 suppresses the adhesion of human U937 monocytes to human endothelial cells (HECs) via adhesion molecule down-regulation. The present study evaluated the expression of vasostatin-1 in human atherosclerotic lesions and its effects on inflammatory responses in HECs and human THP-1 monocyte-derived macrophages, macrophage foam cell formation, migration and proliferation of human aortic smooth muscle cells (HASMCs) and extracellular matrix (ECM) production by HASMCs, and atherogenesis in apolipoprotein E-deficient (ApoE-/-) mice. Vasostatin-1 was expressed around Monckeberg's medial calcific sclerosis in human radial arteries. Vasostatin-1 suppressed lipopolysaccharide (LPS)-induced up-regulation of monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in HECs. Vasostatin-1 suppressed inflammatory M1 phenotype and LPS-induced interleukin-6 (IL-6) secretion via nuclear factor-κB (NF-κB) down-regulation in macrophages. Vasostatin-1 suppressed oxidized low-density lipoprotein (oxLDL)-induced foam cell formation associated with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) and CD36 down-regulation and ATP-binding cassette transporter A1 (ABCA1) up-regulation in macrophages. In HASMCs, vasostatin-1 suppressed angiotensin II (AngII)-induced migration and collagen-3 and fibronectin expression via decreasing ERK1/2 and p38 phosphorylation, but increased elastin expression and matrix metalloproteinase (MMP)-2 and MMP-9 activities via increasing Akt and JNK phosphorylation. Vasostatin-1 did not affect the proliferation and apoptosis in HASMCs. Four-week infusion of vasostatin-1 suppressed the development of aortic atherosclerotic lesions with reductions in intra-plaque inflammation, macrophage infiltration, and SMC content, and plasma glucose level in ApoE-/- mice. These results indicate the inhibitory effects of vasostatin-1 against atherogenesis. The present study provided the first evidence that vasostatin-1 may serve as a novel therapeutic target for atherosclerosis.

Interleukin-32 promotes detachment and activation of human Langerhans cells in a human skin explant model.

Cross-talk between skin keratinocytes (KCs) and Langerhans cells (LCs) plays a fundamental role in the body's first line of immunological defences. However, the mechanism behind the interaction between these two major epidermal cells is unknown. Interleukin (IL)-32 is produced in inflammatory skin disorders. We questioned the role of IL-32 in the epidermis. We aimed to determine the role of IL-32 produced by KCs on surrounding LCs. We used an ex vivo human explant model from healthy donors and investigated the role of IL-32 on LC activation using imaging, flow cytometry, reverse transcriptase quantitative polymerase chain reaction and small interfering (si)RNA treatment. Modified vaccinia virus ankara (MVA) infection induced KC death alongside the early production of the proinflammatory cytokine IL-32. We demonstrated that IL-32 produced by MVA-infected KCs induced modest but significant morphological changes in LCs and downregulation of adhesion molecules, such as epithelial cell adhesion molecule and very late antigen-4, and CXCL10 production. The treatment of KCs with IL-32-specific siRNA, and anti-IL-32 blocking antibody significantly inhibited LC activation, demonstrating the role of IL-32 in LC activation. We also found that some Toll-like receptor ligands induced a very high level of IL-32 production by KCs, which initiated LC activation. We propose, for the first time, that IL-32 is a molecular link between KCs and LCs in healthy skin, provoking LC migration from the epidermis to the dermis prior to their migration to the draining lymph nodes.

Ameliorating effect of TI-1-162, a hydroxyindenone derivative, against TNBS-induced rat colitis is mediated through suppression of RIP/ASK-1/MAPK signaling

The pathogenesis of inflammatory bowel disease (IBD) is associated with production of immense pro-inflammatory cytokines including TNF-α. Once generated, TNF-α stimulates production of various pro-inflammatory cytokines and disrupts mucosal barrier by inducing inflamed mucosal epithelial cell death. In the present study, we investigated inhibitory effects of TI-1-162, a hydroxyindenone derivative, against TNF-α-induced and TNBS-induced colon inflammation. TI-1-162 showed inhibitory effect on the TNF-α-induced adhesion of U937 monocytic cells to HT-29 colonic epithelial cells (IC50 = 0.83 ± 0.12 μM), which is an in vitro model representing the initial step of colitis. In addition, TI-1-162 suppressed TNF-α-stimulated caspase-3 activation and HT-29 cell apoptosis. These in vitro inhibitory activities of TI-1-162 correlated to recovery changes in in vivo colon tissues, such as downregulation of adhesion molecules (ICAM-1, VCAM-1) and chemokines (CCL11, CXCL1, CXCL2, CXCL3, CX3CL1) revealed by gene expression array and Western blot analyses. Such molecular recovery of colon epithelium from TNBS-treated rats corresponded to the recovery in body weight, colon weight/length, and myeloperoxidase level by TI-1-162 (10 and 30 mg/kg/day, orally). In relation to action mechanism, TI-1-162 did not disturb TNF-α binding to its receptor, but suppressed phosphorylation of RIP-1, ASK-1, JNK and p38, and nuclear translocation of NF-kB and AP-1, which corresponded to down regulation of inflammatory cytokines in TNF-α-treated cells (HT-29 and U937) and TNBS-treated rat colon tissues. Taken together, the results indicate that the protective effects of TI-1-162 against colon inflammation and epithelial cell death are associated with its inhibitory action in RIP/ASK-1/MAPK signaling pathway downstream to TNF receptor 1.

Anti-pandemic influenza A (H1N1) virus potential of catechin and gallic acid

BackgroundThe pandemic influenza A (H1N1) virus has spread worldwide and infected a large proportion of the human population. Discovery of new and effective drugs for the treatment of influenza is a crucial issue for the global medical community. According to our previous study, TSL-1, a fraction of the aqueous extract from the tender leaf of Toonasinensis, has demonstrated antiviral activities against pandemic influenza A (H1N1) through the down-regulation of adhesion molecules and chemokine to prevent viral attachment. MethodsThe aim of the present study was to identify the active compounds in TSL-1 which exert anti-influenza A (H1N1) virus effects. XTT assay was used to detect the cell viability. Meanwhile, the inhibitory effect on the pandemic influenza A (H1N1) virus was analyzed by observing plaque formation, qRT-PCR, neuraminidase activity, and immunofluorescence staining of influenza A-specific glycoprotein. ResultsBoth catechin and gallic acid were found to be potent inhibitors in terms of influenza virus mRNA replication and MDCK plaque formation. Additionally, both compounds inhibited neuraminidase activities and viral glycoprotein. The 50% effective inhibition concentration (EC50) of catechin and gallic acid for the influenza A (H1N1) virus were 18.4 μg/mL and 2.6 μg/mL, respectively; whereas the 50% cytotoxic concentrations (CC50) of catechin and gallic acid were >100 μg/mL and 22.1 μg/mL, respectively. Thus, the selectivity indexes (SI) of catechin and gallic acid were >5.6 and 22.1, respectively. ConclusionThe present study demonstrates that catechin might be a safe reagent for long-term use to prevent influenza A (H1N1) virus infection; whereas gallic acid might be a sensitive reagent to inhibit influenza virus infection. We conclude that these two phyto-chemicals in TSL-1 are responsible for exerting anti-pandemic influenza A (H1N1) virus effects.

Dysregulated infertility-related genes in women with endometriosis

A number of mechanisms may be responsible for endometriosis-related infertility, such as inflammatory microenvironment, altered immune response, reduced/absent adhesion molecules and oxidative stress. Many genes play important roles in these processes and their expression levels may be altered in women with endometriosis. A prospective case-control study was conducted to assess expression of dysregulated genes in the eutopic endometrium of women with endometriosis compared to that of women without endometriosis. Eutopic endometrial biopsies from 29 women (N=16 with endometriosis; N=13 without endometriosis) were obtained by Pipelle curette between 1 January 2015 and 31 December 2015 in Akanksha IVF centre. Paralleled gene expression profiling using high density oligonucleotide microarrays was applied to investigate dysregulated genes in eutopic endometrium of women with endometriosis compared to women without endometriosis. Data analysis was conducted by GeneSpring version 4.0.4. T-test using P value >0.05 and a fold-change expression >1.5 was applied to assess statistical significance. Selected dysregulated genes were randomly chosen and validated by RT-PCR. This study has shown that in the eutopic endometrium from women with endometriosis infertility-related genes were significantly dysregulated. Particularly, genes involved in inflammation, immune response and oxidative stress were upregulated whereas genes involved in the expression of adhesion molecules were downregulated. Dysregulation of genes involved in infertility likely contributes to decreased pregnancy rates in women with endometriosis. Increased inflammation in the eutopic endometrium may contribute to increased cytokine production and altered immune environment leading to oxidative stress thereby impairing embryo implantation. Moreover, downregulation of adhesion molecules in the eutopic endometrium may affect the endometrial receptivity in women with endometriosis. An improved understanding of the underlying molecular infertility mechanisms may aid in identifying candidate genes for managing endometriosis-associated infertility.Tabled 1Table 1: Significantly dysregulated genes in eutopic endometrium of women with endometriosis (n=16)Gene SymbolGene nameFunctionFold ChangeCOL16A1Collagen, type XVI, alpha 1Adhesion-1.5ITGA6Integrin, alpha 6Adhesion-1.6GZMBGranzyme BInflammation3.05CXCL14Chemokine (C-X-C-motif) ligand 14Inflammation10.7ERO1LERO1-likeoxidative stress1.65HIST1H2ACHistone cluster 1, H2acoxidative stress2.29MAFFv-Mafmusculoaponeuroticfibrosarcoma oncogene homolog Foxidative stress2.26MAP3K5mitogen-activated protein kinase kinase kinase 5oxidative stress3.15CD68CD68 moleculeImmune response1.53CD58CD58 moleculeImmune response1.69TLR4Toll-like receptor 4Immune response1.75 Open table in a new tab

BPS-001, a complex biologic agent extracted from the medicinal leech (Huridinaria manillensis) for treatment of metastatic castration-resistant prostate cancer.

209 Background: We established that subcutaneous BPS-001 reduced tumor size and inhibited tumor growth in PC3, LNCaP and 22RV-1 mouse xenografts; and, tumor lHC demonstrated an increase in caspase-3 and decreases in expression of P21, Ki-67 and PCNA. We also showed that BPS-001 reduced PSA expression in both In vitro and in In vivo xenografts (LNCaP and 22RV1). We now demonstrate that BPS-001 inhibits angiogenesis in the xenografts; and, In vitro studies demonstrate that BPS-001 affects expression of adhesion molecules and the androgen receptor. In addition, BPS-001 inhibited tumor growth in a TRAMP allograft model. Methods: CD31 expression was measured using IHC of the previous mouse xenografts. In vitro androgen receptor and adhesion molecule expression were assessed by Western blot. Transplantable TRAMP-C2 mouse prostate cancer in B57CL allografts were grown to 5 mm prior to initiation of treatment. Immune markers in TRAMP allograft tumors were measured by IHC. Results: Mechanistic studies show BPS-001 acts by blocking angiogenesis demonstrated by a &gt; 50% decrease in expression of CD31 in the LNCaP and 22RV-1 xenografts. Furthermore, preliminary toxicology studies show similar efficacy to docetaxel with no weight loss in the longer-term PC3 xenograft study as was observed with docetaxel. Preliminary toxicology studies of BPS-001 also support a favorable toxicity profile, and the therapeutic dose (1-5 mg/Kg, QW or BIW) is well within the margin of safety. Dose limiting toxicity (bleeding) was observed at doses of 20mg/Kg daily. In vitro studies showed BPS-001 decreased expression of both N- and P- cadherins and the androgen receptor. BPS-001 inhibited tumor growth in the TRAMP allograft models. IHC analyses of the TRAMP allograft tumors suggest immunomodulation. Conclusions: BPS-001 exhibits anticancer activity with no observed toxicity in the therapeutic range. Effects of BPS-001 include inhibition of angiogenesis, downregulation of adhesion molecules associated with advanced prostate cancer and inhibition of androgen receptor signaling. Elucidation of the mechanism of action is ongoing.

Fenofibrate in cancer: mechanisms involved in anticancer activity

Objective: To review the mechanisms of anti-cancer activity of fenofibrate (FF) and other Peroxisome Proliferator Activator Receptor α (PPARα) agonists based on evidences reported in the published literature.Methods: We extensively reviewed the literature concerning FF as an off target anti-cancer drug. Controversies regarding conflicting findings were also addressed.Results:The main mechanism involved in anti-cancer activity is anti-angiogenesis through down-regulation of Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor Receptor (VEGFR) and Hypoxia Inducible factor-1 α (HIF-1α), inhibition of endothelial cell migration, up-regulation of endostatin and thrombospondin-1, but there are many other contributing mechanisms like apoptosis and cell cycle arrest, down-regulation of Nuclear Factor Kappa B (NF-kB) and Protein kinase B (Akt) and decrease of cellular energy by impairing mitochondrial function. Growth impairment is related to down-regulation of Phospho-Inositol 3 Kinase (PI3K)/Akt axis and down-regulation of the p38 map kinase (MAPK) cascade. A possible role should be assigned to FF stimulated over-expression of Tribbles Homolog-3 (TRIB3) which inhibits Akt phosphorylation. Important anti-cancer and anti-metastatic activities are due to down-regulation of MCP-1 (monocyte chemotactic protein-1), decreased Metalloprotease-9 (MMP-9) production, weak down-regulation of adhesion molecules like E selectin, intercellular adhesion molecules (ICAM) and Vascular Endothelial Adhesion Molecules (VCAM), and decreased secretion of chemokines like Interleukin-6 (IL-6), and down-regulation of cyclin D-1. There is no direct link between FF activity in lipid metabolism and anticancer activity, except for the fact that many anticancer actions are dependent from PPARα agonism. FF exhibits also PPARα independent anti-cancer activities.Conclusions: There are strong evidences indicating that FF can disrupt growth-related activities in many different cancers, due to anti-angiogenesis and anti-inflammatory effects. Therefore FF may be useful as a complementary adjunct treatment of cancer, particularly included in anti-angiogenic protocols like those currently increasingly used in glioblastoma. There are sound reasons to initiate well planned phase II clinical trials for FF as a complementary adjunct treatment of cancer.

Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment.

The low cell engraftment after transplantation limits the successful application of stem cell therapy and the exact pathway leading to acute donor cell death following transplantation is still unknown. Here we investigated if processes involved in cell preparation could initiate downregulation of adhesion-related survival signals, and further affect cell engraftment after transplantation. Human embryonic stem cell-derived endothelial cells (hESC-ECs) were suspended in PBS or Matrigel and kept at 4 °C. Quantitative RT-PCR analysis was used to test the adhesion and apoptosis genes’ expression of hESC-ECs. We demonstrated that cell detachment can cause downregulation of cell adhesion and extracellular matrix (ECM) molecules, but no obvious cell anoikis, a form of apoptosis after cell detachment, was observed. The downregulation of adhesion and ECM molecules could be regained in the presence of Matrigel. Finally, we transplanted hESC-ECs into a mouse myocardial ischemia model. When transplanted with Matrigel, the long-term engraftment of hESC-ECs was increased through promoting angiogenesis and inhibiting apoptosis, and this was confirmed by bioluminescence imaging. In conclusion, ECM could rescue the functional genes expression after cell detached from culture dish, and this finding highlights the importance of increasing stem cell engraftment by mimicking stem cell niches through ECM application.

Senescent Stromal Fibroblasts Secrete MMP-2 Which Promotes The Invasion of OSCC Keratinocyte Cells

regulation of a broad spectrum of cell-cell adhesion molecules including Desmoglein 1 and 3, Desmoplakin, b-catenin, Plakophilin and Desmocolin. The induction of EMT and down-regulation of adhesion molecules was associated with a less cohesive and more invasive epithelial phenotype. Inhibition of CAFderived TGF-b reduced the ability of senescent fibroblasts to promote keratinocyte tumour cell dissociation and invasion in vitro. Conclusions: Senescent stromal fibroblasts promote tumour cell invasion by the downregulation of cell-cell adhesion molecules and the induction of keratinocyte discohesion, a heterotypic cell interaction that occurs in a TGF-b dependent manner.