Cell Motility Processes Research Articles

Studying ABI3 variant immune contributions to Alzheimer’s disease in a transgenic mouse model

AbstractBackgroundAlzheimer’s Disease (AD) is a progressive disorder populating in a world‐wide range, which causes dementia with accumulation of amyloid‐beta (Aβ) and hyperphosphorylated tau. AD destroys memory and disrupts cognition, as well as impairs thinking, behaving and person’s ability to function independently. Numbers of genes now have been confirmed through large scale genetic studies as significant AD risk genes, including ABI3. Abelson interactor (ABI) family is an adaptor protein family commonly participating in regulation of actin cytoskeleton in cell. As a member of ABI family, ABI3 can modulate cell motility process like inhibiting tumor metastasis and migration. Genetic variants of ABI3 have been found to be strongly associated with late onset AD (LOAD) in various human populations, but the molecular mechanisms of how they influence AD pathogenesis remain unknown.MethodIn order to further study the pathological mechanisms of ABI3 in AD in vivo, we created an Abi3 transgenic mouse model on C57BL/6 background carrying a human ABI3 mutation (S209F) by CRISPER/Cas‐mediated genome engineering. ABI3 S209F mutation is encoded by a very rare variant rs616338 (MAF=0.008, OR = 1.43 for AD with P = 4.56 × 10−10). In this transgenic model, a corresponding mouse S212F mutation was introduced into Exon 5 on Chromosome 11 by homology‐directed repair, and homozygous Abi3F/F mice were generated by crossing the F1 heterozygotes. In addition, we also crossed Abi3F/F with the 5xFAD model of AD to determine the influence of this ABI3 mutation on AD pathology and cognitive functions in vivo.ResultWe hypotheses 5xFAD mice who carrying the ABI3 mutation may exhibit exacerbated AD pathologies compared with 5xFAD with wildtype ABI3, including accelerate Aβ aggregation and neuroinflammation. As ABI3 is exclusively expressed in immune cells in the brain and peripheral, this new Abi3F/F model will be an effective method for us to understand the immune contribution in AD pathogenesis and perhaps the crosstalk between peripheral and central in the disease as well.ConclusionWe believe that studying AD with ABI3 Tg mouse models can allow us to have better understanding on the immune contribution to AD pathogenesis and provide valuable strategies on clinical therapy.

Network pharmacology-based study of chinese herbal qixiong formula in treating oligoasthenospermia

Objective: The objective is to study the network pharmacology of Qixiong formula (QXF) and explore the mechanism of QXF in the treatment of oligoasthenospermia. Materials and Methods: Using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-traditional Chinese medicine), and an encyclopaedia of traditional Chinese medicine (ETCM) databases as well as data from relevant studies, the effective components and targets of QXF were obtained. Genes associated with oligospermia were screened using GeneCards, OMIM, DisGeNet, DrugBank, and GAD databases. The intersection target was obtained by mapping the target to the gene, and the protein interaction network was created using the STRING database to screen the core target of QXF in the treatment of oligospermia. The intersection target was enriched using gene ontology (GO) and the Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis with the DAVID database. The network of the disease drug target pathway was drawn using Cytoscape software. Results: Overall, 536 active components of QXF and 40 core targets for the treatment of oligoasthenozoospermia were obtained. The analysis of GO and KEGG showed that QXF is mainly involved in oxidative stress, cell motility, nutritional response, and other biological processes. Through the regulation of FOXO, p53, PI3K/Akt, MAPK, mammalian target of rapamycin, Foxo, Wnt, and other signaling pathways, QXF played a role in the treatment of oligoasthenospermia. Conclusion: QXF has multi-component, multi-target, and multi-channel characteristics, providing a new way to study the mechanism of QXF in the treatment of oligoasthenospermia.

Anti-Tumor Mechanisms of Novel 3-(4-Substituted Benzyl)-5-Isopropil-5- Phenylhydantoin Derivatives in Human Colon Cancer Cell Line.

Hydantoin and its newly synthesized derivatives have recently become a focus of interest due to their numerous biological activities and newly emerging beneficial effects in different pathological conditions, including cancer. The aim of this study was to evaluate the possible anti-tumor mechanisms of a series of newly synthesized 3-(4-substituted benzyl)-5-isopropyl-5-phenylhydantoin derivatives in different aspects of cell physiology of human colon cancer cell line, HCT-116. The increasing concentrations of derivatives (0.01µM up to 100µM) were applied to cells during 24h, 48h, and 72h after which the evaluation of proliferation, apoptosis, oxidative/anti-oxidative status, nitrite production, and migration/invasion potential of treated cells was performed. All tested compounds expressed the dose- and time-dependent anti-proliferative and pro-apoptotic activities against HCT-116 cells. The investigated derivatives induced a decrease in levels of oxidative stress parameters and an increase in levels of nitrite production by treated cells suggesting their significant antioxidative effects. The cell migration index and expression level of tumor invasion-promoting metalloproteinase- 9 (MMP-9) gene were significantly decreased after treatment with the tested hydantoin derivatives implicating their inhibitory role in colon cancer cell motility and invasion processes. The mRNA level of cyclooxygenase-2 (COX-2) gene as a pro-inflammatory gene related to colorectal carcinogenesis was reduced compared to values in the non-treated control cells indicating the significant anti-inflammatory/anti-tumor effects of these compounds. The obtained results show the significant anti-tumor potential of tested derivatives, especially 3- benzyl-5-isopropyl-5-phenylhydantoin and 3-(4-chlorobenzyl)-5-isopropyl-5-phenylhydantoin, suggesting their potential usage in the development of more effective chemotherapies.

Notch signaling coordinates ommatidial rotation in the Drosophila eye via transcriptional regulation of the EGF-Receptor ligand Argos

In all metazoans, a small number of evolutionarily conserved signaling pathways are reiteratively used during development to orchestrate critical patterning and morphogenetic processes. Among these, Notch (N) signaling is essential for most aspects of tissue patterning where it mediates the communication between adjacent cells to control cell fate specification. In Drosophila, Notch signaling is required for several features of eye development, including the R3/R4 cell fate choice and R7 specification. Here we show that hypomorphic alleles of Notch, belonging to the Nfacet class, reveal a novel phenotype: while photoreceptor specification in the mutant ommatidia is largely normal, defects are observed in ommatidial rotation (OR), a planar cell polarity (PCP)-mediated cell motility process. We demonstrate that during OR Notch signaling is specifically required in the R4 photoreceptor to upregulate the transcription of argos (aos), an inhibitory ligand to the epidermal growth factor receptor (EGFR), to fine-tune the activity of EGFR signaling. Consistently, the loss-of-function defects of Nfacet alleles and EGFR-signaling pathway mutants are largely indistinguishable. A Notch-regulated aos enhancer confers R4 specific expression arguing that aos is directly regulated by Notch signaling in this context via Su(H)-Mam-dependent transcription.

Immediate Effects of Dasatinib on the Migration and Redistribution of Naïve and Memory Lymphocytes Associated With Lymphocytosis in Chronic Myeloid Leukemia Patients.

Introduction: Dasatinib is a dual SRC/ABL tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML) that is known to have unique immunomodulatory effects. In particular, dasatinib intake typically causes lymphocytosis, which has been linked to better clinical response. Since the underlying mechanisms are unknown and SRC family kinases are involved in many cell motility processes, we hypothesized that the movement and migration of lymphocytes is modulated by dasatinib. Patients, Materials and Methods: Peripheral blood samples from CML patients treated with second-line dasatinib were collected before and 2 h after the first dasatinib intake, and follow-up samples from the same patients 3 and 6 months after the start of therapy. The migratory capacity and phenotype of lymphocytes and differential blood counts before and after drug intake were compared for all study time-points. Results: We report here for the first time that dasatinib intake is associated with inhibition of peripheral blood T-cell migration toward the homeostatic chemokines CCL19 and CCL21, which control the trafficking toward secondary lymphoid organs, mainly the lymph nodes. Accordingly, the proportion of lymphocytes in blood expressing CCR7, the chemokine receptor for both CCL19 and CCL21, decreased after the intake including both naïve CD45RA+ and central memory CD45RO+ T-cells. Similarly, naïve B-cells diminished with dasatinib. Finally, such changes in the migratory patterns did not occur in those patients whose lymphocyte counts remained unchanged after taking the drug. Discussion: We, therefore, conclude that lymphocytosis induced by dasatinib reflects a pronounced redistribution of naïve and memory populations of all lymphocyte subsets including CD4+ and CD8+ T-cells and B-cells.

The effect of medium supplementation and serial passaging on the transcriptome of human adipose-derived stromal cells expanded in vitro

BackgroundFor adipose-derived stromal cells (ASCs) to be safe for use in the clinical setting, they need to be prepared using good manufacturing practices (GMPs). Fetal bovine serum (FBS), used to expand ASCs in vitro in some human clinical trials, runs the risk of xenoimmunization and zoonotic disease transmission. To ensure that GMP standards are maintained, pooled human platelet lysate (pHPL) has been used as an alternative to FBS. ASCs proliferate more rapidly in pHPL than in FBS, with no significant change in immunophenotype and differentiation capacity. However, not much is known about how pHPL affects the transcriptome of these cells.MethodsThis study investigated the effect of pHPL and FBS on the ASC transcriptome during in vitro serial expansion from passage 0 to passage 5 (P0 to P5). RNA was isolated from ASCs at each passage and hybridized to Affymetrix HuGene 2.0 ST arrays for gene expression analysis.ResultsWe observed that the transcriptome of ASCs expanded in pHPL (pHPL-ASCs) and FBS (FBS-ASCs) had the greatest change in gene expression at P2. Gene ontology revealed that genes upregulated in pHPL-ASCs were enriched for cell cycle, migration, motility, and cell-cell interaction processes, while those in FBS-ASCs were enriched for immune response processes. ASC transcriptomes were most homogenous from P2 to P5 in FBS and from P3 to P5 in pHPL. FBS- and pHPL-gene-specific signatures were observed, which could be used as markers to identify cells previously grown in either FBS or pHPL for downstream clinical/research applications. The number of genes constituting the FBS-specific effect was 3 times greater than for pHPL, suggesting that pHPL may be a milder supplement for cell expansion. A set of genes were expressed in ASCs at all passages and in both media. This suggests that a unique ASC in vitro transcriptomic profile exists that is independent of the passage number or medium used.ConclusionsGO classification revealed that pHPL-ASCs are more involved in cell cycle processes and cellular proliferation when compared to FBS-ASCs, which are involved in more specialized or differentiation processes like cardiovascular and vascular development. This makes pHPL a potential superior supplement for expanding ASCs as they retain their proliferative capacity, remain untransformed and pHPL does not affect the genes involved in differentiation in specific developmental processes.

Identification of global mRNA expression profiles and comprehensive bioinformatic analyses of abnormally expressed genes in cholestatic liver disease

Cholestatic liver disease (CLD) is a highly heterogeneous hepatobiliary disease with various causes. The purpose of this research was to explore the gene expression changes throughout the course of CLD revealing potential causative molecular mechanisms and therapeutic targets. We established two animal models of cholestasis: 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding for 2, 4 and 6 weeks and bile duct ligation for 14 days. Using these two models, we identified differentially expressed genes (DEGs) by RNA-Seq analysis and used the newly-found knowledge of DEGs in comprehensive bioinformatic analyses to investigate key molecular events. Sequencing results were confirmed by experimental verification. Our study detected overlapping DEGs in the two models, of these 568 genes were upregulated and 117 genes were downregulated. Gene Ontology analysis demonstrated that the upregulated genes were associated with the biological processes of cell adhesion, cell migration and cell motility, while the metabolic processes of various substances were enriched for the downregulated genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the upregulated pathways were mainly distributed in focal adhesion, ECM-receptor interaction and amoebiasis, while downregulated pathways focused on peroxisome proliferator-activated receptor signaling pathway, metabolic pathways and primary bile acid biosynthesis. These findings were further confirmed by protein-protein interaction network modeling. Hub genes Src, Pdgfb, Col15a1, Mmp9, Egfr were selected using centralities analyses and verified by qRT-PCR. We profiled a global mRNA landscape in CLD to promote a complete understanding of transcriptomic events of this disease, offering candidate biomarkers and therapeutic targets for the clinic.

NF-\u03baB upregulates glutamine-fructose-6-phosphate transaminase 2 to promote migration in non-small cell lung cancer

BackgroundEpithelial-to-mesenchymal transition (EMT) results in changes that promote de-differentiation, migration, and invasion in non-small cell lung cancer (NSCLC). While it is recognized that EMT promotes altered energy utilization, identification of metabolic pathways that link EMT with cancer progression is needed. Work presented here indicates that mesenchymal NSCLC upregulates glutamine-fructose-6-phosphate transaminase 2 (GFPT2). GFPT2 is the rate-limiting enzyme in the synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is the obligate activator of O-linked N-acetylglucosamine transferase (OGT).MethodsAnalysis of our transcriptomic data indicates that GFPT2 is one of the most significantly upregulated metabolic genes in mesenchymal NSCLC. Ectopic GFPT2 expression, as well as gene silencing strategies were used to determine the importance of this metabolic enzyme in regulating EMT-driven processes of cell motility and invasion.ResultsOur work demonstrates that GFPT2 is transcriptionally upregulated by NF-κB and repressed by the NAD+-dependent deacetylase SIRT6. Depletion of GFPT2 expression in NSCLC highlights its importance in regulating cell migration and invasion during EMT.ConclusionsConsistent with GFPT2 promoting cancer progression, we find that elevated GFPT2 expression correlates with poor clinical outcome in NSCLC. Modulation of GFPT2 activity offers a potentially important therapeutic target to combat NSCLC disease progression.

Gene expression and DNA methylation are extensively coordinated with MRI-based brain microstructural characteristics

Cognitive function relies on both molecular levels and cellular structures. However, systematic relationships between these two components of cognitive function, and their joint contribution to disease, are largely unknown. We utilize postmortem neuroimaging in tandem with gene expression and DNA methylation, from 222 deeply-phenotyped persons in a longitudinal aging cohort. Expression of hundreds of genes and methylation at thousands of loci are related to the microstructure of extensive regions of this same set of brains, as assessed by MRI. The genes linked to brain microstructure perform functions related to cell motility, transcriptional regulation and nuclear processes, and are selectively associated with Alzheimer’s phenotypes. Similar methodology can be applied to other diseases to identify their joint molecular and structural basis, or to infer molecular levels in the brain on the basis of neuroimaging for precision medicine applications.

GMF as an Actin Network Remodeling Factor.

Glia maturation factor (GMF) has recently been established as a regulator of the actin cytoskeleton with a unique role in remodeling actin network architecture. Conserved from yeast to mammals, GMF is one of five members of the ADF-H family of actin regulatory proteins, which includes ADF/cofilin, Abp1/Drebrin, Twinfilin, and Coactosin. GMF does not bind actin, but instead binds the Arp2/3 complex with high affinity. Through this association, GMF catalyzes the debranching of actin filament networks and inhibits actin nucleation by Arp2/3 complex. Here, we discuss GMF's emerging role in controlling actin filament spatial organization and dynamics underlying cell motility, endocytosis, and other biological processes. Further, we attempt to reconcile these functions with its earlier characterization as a cell differentiation factor.

Etoposide-induced DNA damage affects multiple cellular pathways in addition to DNA damage response.

DNA damage response (DDR) coordinates lesion repair and checkpoint activation. DDR is intimately connected with transcription. However, the relationship between DDR and transcription has not been clearly established. We report here RNA-sequencing analyses of MCF7 cells containing double-strand breaks induced by etoposide. While etoposide does not apparently cause global changes in mRNA abundance, it altered some gene expression. At the setting of fold alteration ≥ 2 and false discovery rate (FDR) ≤ 0.001, FDR < 0.05, or p < 0.05, etoposide upregulated 96, 268, or 860 genes and downregulated 41, 133, or 503 genes in MCF7 cells. Among these differentially expressed genes (DEGs), the processes of biogenesis, metabolism, cell motility, signal transduction, and others were affected; the pathways of Ras GTPase activity, RNA binding, cytokine-mediated signaling, kinase regulatory activity, protein binding, and translation were upregulated, and those pathways related to coated vesicle, calmodulin binding, and microtubule-based movement were downregulated. We further identified RABL6, RFTN2, FAS-AS1, and TCEB3CL as new DDR-affected genes in MCF7 and T47D cells. By metabolic labelling using 4-thiouridine, we observed dynamic alterations in the transcription of these genes in etoposide-treated MCF7 and T47D cells. During 0-2 hour etoposide treatment, RABL6 transcription was robustly increased at 0.5 and 1 hour in MCF7 cells and at 2 hours in T47D cells, while FAS-AS1 transcription was dramatically and steadily elevated in both cell lines. Taken together, we demonstrate dynamic alterations in transcription and that these changes affect multiple cellular processes in etoposide-induced DDR.

Abstract P1-07-35: Adipokine receptor CAP1 expression as predictor of survival outcome among breast cancer patients

Abstract Background: Obesity is a challenging health concern in breast cancer. Not only are obese women at higher risk of breast cancer, but if diagnosed with the disease, obesity is also impairing the prognosis of a breast cancer. Understanding the underlying biology behind these associations are key to improve survival rates in breast cancer. In obesity, the more abundant, activated adipose tissue may lead to insulin resistance and secretion of soluble mediators that may contribute to impaired breast cancer outcomes observed among obese women. The adipokine resistin is implied to be an important link between obesity and insulin resistance, and to be elevated among breast cancer patients. Yet, the expression of the newly identified resistin receptor CAP1 and its impact on breast cancer prognosis is poorly understood. Purpose: To evaluate the impact by the adipokine receptor CAP1 tumor expression on breast cancer outcomes. Experimental Design: CAP1 mRNA expression was explored among 1,881 primary breast tumors. Associations between CAP1 expression, categorized into tertiles, and survival outcomes were estimated using the Kaplan-Meier method and log-rank test, and by multivariable Cox regression models providing adjusted hazard ratios (HRadj) with 95% confidence intervals (CI). Independent biological network interaction analyzes were conducted in a subset of 1,105 invasive breast carcinomas within The Cancer Genome Atlas (TCGA) project. Results: CAP1 was linked to poor tumor characteristics with higher CAP1 expression found among estrogen receptor (ER)-negative tumors, relative to ER-positive tumors, as well as with increasing tumor grade (P=0.025). High CAP1 tumor expression was further associated with shorter overall survival (HRadj 1.54; 95% CI, 1.11-2.13) and relapse-free survival (HRadj 1.47; 95% CI, 1.10-1.96), compared with low or intermediate CAP1 expression, particularly among ER-positive tumors or lymph node positive tumors. Additionally, CAP1 was identified in complex with genes involved in growth promoting and cell motility processes. Conclusion: These results highlight the potential role of the resistin receptor CAP1 regarding breast cancer outcomes. Further investigations are needed to elucidate the biological and clinical implication of the resistin-CAP1 link in obesity-associated breast cancer. Citation Format: Rosendahl AH, Bergqvist M, Kimbung S, Borgquist S. Adipokine receptor CAP1 expression as predictor of survival outcome among breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-35.

Abstract B56: RNA-seq analysis of prostate cancer samples from Hispanic patients reveal progressive characteristics in Gleason patterns

Abstract Background. One of the prognostic markers widely used in clinic to predict survival in prostate cancer (PC) patients is the Gleason score. Differences have been observed in biochemical progression-free and overall survival in patients with different Gleason scores. Because each Gleason represents a different state of tumor progression, we decided to characterize expression profiles and pathways in Gleason 3, 4 and 5 in order to explore the tumor biology context in each pattern, and thus determine possible prognosis biomarkers in PC. Methods. We extracted RNA from normal and malignant formalin-fixed paraffin-embedded tissues from radical prostatectomy samples of Colombian patients and analyzed expression using data generated with next generation sequencing methods. Pathway analysis was done with MetaCore. Results. Eleven gene networks (7 genes) were common between all Gleason scores and represented cellular processes possibly involved with damage of the prostatic tissue including: cell adhesion, cytoskeleton remodeling and metabolism of phosphatidyl compounds. According to the analysis in Gleason pattern 3, the principal pathways were associated with cytoskeleton remodeling, gap junctions signaling and regulation of cell adhesion. These pathways could contribute to de-differentiation process experienced by cells towards tumor transformation in early stages of progression. Also, signaling of epithelial-to-mesenchymal transition (EMT) were observed, this findings could suggests not only cytoskeleton remodeling during de-differentiation, but also appearance of metastatic cells in this Gleason pattern. On the other hand, the action of the Androgen Receptor (AR) is suggested by protein-protein interactions characterized by rapid activation of signaling cascades of proliferation, cell growth and survival, via MAPK and PI3K/AKT. The latter in turn induces AR overexpression through control of regulatory protein MDM-2 and optimizes binding of AR to promoter/enhancer regions over its target genes. Also, AR inhibits WNT signaling via interaction with B-catenin. However, AR is a LEF-1/TCF transcriptional target, which is activated by WNT/B-catenin signaling. The pathways identified in Gleason pattern 4 were similar to those of Gleason 3, with a more significant participation of pathways leading to cytoskeleton remodeling, regulation of cell adhesion molecules and of EMT. In addition, there is ligand-independent activation of AR, wherein activating mutations, splicing variants and activation by other signaling cascades, may lead to directed transactivation of target genes in a deregulated manner. Further, NGF/TrkA/PI3K-mediated signaling may lead to inhibition of apoptosis and survival of these cells. Finally, pathways analysis in Gleason pattern 5 showed that the most significant was cell adhesion and ECM remodeling, which favors the metastatic process and regulation of cell motility and adhesion, processes aided by migration and angiogenesis signaling pathways. Likewise, AR activation and downstream signaling has an important role, transactivating transcription factors, which together lead to cell proliferation, inhibition of apoptosis, expression of tumor growth factors, regulation of cell adhesion molecules and from inflammatory response. The latter is reflected in another pathway identified, as is the antigen presentation by MHC class II. Conclusion. The pathways identified by different Gleason patterns exhibit progression of localized prostate tumors into metastatic ones, where there is cell growth and dysregulation in signaling pathways at the beginning, and then, as they progress, they acquire the ability to migrate and to regulate AR signaling through different mechanisms so that allows AR to be a central regulator in PC. Citation Format: Natalia L. Acosta, Melody C. Baddoo, Alba L. Combita, Rodolfo Varela, Jorge Mesa, Maria C. Sanabria-Salas, Jovanny Zabaleta. RNA-seq analysis of prostate cancer samples from Hispanic patients reveal progressive characteristics in Gleason patterns. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B56.

Subcellular Spatial Control of Non-Muscle Myosin 2 Redistribution and Stress Fiber Strain by Molecular Tattoo

The cellular distribution of the motor protein non-muscle myosin 2 (NM2) leads to different forms of intracellular strain driving cell motility, cytokinesis and morphogenetic processes including axonal growth and retraction. However, it remains elusive how these cellular processes are governed by the dynamic changes in NM2 localization and supramolecular assembly. To address this problem, we determined the effect of different types of NM2 inhibition on the dynamics of load-bearing stress fibers and unloaded inner cytoplasmatic NM2 structures in live HeLa cells. We followed NM2 redistribution via FRAP, applied also in combination with our recently developed optopharmacological tool, Molecular Tattoo, which allows subcellular confinement of drug effects via 2-photon induced photocrosslinking to targets. We found that the Rho-kinase inhibitor, Y-27632, dramatically accelerates NM2 redistribution and induces stress fiber dissolution, due to NM2 filament disassembly resulting from myosin light chain dephosphorylation. When NM2 was inhibited by para-nitroblebbistatin (pNBleb) or locally by tattooed azidoblebbistatin, in the stress fibers a significant acceleration and suppression of NM2 redistribution was detected at moderate and high inhibitor concentrations, respectively. The observed effects were local and specific for load-bearing peripheral stress fibers, implying the role of mechanical load in NM2 redistribution. Furthermore, in these tests stress fibers remained intact, contrary to that seen upon Rho-kinase inhibition. These results highlight that variations in the localization and/or pharmacological mechanism of NM2 inhibition produce distinct effects on intracellular strain and cellular morphogenesis.Supported by Hungarian Research and Innovation Fund (VKSZ_14-1-2015-0052).

PRL-3 promotes cell adhesion by interacting with JAM2 in colon cancer.

Phosphatase of regenerating liver-3 (PRL-3), also termed PTP4A3, is a metastasis-related protein tyrosine phosphatase. Its expression levels are significantly correlated with the progression and survival of a wide range of malignant tumors. However, the mechanism by which PRL-3 promotes tumor invasion and metastasis is not clear. In the present study, the functions of PRL-3 were systemically analyzed in the key events of metastasis including, motility and adhesion. A cell wounding assay, cell spread assay and cell-matrix adhesion assay were carried out to analyze the cell movement and cell adhesion ability of colon cancer, immunoprecipitation and immunofluorescence assay was confirmed the interaction of PRL-3 and JAM2. It was demonstrated that PRL-3 promoted the motility of Flp-In-293 and LoVo colon cancer cells and increased the distribution of cell skeleton proteins on the cell protrusions. In addition, stably expressing PRL-3 reduced the spreading speed of colon cancer cells and cell adhesion on uncoated, fibronectin-coated and collagen I-coated plates. Mechanistically, junction adhesion molecular 2 (JAM2) was identified as a novel interacting protein of PRL-3. The findings of the present study revealed the roles of PRL-3 in cancer cell motility and adhesion process, and provided information on the possibility of PRL-3 increase cell-cell adhesion by associating with JAM2.

Research Article: Mycobacterium bovis-BCG regulates macrophage vasodilator-stimulated phosphoprotein

Abstract Vasodilator-stimulated phosphoprotein (VASP) is a processive actin polymerase that regulates membrane architecture, cell motility, and pathophysiologic processes such as metastatic invasion. VASP is also a putative regulator of host-pathogen interactions. Intracellular pathogens such as Listeria monocytogenes, Cryptosporidium parvum, and Mycobacterium marnium are known to utilize the host's actin network to facilitate entry into and motility within host cells. In order to determine if VASP is involved in mediating cytoskeletal rearrangement in cells infected with Mycobacterium bovis-BCG, a model organism for the study of Mycobacterium tuberculosis, murine macrophages were infected with the bacterium and analyzed by immunofluorescence. Antibodies against VASP were used to determine its localization. Results indicate that VASP is present on the membrane of murine macrophages in an evenly distributed fashion. Macrophages infected with M. bovis-BCG showed punctate VASP structures. Activated macrophag...

Human recombinant RNASET2: A potential anti-cancer drug.

The roles of cell motility and angiogenetic processes in metastatic spread and tumor aggressiveness are well established and must be simultaneously targeted to maximize antitumor drug potency. This work evaluated the antitumorigenic capacities of human recombinant RNASET2 (hrRNASET2), a homologue of the Aspergillus niger T2RNase ACTIBIND, which has been shown to display both antitumorigenic and antiangiogenic activities. hrRNASET2 disrupted intracellular actin filament and actin-rich extracellular extrusion organization in both CT29 colon cancer and A375SM melanoma cells and induced a significant dose-dependent inhibition of A375SM cell migration. hrRNASET2 also induced full arrest of angiogenin-induced tube formation and brought to a three-fold lower relative HT29 colorectal and A375SM melanoma tumor volume, when compared to Avastin-treated animals. In parallel, mean blood vessel counts were 36.9% lower in hrRNASET2-vs. Avastin-treated mice and survival rates of hrRNASET2-treated mice were 50% at 73 days post-treatment, while the median survival time for untreated animals was 22 days. Moreover, a 60-day hrRNASET2 treatment period reduced mean A375SM lung metastasis foci counts by three-fold when compared to untreated animals. Taken together, the combined antiangiogenic and antitumorigenic capacities of hrRNASET2, seemingly arising from its direct interaction with intercellular and extracellular matrices, render it an attractive anticancer therapy candidate.

The influence of the WWOX gene on the regulation of biological processes during endometrial carcinogenesis

The purpose of the present study was to investigate the role of WW domain containing oxidoreductase (WWOX) downregulation in biological cancer-related processes in normal (non-malignant) and cancer endometrial cell lines. We created an in vitro model using the normal endometrial cell line, THESC, and 2 endometrial cancer cell lines with varying degrees of differentiation, the Ishikawa (well-differentiated) and the MFE296 (moderately differentiated) cells, in which the WWOX tumor suppressor gene was silenced using Gipz lentiviral shRNA. In this model, we examined the changes in invasiveness via biological assays, such as zymography, migration through a basement membrane, the adhesion of cells to extracellular matrix proteins, anchorage-independent growth and colony formation assay. We also evaluated the correlation between the mRNA expression of the WWOX gene and genes involved in the processes of carcinogenesis, namely catenin beta-1 (CTNNB1) and zinc finger E-box binding homeobox 1 (ZEB1) (gene transcription), cadherin 1 (CDH1) and ezrin (EZR) (cell adhesion), vimentin (VIM) (structural proteins), as well as phosphatase and tensin homolog (PTEN) (tumor suppression) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) (SPARC) (cell growth regulation) by RT-qPCR. Downregulation of the WWOX gene in the moderately differentiated MFE296 cell line caused decreased migratory capacity, and a reduction of matrix metalloproteinase-2 (MMP-2) activity. However, these cells grew in semisolid medium and exhibited higher expression of CDH1 and EZR (cell adhesion) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) (cell growth regulation). Moreover, in the well-differentiated endometrial cancer (Ishikawa) cell line, WWOX gene silencing resulted in an increased ability of the cells to proliferate indefinitely. Additionally, WWOX regulated changes in adhesion potential in both the normal and cancer cell lines. Our results suggest that the WWOX tumor suppressor gene modulated the processes of cell motility, cell adhesion, gene expression and remodeling in endometrial cell lines.

Hepatic miRNA profiles and thyroid hormone homeostasis in rats exposed to dietary potassium perfluorooctanesulfonate (PFOS).

Perfluorooctanesulfonate (PFOS) has been widely used in a variety of industrial and commercial applications as a surfactant and stain repellent. PFOS causes liver damage (including liver tumors) in experimental animals, primarily via interaction with PPARα and CAR/PXR. We investigated the involvement of microRNAs (miRNAs) in PFOS-induced hepatotoxicity, and mechanisms involved in abnormal thyroid hormone (TH) homeostasis, in the livers of adult male rats exposed in feed to 50mg PFOS/kg diet for 28 days. PFOS-treated rats exhibited expected histopathological and clinical chemistry changes, and global gene expression changes consistent with the involvement of PPARα and CAR/PXR. Thirty-eight miRNAs were significantly altered. Three members of the miR-200 family were the most increased, while miR-122-5p and miR-21-5p were the most decreased, in PFOS-treated rats. Expression of the miR-23b-3p/27b-3p/24-3p cluster also decreased in PFOS-treated animals. Pathway analysis of miRNAs and associated gene expression changes suggests involvement of epithelial to mesenchymal transition (EMT), which is a primary process of tumor cell motility and cancer metastasis. Our analysis also revealed transcripts that may mediate PFOS-induced effects on TH homeostasis including: activation of the CAR/PXR pathway, phase II/III enzymes, and deiodinase. These changes are consistent with low serum TH due to enhanced metabolic clearance of TH. However, most TH hepatic target genes were not altered in a manner consistent with reduced TH signaling, suggesting that PFOS exposure did not induce functional hypothyroidism. Collectively, the study suggests an important role for miRNAs in PFOS-induced hepatotoxicity and provides insight into the effects of PFOS on TH homeostasis.

Proteomic analysis of mature and immature ejaculated spermatozoa from fertile men

Dysfunctional spermatozoa maturation is the main reason for the decrease in sperm motility and morphology in infertile men. Ejaculated spermatozoa from healthy fertile men were separated into four fractions using three-layer density gradient. Proteins were extracted and bands were digested on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. Western blotting was performed to verify the expression levels of the proteins of interest. 1469 proteins were identified in four fractions of spermatozoa. The number of detected proteins decreased according to the maturation level of spermatozoa. During spermatozoa maturation, proteins involved in gamete generation, cell motility, energy metabolism and oxidative phosphorylation processes showed increasing expression levels and those involved in protein biosynthesis, protein transport, protein ubiquitination, and response to oxidative stress processes showed decreasing expression levels. We validated four proteins (HSP 70 1A, clusterin, tektin 2 and tektin 3) by Western blotting. The study shows protein markers that may provide insight into the ejaculated spermatozoa proteins in different stages of sperm maturation that may be altered or modified in infertile men.