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cell migration - Top 30 Publications

Syndecan-1 in mechanosensing of nanotopological cues in engineered materials.

The cells of the vascular system are highly sensitive to biophysical cues from their local cellular microenvironment. To engineer improved materials for vascular devices and delivery of cell therapies, a key challenge is to understand the mechanisms that cells use to sense biophysical cues from their environment. Syndecans are heparan sulfate proteoglycans (HSPGs) that consist of a protein core modified with heparan sulfate glycosaminoglycan chains. Due to their presence on the cell surface and their interaction with cytoskeletal and focal adhesion associated molecules, cell surface proteoglycans are well poised to serve as mechanosensors of the cellular microenvironment. Nanotopological cues have become recognized as major regulators of cell growth, migration and phenotype. We hypothesized that syndecan-1 could serve as a mechanosensor for nanotopological cues and can mediate the responsiveness of vascular smooth muscle cells to nanoengineered materials. We created engineered substrates made of polyurethane acrylate with nanogrooves using ultraviolet-assisted capillary force lithography. We cultured vascular smooth muscle cells with knockout of syndecan-1 on engineered substrates with varying compliance and nanotopology. We found that knockout of syndecan-1 reduced alignment of vascular smooth muscle cells to the nanogrooves under inflammatory treatments. In addition, we found that loss of syndecan-1 increased nuclear localization of Yap/Taz and phospho-Smad2/3 in response to nanogrooves. Syndecan-1 knockout vascular smooth muscle cells also had elevated levels of Rho-associated protein kinase-1 (Rock1), leading to increased cell stiffness and an enhanced contractile state in the cells. Together, our findings support that syndecan-1 knockout leads to alterations in mechanosensing of nanotopographical cues through alterations of in rho-associated signaling pathways, cell mechanics and mediators of the Hippo and TGF-β signaling pathways.

Targeting FGFR pathway in breast cancer.

Developments in breast cancer biology over the last years have permitted deconstructing the molecular profile of the most relevant breast cancer subtypes. This has led to an increase in therapeutic options, including more effective personalized therapy for breast cancer and substantial improvements in patient outcomes. Although currently there are only a few targeted therapies approved for metastatic breast cancer, the discovery of druggable kinase gene alterations has radically changed cancer treatment by providing novel and successfully actionable drug targets. Fibroblast growth factors and their receptors (FGFRs) participate in different physiologic processes and also play an essential role in cancer cell proliferation, survival, differentiation, migration, and apoptosis. This article summarizes the main molecular alterations of FGFRs, as well as the available preclinical and clinical data with FGFR inhibitors in breast cancer, and discusses new opportunities for the clinical development of these agents in patients with breast cancer.

Genetic profiling and surface proteome analysis of human atrial stromal cells and rat ventricular epicardium-derived cells reveals novel insights into their cardiogenic potential.

Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73, CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.

A microfabricated 96-well wound-healing assay.

This article presents a microfabricated 96-well wound-healing assay enabling high-throughput measurement of cellular migration capabilities. Within each well, the middle area is the wound region, made of microfabricated gold surface with self-assembled PEG repellent for cell seeding. After the formation of a cellular confluent monolayer around the wound region, collagen solution was applied to form three-dimensional matrix to cover the PEG surface, initiating the wound-healing process. By interpreting the numbers of migrated cells into the wound regions as a function of specific stimuli with different concentrations, EC50 (half-maximal effective concentration) was obtained. Using H1299 as a model, values of EC50 were quantified as 8% and 160 ng/ml for fetal bovine serum and CXCL12, respectively. In addition, the values of EC50 were demonstrated not to be affected by variations in compositions of extracellular matrix and geometries of wounds, which can thus be regarded as an intrinsic marker. Furthermore, the migration capabilities of a second cell type (HeLa) were characterized by the developed wound-healing assay, producing EC50 of 2% when fetal bovine serum was used as the stimuli. These results validated the proposed high-throughput wound-healing assay, which may function as an enabling tool in studying cellular capabilities of migration and invasion. © 2017 International Society for Advancement of Cytometry.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons.

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo.

Chemotaxis is migration along a specific chemical gradient(1). Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity(2). Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Modeling Persistent Mycoplasma pneumoniae Infection of Human Airway Epithelium.

Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extra-pulmonary spread. The mechanisms of persistence and migration beyond the conducting airways, however, remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture, showing that M. pneumoniae gliding motility is essential for initial colonization and subsequent spread, including localization to epithelial cell junctions. We extended those observations here, characterizing M. pneumoniae infection of NHBE for up to four weeks. Colonization of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation, barrier function remained largely intact. Desquamation was accompanied by epithelial remodeling that included cytoskeletal reorganization and development of deep furrows in the epithelium. Finally, M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology, consistent with contrasting virulence in experimentally infected mice. In summary, this study reports pericellular invasion, NHBE cytoskeletal reorganization, and tissue remodeling with persistent infection in a human airway epithelium model, providing clear insight into the likely route for extra-pulmonary spread.

Targeting ALDH2 with disulfiram/copper reverses the resistance of cancer cells to microtubule inhibitors.

Disulfiram (DSF) in combination with copper (Cu) has been reported to override drug resistance in cancer cells, and DSF combined with chemotherapy based on the microtubule inhibitor vinorelbine appears to prolong survival in non-small cell lung cancer patients. Here, we investigated the mechanisms underlying these findings. DSF/Cu reversed the microtubule inhibitor resistance in A549/Taxol and KB/VCR cells in vitro, and had anti-tumor effects in A549/Taxol and KB/VCR xenograft mice. DSF/Cu and DSF reduced the cancer stem cell (CSC) characteristics of drug-resistant A549/Taxol and KB/VCR cells, including sphere formation, colony generation and migration, and DSF/Cu was more effective than DSF alone. DSF/Cu also decreased the aldehyde dehydrogenase (ALDH) activity and the expression of P-gp and stem cell transcription factors in A549/Taxol and KB/VCR cells. Knockdown of ALDH2 attenuated the CSC characteristics of resistant cancer cells and enhanced their sensitivity to Taxol or VCR. Importantly, DSF/Cu treatment inhibited the expression of ALDH2 in vitro and in vivo. Our findings suggest that DSF/Cu reverses microtubule inhibitor resistance in cancer cells by suppressing ALDH2 expression, and Cu improves the activity of DSF.

Knockdown of pyruvate kinase type M2 suppresses tumor survival and invasion in osteosarcoma cells both in vitro and in vivo.

Osteosarcoma (OS) is the mostly diagnosed primary bone malignancy. Emerging evidence indicates that the activity of pyruvate kinase M2 (PKM2) isoform is crucial for the survival of tumor cells. In the present study, the effect of PKM2 knockdown on the proliferation and migration of OS cells were assessed both in vitro and in vivo. Small hairpin RNA (shRNA) technology were employed to suppresse the expression of PKM2 in MG-63 and Saos-2 cell lines. In vitro, shRNA-mediated knockdown of PKM2 efficiently inhibited cell proliferation, and induced G1 cell cycle arrest and apoptosis in both cell lines, which was associated with decreased expressions of cyclin D1 and Bcl-2 as well as increased expressions of Bax, cleaved-caspase-3, and cleaved-PARP. The invasion and migration potential of OS cell lines were also inhibited by PKM2 knockdown through the regulating effect of PKM2 on MMP-2 and VEGF signaling. In vivo, knockdown of PKM2 decelerated tumor growth rate and induced structure deterioration in tumor tissues. The current study for the first time showed that the activity of PKM2 was indispensable for the development and metastasis of OS, thereby providing the basic information for the future development of PKM2-based anti-OS therapies.

HDAC inhibitor suppresses proliferation and invasion of breast cancer cells through regulation of miR-200c targeting CRKL.

Although histone deacetylase (HDAC) inhibitors have been shown to effectively induce the inhibition of proliferation and migration in breast cancer, the anticancer mechanism remains poorly understood. Our studies show that miR-200c was significantly downregulated in breast cancer cell lines compared to normal cell lines and inversely correlated with the levels of class IIa HDACs and CRKL. HDAC inhibitors and the ectopic expression of miR-200c as tumor suppressors inhibited the proliferation, invasion, and migration of breast cancer cells by downregulating CRKL. These results indicate that the anticancer mechanism of HDAC inhibitor was realized partially by regulating miR-200c via CRKL targeting. Our findings suggest that the HDAC-miR200c-CRKL signaling axis could be a novel diagnostic marker and potential therapeutic target in breast cancer.

Targeting of NT5E by miR-30b and miR-340 attenuates proliferation, invasion and migration of gallbladder carcinoma.

MicroRNAs (miRNAs) have been closely associated with the proliferation, invasion and migration of various cancers, including gallbladder carcinoma (GBC). Previous studies have revealed dysregulation of miR-30b and miR-340 in many types of cancer. However, the role of miR-30b and miR-340 in the development and progression of GBC remains unclear. Moreover, epithelial-to-mesenchymal transition (EMT) has been gradually viewed as a significant contributor to tumor metastasis. In this study, the cell line GBC-SD was used and we explored that EMT promoted GBC cells invasion and migration and inhibited the expression level of miR-30b and miR-340 compared with the control. We showed that overexpression of miR-30b and miR-340 suppressed GBC cells proliferation, invasion and migration, as well as the expression of EMT-associated genes. In addition, we identified ecto-5'-nucleotidase (NT5E) as a common target of miR-30b and miR-340 using bioinformatics analysis and a luciferase assay. Further experiments found that exogenous expression of NT5E in GBC cells could partially reverse the inhibitory effect of miR-30b and miR-340 on cell proliferation, invasion and migration. Our findings suggest that NT5E-targeting miRNAs (miR-30b and miR-340) function as tumor suppressors and may represent promising therapeutic targets for GBC.

Anti-inflammatory, antinociceptive and antioxidant properties of Schinopsis brasiliensis bark.

Schinopsis brasiliensis is a native plant from Brazil, popularly used in folk medicine to relieve pain and treat inflammation. This study evaluated the antinociceptive and anti-inflammatory activities and antioxidant properties of the hydroethanol extract (HEE) and ethyl acetate fraction (EAF) obtained from S. brasiliensis bark.

GV1001 Induces Apoptosis by Reducing Angiogenesis in RCC Cells Both in Vitro and in Vivo.

Objective To investigate the anti-cancer effects of GV1001 and its biological mechanism of action in renal cell carcinoma (RCC). Methods The effects of GV1001 on cell survival and apoptosis in RCC cells were examined in vitro using cell viability assay, fluorescence-activated cell sorting (FACS), and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. To evaluate the effect of GV1001 on migration, invasion and angiogenesis, we used wound healing, invasion, endothelial cell tube formation assay and western blot analysis. Furthermore, we used an RCC xenograft model with either PBS or GV1001 to confirm the anti-cancer effect of GV1001 in vivo. Tumor volume was monitored during treatment, and tumor weight was measured after animal sacrifice. Apoptosis and angiogenesis of the tumor tissue were assessed using H&E staining, immunohistochemistry and western blot analysis. Results GV1001 reduced cell viability and induced apoptosis in RCC cells in vitro. Furthermore, GV 1001 suppressed the migration and invasion of RCC cells through regulation of MMPs and TIMPs. In addition, GV1001 reduced angiogenesis via regulation of HIF-1α. In xenograft mouse model experiment, GV1001 reduced tumor growth and induced apoptosis. As in the in vitro results, GV1001 significantly reduced angiogenesis through regulation of HIF-1α in vivo. Conclusion Our data demonstrated that GV1001 induced apoptosis through suppression of angiogenesis in RCCs both in vitro and in vivo, which suggests that GV1001 may be a potential therapeutic target for RCC.

In vitro mesenchymal-epithelial transition in NIH3T3 fibroblasts results in onset of low-dose radiation hypersensitivity coupled with attenuated connexin-43 response.

Mesenchymal-to-epithelial transition (MET) is associated with altered cell adhesion patterns. Independent studies showed that cellular adhesion regulates low-dose hyper-radiosensitivity (HRS), a phenomenon reported widely in tumour cells. Therefore, present study aimed to investigate whether MET and associated cellular adhesion alterations affect cellular radiosensitivity.

Cancer Stem Cell-Like Population is Preferentially Suppressed by EGFR-TKIs in EGFR-mutated PC-9 Tumor Models.

Although the epidermal growth factor receptor (EGFR) and Wnt/β-catenin signaling systems synergistically regulate many essential developmental and regenerative processes in lung cancer, the mechanisms of their crosstalk remain poorly defined. Our study aimed to investigate an interaction between EGFR and the β-catenin signal.

Intracrine prostaglandin E2 pro-tumoral actions in prostate epithelial cells originate from non-canonical pathways.

Prostaglandin E2 (PGE2 ) increases cell proliferation and stimulates migratory and angiogenic abilities in prostate cancer cells. However, the effects of PGE2 on non-transformed prostate epithelial cells are unknown, despite the fact that PGE2 overproduction has been found in benign hyperplastic prostates. In the present work we studied the effects of PGE2 in immortalized, non-malignant prostate epithelial RWPE-1 cells and found that PGE2 increased cell proliferation, cell migration, and production of vascular endothelial growth factor-A, and activated in vitro angiogenesis. These actions involved a non-canonic intracrine mechanism in which the actual effector was intracellular PGE2 (iPGE2 ) instead of extracellular PGE2 : inhibition of the prostaglandin uptake transporter (PGT) or antagonism of EP receptors prevented the effects of PGE2 , which indicated that PGE2 activity depended on its carrier-mediated translocation from the outside to the inside of cells and that EP receptors located intracellularly (iEP) mediated the effects of PGE2 . iPGE2 acted through transactivation of epidermal growth factor-receptor (EGFR) by iEP, leading to increased expression and activity of hypoxia-inducible factor-1α (HIF-1α). Interestingly, iPGE2 also mediates the effects of PGE2 on prostate cancer PC3 cells through the axis iPGE2 -iEP receptors-EGFR-HIF-1α. Thus, this axis might be responsible for the growth-stimulating effects of PGE2 on prostate epithelial cells, thereby contributing to prostate proliferative diseases associated with chronic inflammation. Since this PGT-dependent non-canonic intracrine mechanism of PGE2 action operates in both benign and malignant prostate epithelial cells, PGT inhibitors should be tested as a novel therapeutic modality to treat prostate proliferative disease.

PRR11 immunoreactivity is a weak prognostic factor in non-mucinous invasive adenocarcinoma of the lung.

Proline-rich protein 11 (PRR11) functions in the progression of cell cycle, and silencing the PRR11 gene in lung cancer cells results in the inhibition of cellular proliferation, cell cycle progression, cell migration, invasion and colony formation. PRR11 may therefore be a therapeutic target in lung cancer.

Clinical presentation of anti-N-methyl-d-aspartate receptor and anti-voltage-gated potassium channel complex antibodies in children: A series of 24 cases.

The symptomatology and paraclinical findings of antibody-mediated encephalitis, a relatively novel disorder, are still being characterized in adults and children. A high index of suspicion is needed in order to identify these cases among children presenting with various neurological symptoms. The aim of this study is to examine the clinical, demographic and laboratory findings and outcome of children with anti-NMDAR and anti-VGKC encephalitis for any typical or distinctive features.

Efficient Induction of Syncytiotrophoblast Layer II Cells from Trophoblast Stem Cells by Canonical Wnt Signaling Activation.

The syncytiotrophoblast layer is the most critical and prominent tissue in placenta. SynT cells are differentiated from trophoblast stem cells (TSCs) during early embryogenesis. Mouse TSCs can spontaneously differentiate into cells of mixed lineages in vitro upon withdrawal of stemness-maintaining factors. However, differentiation into defined placental cell lineages remains challenging. We report here that canonical Wnt signaling activation robustly induces expression of SynT-II lineage-specific genes Gcm1 and SynB and suppresses markers of other placental lineages. In contrast to mouse TSCs, the induced SynT-II cells are migratory. More importantly, the migration depends on hepatocyte growth factor (HGF) and the c-MET signaling axis. Furthermore, HGF-expressing cells lie adjacent to SynT-II cells in developing murine placenta, suggesting that HGF/c-MET signaling plays a critical role in SynT-II cell morphogenesis during the labyrinth branching process. The availability of SynT-II cells in vitro will facilitate molecular understanding of labyrinth layer development.

Establishing and transducing cell polarity: common themes and variations.

All cells in vivo have a primary axis of polarity that controls many aspects of their behaviour, such as the direction of protein secretion and signalling, the orientation of cell division and directed cell movement and morphogenesis. Cell polarise in response to extracellular cues or intracellular landmarks that initiate a signal transduction process that establishes complementary cortical domains of conserved polarity factors. These cortical domains then transmit this polarity to the rest of the cell by regulating the organisation of the cytoskeleton and membrane trafficking systems. Here I review work over the past couple of years that has elucidated many key features of how polarity is established and transduced in different systems, but has also revealed unexpected variations in polarity mechanisms depending on context.

Cofilin signaling in hemin-induced microglial activation and inflammation.

Intracerebral hemorrhage (ICH) is the most severe form of stroke and is further exacerbated by the secondary injury involving inflammatory response due to the activation of microglia. This secondary injury is partly due to the toxic effects of hemin, an endogenous breakdown product of hemoglobin. Cofilin, an actin depolymerizing factor, controls actin dynamics and has been previously shown to be involved in mediating neuronal cell death in ischemic conditions and during bacterial lipopolysaccharide induced microglial activation. There are limited studies regarding the deleterious effects of extremely high concentrations of hemin released during ICH and its effects on microglia and subsequent cofilin response. Therefore, investigations were conducted to study the effects of hemin on microglial activation induced inflammation and the critical role of cofilin in mediating the response. We observed that hemin treated microglia had a concentration dependent increase in cofilin expression and NO production. There were increased levels of iNOS, TNF-α, HO1, Nrf2, Wfs-1, XBP-1 and spliced XBP-1 observed in response to hemin treatment and the signaling was found to be partly mediated by cofilin. Acute hemin treatment did not evoke Ca(2+) signaling and long-term treatment of hemin also resulted in the failure of microglial response to acetylcholine-evoked Ca(2+) signaling. Knockdown of cofilin by siRNA also reduced acetylcholine-evoked Ca(2+) signaling. These studies demonstrate that cofilin signaling is important in hemin-induced inflammation, oxidative stress, ER stress, microglial migration, and the ability to evoke Ca(2+) signaling. Therefore, cofilin inhibition could be a potential therapy in brain injuries triggered by hemin toxicity in conditions like ICH.

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces.

The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends in vitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity in vitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation.

Differences between chronic lymphocytic leukaemia and small lymphocytic lymphoma cells by proteomic profiling and SNP microarray analysis.

The majority of malignant cells in chronic lymphocytic leukaemia (CLL) circulate in the peripheral blood whereas small lymphocytic lymphoma (SLL) cells reside in tissues. The aim of this study was to detect differences in chemokine receptor expression, DNA single nucleotide polymorphism (SNP) microarray analysis and proteomic profiling to help elucidate why the cells remain in their respective environments. We identified by flow cytometric studies of chemokine receptors and DNA SNP microarray analysis significant differences between cells from CLL and SLL patients. Proteomic analysis revealed two potential markers (m/z 3091 and 8707) to distinguish the two disorders. There was a significantly greater expression of leucocyte trafficking receptor CXCR3 (CD183) and migration and homing receptor CXCR4 (CD184), and significantly lower expression of cell adhesion molecule integrin α4 chain (CD49d), on CLL cells, compared with SLL cells. Conversely, SNP microarrays revealed greater numbers of copy-neutral loss of heterozygosity chromosomal aberrations, as well as gross chromosomal aberrations, in the SLL group, compared with the CLL group. These findings revealed that there was a significantly greater expression of trafficking, migration and homing receptors and significantly lower expression of adhesion molecules on CLL cells than on SLL cells, and that SLL may be a more progressive disease than CLL, with a more complex genotype.

MicroRNA‑137 has a suppressive role in liver cancer via targeting EZH2.

A variety of microRNAs (miRs) have been demonstrated to be associated with the development and malignant progression of human cancer; however, the regulatory mechanism of miR‑137 underlying hepatocellular carcinoma (HCC) growth and metastasis still remains to be fully revealed. In the present study, reverse transcription‑quantitative polymerase chain reaction and western blot were used to examine mRNA and protein expression. MTT assay, wound healing assay and Transwell assay were performed to determine cell proliferation, migration and invasion. Luciferase reporter assay was conducted to confirm the targeting relationship. miR‑137 was significantly downregulated in HCC tissues compared to adjacent normal tissues. Low expression of miR‑137 was significantly associated with lymph node metastasis, vein invasion, advanced clinical stage and poor prognosis in HCC. In addition, miR‑137 was also downregulated in several liver cancer cell lines compared with normal liver epithelial cells. Overexpression of miR‑137 led to a significant reduction in cell proliferation, migration and invasion of HepG2 cells. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) was further identified as a direct target gene of miR‑137, and the protein expression of EZH2 was negatively regulated by miR‑137 in HepG2 cells. Additionally, EZH2 was significantly upregulated in HCC tissues and liver cancer cell lines. Furthermore, overexpression of EZH2 significantly eliminated the inhibitory effects of miR‑137 on the malignant phenotypes of HepG2 cells. Therefore, the findings suggest that miR‑137 may have a suppressive role in HCC growth and metastasis via targeting EZH2.

Melatonin inhibits colon cancer RKO cell migration by downregulating Rho‑associated protein kinase expression via the p38/MAPK signaling pathway.

Melatonin is predominately produced and secreted by the pineal gland, and inhibits cell growth in various cancer cell lines such as colorectal cancer. However, the precise mechanisms involved have not been fully elucidated. In the present study, the potential molecular mechanism underlying the efficacy of melatonin on migration in RKO colon cancer cells was investigated. The effects of melatonin and H‑1152, a selective inhibitor of Rho‑associated protein kinase (ROCK), on the migration of RKO cells were analyzed by an in vitro wound healing assay. The localization of zonula occludens‑1 (ZO‑1) and occludin were observed by immunofluorescence. Reverse transcription‑quantitative polymerase chain reaction (qPCR) was performed to analyze the relative mRNA levels of ROCK, ZO‑1 and occludin. In addition, western blot analysis was implemented to examine the expression of ROCK, phospho (p)‑myosin phosphatase targeting subunit 1 (MYPT1), p‑myosin light chains (MLC) and p‑p38. The results revealed that the expression levels of ROCK2, p‑MYPT1 and p‑MLC in RKO cells were decreased, and the membrane protein expression of ZO‑1 and occludin increased when the cells were treated with melatonin. qPCR demonstrated that melatonin downregulated ROCK2 gene expression, and upregulated the expression of the ZO‑1 and occludin genes. The levels of ZO‑1 and occludin localized in the tight junctions were markedly increased in the immunofluorescence assay. In addition, the phosphorylation levels of p38 were reduced when the cells were treated with melatonin, and treatment with H‑1152 downregulated p38 phosphorylation. The results indicated that melatonin may inhibit the migration of RKO colon cancer cells by downregulating ROCK expression via the p38/mitogen‑activated protein kinase signaling pathway.

HMGA1 participates in MHCC97H cell proliferation and invasion through the ILK/Akt/GSK3β signaling pathway.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer‑related mortality, and the prognosis of HCC patients is unsatisfactory. It is known that the occurrence and development of HCC involves numerous genes, as well as various steps and stages in the pathological process. High mobility group AT‑hook 1 (HMGA1) and integrin‑linked kinase (ILK) may be overexpressed in HCC and may serve important roles in the development of cancer; however, the relationship between HMGA1 and ILK in HCC has not been examined. The present study demonstrated that inhibition of HMGA1 expression significantly decreased the levels of expression of ILK and the downstream elements phosphorylated (p)‑Akt, p‑glycogen synthase kinase 3β (GSK3β), matrix metalloproteinase (MMP)2, MMP9, CyclinD1 and c‑Myc. Transfection with an ILK expression vector was able to recover the decreased expression of these downstream genes, and affected cell proliferation and apoptosis. In addition, results from Transwell and wound‑healing experiments indicated that HMGA1 participates cell invasion and migration through the ILK/Akt/GSK3β pathway. The present study aimed to improve our understanding about the regulatory pathway involved in HCC and provides the basis for exploring HMGA1 inhibition as a therapy for patients with HCC and a new treatment strategy to prevent the development of HCC.

Inhibition of Retrograde Transport Limits Polyomavirus Infection In Vivo.

Polyomaviruses (PyVs) silently infect most humans, but they can cause life-threatening diseases in immunocompromised individuals. The JC polyomavirus (JCPyV) induces progressive multifocal leukoencephalopathy, a severe demyelinating disease in multiple sclerosis patients receiving immunomodulatory therapy, and BK polyomavirus (BKPyV)-associated nephropathy is a major cause of kidney allograft failure. No effective anti-PyV agents are available. Several compounds have been reported to possess anti-PyV activity in vitro, but none have shown efficacy in clinical trials. Productive PyV infection involves usurping the cellular retrograde vesicular transport pathway to enable endocytosed virions to navigate to the endoplasmic reticulum where virion uncoating begins. Compounds inhibiting this pathway have been shown to reduce infection by simian virus 40 (SV40), JCPyV, and BKPyV in tissue culture. In this study, we investigated the potential of Retro-2.1, a retrograde transport inhibitor, to limit infection by mouse polyomavirus (MuPyV) in vivo. We found that Retro-2.1 significantly reduced MuPyV levels in the kidney during acute infection without affecting renal function or the MuPyV-specific CD8 T cell response. To approximate the clinical setting of PyV resurgence in immunocompromised hosts, we showed that antibody-mediated depletion of T cells in persistently infected mice elevated MuPyV levels in the kidney and that Retro-2.1 blunted this increase in virus levels. In summary, these data indicate that inhibition of retrograde vesicular transport in vivo controls infection in a natural PyV mouse model and supports development of these compounds as potential therapeutic agents for individuals at risk for human PyV-associated diseases. IMPORTANCE PyVs can cause significant morbidity and mortality in immunocompromised individuals. No clinically efficacious anti-PyV therapeutic agents are available. A recently identified inhibitor of retrograde transport, Retro-2(cycl), blocks movement of PyV virion-containing vesicles from early endosomes to the endoplasmic reticulum, an early step in the PyV life cycle. Retro-2(cycl) and its derivatives have been shown to inhibit infection by human PyVs in tissue culture. Here, we demonstrate that a derivative of Retro-2(cycl), Retro-2.1, reduces infection by MuPyV in the kidneys of acutely infected mice. Mimicking the common clinical scenario of PyV resurgence, we further show that MuPyV levels increase in the kidneys of immunocompromised, persistently infected mice and that this increase is inhibited by Retro-2.1. These data provide the first evidence for control of a natural PyV infection in vivo by administration of an inhibitor of retrograde transport.

Zinc-finger proteins in health and disease.

Zinc-finger proteins (ZNFs) are one of the most abundant groups of proteins and have a wide range of molecular functions. Given the wide variety of zinc-finger domains, ZNFs are able to interact with DNA, RNA, PAR (poly-ADP-ribose) and other proteins. Thus, ZNFs are involved in the regulation of several cellular processes. In fact, ZNFs are implicated in transcriptional regulation, ubiquitin-mediated protein degradation, signal transduction, actin targeting, DNA repair, cell migration, and numerous other processes. The aim of this review is to provide a comprehensive summary of the current state of knowledge of this class of proteins. Firstly, we describe the actual classification of ZNFs, their structure and functions. Secondly, we focus on the biological role of ZNFs in the development of organisms under normal physiological and pathological conditions.