Transcription factor Sp1 induces ADAM17 and contributes to tumor cell invasiveness under hypoxia

<p>Abstract</p> <p>Background</p> <p>Expression of the Sp1 transcription factor is induced by hypoxia, and the ADAM17 promoter contains predicted Sp1 binding sites. ADAM17 contributes to hypoxic-induce invasiveness of glioma. In this study, we investigated whether Sp1 transcription factor induces ADAM17 and/or contributes to tumor cell invasiveness in hypoxia.</p> <p>Methods</p> <p>Employing RT-PCR and Western blot, we examined the role of Sp1 in ADAM17 transcription/expression under normoxic and hypoxic conditions, and whether it binds to the ADAM17 GC-rich promoter region using a chromatin immunoprecipitation assay. Additionally, we tested the effect of Sp1 suppression in tumor cell invasion and migration, using Matrigel basement membrane invasion chambers, a scratch wound-healing assay, and small interfering RNA.</p> <p>Results</p> <p>Here, we found that Sp1 binds to the ADAM17 promoter, and that Sp1 regulates ADAM17 expression under hypoxia. Furthermore, suppression of Sp1 decreases invasiveness and migration in U87 tumor cells.</p> <p>Conclusion</p> <p>Our findings suggest the Sp1 transcription factor mediates ADAM17 expression under hypoxia, regulates glioma invasiveness, and thus, may be a target for anti-invasion therapies.</p

Overexpression of miR‑145 in U87 cells reduces glioma cell malignant phenotype and promotes survival after in vivo implantation

In the present study, we sought to elucidate the effect of miR‑145 on glioma cell progression and its mechanisms of action. We examined the effects of miR‑145 on proliferation and invasion of U87 glioma cells and on capillary tube formation. Our data show that restoration of miR‑145 in U87 glioma cells significantly reduced their in vitro proliferation, invasion and angiogenesis. However, decreased miR‑145 expression promoted U87 glioma cell proliferation, invasion and angiogenesis, and reduced-expression of miR‑145 increased ADAM17 and EGFR expression in U87 cells. Overexpression of miR‑145 reduced ADAM17 and EGFR expression. VEGF secretion and VEGF expression were decreased by increased miR‑145 expression in U87 cells and were reversed by miR‑145 downregulation in vitro. Nude mice with intracerebral implantation of U87 overexpressing miR‑145 cells exhibited significantly reduced tumor growth and promoted survival compared with control groups. Taken together, these results suggest a role for miR‑145 as a tumor suppressor which inhibits glioma cell proliferation, invasion and angiogenesis in vitro and reduces glioma growth in vivo

Modeling Elasticity in Crystal Growth

A new model of crystal growth is presented that describes the phenomena on atomic length and diffusive time scales. The former incorporates elastic and plastic deformation in a natural manner, and the latter enables access to times scales much larger than conventional atomic methods. The model is shown to be consistent with the predictions of Read and Shockley for grain boundary energy, and Matthews and Blakeslee for misfit dislocations in epitaxial growth.Comment: 4 pages, 10 figure

Homo sapiens Systemic RNA Interference-defective-1 Transmembrane Family Member 1 (SIDT1) Protein Mediates Contact-dependent Small RNA Transfer and MicroRNA-21-driven Chemoresistance

Locally initiated RNA interference (RNAi) has the potential for spatial propagation, inducing posttranscriptional gene silencing in distant cells. In Caenorhabditis elegans, systemic RNAi requires a phylogenetically conserved transmembrane channel, SID-1. Here, we show that a human SID-1 orthologue, SIDT1, facilitates rapid, contact-dependent, bidirectional small RNA transfer between human cells, resulting in target-specific non-cell-autonomous RNAi. Intercellular small RNA transfer can be both homotypic and heterotypic. We show SIDT1-mediated intercellular transfer of microRNA-21 to be a driver of resistance to the nucleoside analog gemcitabine in human adenocarcinoma cells. Documentation of a SIDT1-dependent small RNA transfer mechanism and the associated phenotypic effects on chemoresistance in human cancer cells raises the possibility that conserved systemic RNAi pathways contribute to the acquisition of drug resistance. Mediators of non-cell-autonomous RNAi may be tractable targets for novel therapies aimed at improving the efficacy of current cytotoxic agents

Exosomes as Tools to Suppress Primary Brain Tumor

Exosomes are small microvesicles released by cells that efficiently transfer their molecular cargo to other cells, including tumor. Exosomes may pass the blood-brain barrier and have been demonstrated to deliver RNAs contained within to brain. As they are non-viable, the risk profile of exosomes is thought to be less than that of cellular therapies. Exosomes can be manufactured at scale in culture, and exosomes can be engineered to incorporate therapeutic miRNAs, siRNAs, or chemotherapeutic molecules. As natural biological delivery vehicles, interest in the use of exosomes as therapeutic delivery agents is growing. We previously demonstrated a novel treatment whereby mesenchymal stromal cells were employed to package tumor-suppressing miR-146b into exosomes, which were then used to reduce malignant glioma growth in rat. The use of exosomes to raise the immune system against tumor is also drawing interest. Exosomes from dendritic cells which are antigen-presenting, and have been used for treatment of brain tumor may be divided into three categories: (1) exosomes for immunomodulation-based therapy, (2) exosomes as delivery vehicles for anti-tumor nucleotides, and (3) exosomes as drug delivery vehicles. Here, we will provide an overview of these three applications of exosomes to treat brain tumor, and examine their prospects on the long road to clinical use

Density-Dependent Regulation of Glioma Cell Proliferation and Invasion Mediated by miR-9

The phenotypic axis of invasion and proliferation in malignant glioma cells is a well-documented phenomenon. Invasive glioma cells exhibit a decreased proliferation rate and a resistance to apoptosis, and invasive tumor cells dispersed in brain subsequently revert to proliferation and contribute to secondary tumor formation. One miRNA can affect dozens of mRNAs, and some miRNAs are potent oncogenes. Multiple miRNAs are implicated in glioma malignancy, and several of which have been identified to regulate tumor cell motility and division. Using rat 9 L gliosarcoma and human U87 glioblastoma cell lines, we investigated miRNAs associated with the switch between glioma cell invasion and proliferation. Using micro-dissection of 9 L glioma tumor xenografts in rat brain, we identified disparate expression of miR-9 between cells within the periphery of the primary tumor, and those comprising tumor islets within the invasive zone. Modifying miR-9 expression in in vitro assays, we report that miR-9 controls the axis of glioma cell invasion/proliferation, and that its contribution to invasion or proliferation is biphasic and dependent upon local tumor cell density. In addition, immunohistochemistry revealed elevated hypoxia inducible factor 1 alpha (HIF-1α) in the invasive zone as compared to the primary tumor periphery. We also found that hypoxia promotes miR-9 expression in glioma cells. Based upon these findings, we propose a hypothesis for the contribution of miR-9 to the dynamics glioma invasion and satellite tumor formation in brain adjacent to tumor

Modeling elasticity in crystal growth

A new model of crystal growth is presented that describes the phenomena on atomic length and diffusive time scales. The former incorporates elastic and plastic deformation in a natural manner, and the latter enables access to time scales much larger than conventional atomic methods. The model is shown to be consistent with the predictions of Read and Shockley for grain boundary energy, and Matthews and Blakeslee for misfit dislocations in epitaxial growth. DOI: 10.1103/PhysRevLett.88.245701 PACS numbers: 64.60.Cn, 05.70.Ln, 64.60.My, 81.30.Hd The appearance and growth of crystal phases occurs in many technologically important processes including epitaxial growth and zone refinement. While a plethora of models have been constructed to examine various aspects of these phenomena, it has proven difficult to develop a computationally efficient model that can be used for a wide range of applications. For example, standard molecular dynamics simulations include the necessary physics but are limited by atomic sizes (Å) and phonon time scales (ps). Conversely, continuum field theories can access longer length (i.e., correlation length) and time (i.e., diffusive) scales, but are difficult to incorporate with the appropriate physics. In this paper a new model is presented that includes the essential physics and is not limited by atomic time scales. To illustrate the features that must be incorporated, it is useful to consider two examples. First, consider the nucleation and growth of crystals from a pure supercooled liquid or vapor phase. In such a process, small crystallites nucleate (heterogeneously or homogeneously) and grow in arbitrary locations and orientations. Eventually, the crystallites impinge on one another and grain boundaries are formed. Further growth is then determined by the evolution of grain boundaries. Now consider the growth of a thin crystal film on a substrate of a different crystal structure. The substrate stresses the overlying film which can destabilize the growing film and cause an elastic defect-free morphological deformation [1,2], plastic deformation involving misfit dislocations [3], or a combination of both. Thus, the model must be able to nucleate crystallites at arbitrary locations and orientations and contain elastic and plastic deformations. While all these features are naturally incorporated in atomistic descriptions, they are much more difficult to include in continuum or phase field models. Historically, many continuum models have been developed to describe certain aspects of crystal growth and liquid/solid transitions in general. At the simplest level, &quot;model A&quot; in the Halperin and Hohenberg [4] classification scheme has been used to describe liquid/solid transitions. This model treats all solids equivalently and does not introduce any crystal anisotropy. Extensions to this basic model have been developed to incorporate a solid phase that has multiple states that represent multiple orientations In this work, these limitations are overcome by considering a free energy that is minimized by a periodic hexagonal (i.e., solid) state. Such free energies have arisen in many other physical systems and ≠c͞≠t = 2 ͑dF ͞dc͒ 1 h , where h is a stochastic noise, with zero mean and correlations ͗h͑ r, t͒h͑ r 0 , t 0 ͒͘ 2G= 2 d͑ r 2 r 0 ͒d͑t 2 t 0 ͒, and G 0 hereafter, q o and e are constants. The field c represents the local mass density. The specific free energy given in Eq. (1) was chosen as it is the simplest form that produces periodic states. The simplicity is important, as it leads to computationally efficient numerical schemes. Producing periodic structures is crucial, as such states naturally allow for elastic and plastic deformations, multiple orientations, and can accommodate free surfaces, as required for a crystal phase. The dynamical equation of motion, Eq. (2), was chosen to conserve the local mass density. The focus of this paper is on two dimensions (2D); it is straightforward to extend these calculations to 3D. In 2D, this free energy is minimized by striped ͑c s ͒, hexagonal ͑c h ͒, and constant ͑c c ͒ states depending on the average value,c, of c. To estimate the phase diagram, these states 245701-1 0031-9007͞02͞ 88(24)͞245701(4)$20.0

Effect of exosomes derived from multipluripotent mesenchymal stromal cells on functional recovery and neurovascular plasticity in rats after traumatic brain injury

OBJECT: Transplanted multipotent mesenchymal stromal cells (MSCs) improve functional recovery in rats after traumatic brain injury (TBI). In this study the authors tested a novel hypothesis that systemic administration of cell-free exosomes generated from MSCs promotes functional recovery and neurovascular remodeling in rats after TBI. METHODS: Two groups of 8 Wistar rats were subjected to TBI, followed 24 hours later by tail vein injection of 100 μg protein of exosomes derived from MSCs or an equal volume of vehicle (phosphate-buffered saline). A third group of 8 rats was used as sham-injured, sham-treated controls. To evaluate cognitive and sensorimotor functional recovery, the modified Morris water maze, modified Neurological Severity Score, and foot-fault tests were performed. Animals were killed at 35 days after TBI. Histopathological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. RESULTS: Compared with the saline-treated group, exosome-treated rats with TBI showed significant improvement in spatial learning at 34-35 days as measured by the modified Morris water maze test (p \u3c 0.05), and sensorimotor functional recovery (i.e., reduced neurological deficits and foot-fault frequency) was observed at 14-35 days postinjury (p \u3c 0.05). Exosome treatment significantly increased the number of newly generated endothelial cells in the lesion boundary zone and dentate gyrus and significantly increased the number of newly formed immature and mature neurons in the dentate gyrus as well as reducing neuroinflammation. CONCLUSIONS: The authors demonstrate for the first time that MSC-generated exosomes effectively improve functional recovery, at least in part, by promoting endogenous angiogenesis and neurogenesis and by reducing inflammation in rats after TBI. Thus, MSC-generated exosomes may provide a novel cell-free therapy for TBI and possibly for other neurological diseases

Exosomes Derived from Mesenchymal Stromal Cells Promote Axonal Growth of Cortical Neurons

Treatment of brain injury with exosomes derived from mesenchymal stromal cells (MSCs) enhances neurite growth. However, the direct effect of exosomes on axonal growth and molecular mechanisms underlying exosome-enhanced neurite growth are not known. Using primary cortical neurons cultured in a microfluidic device, we found that MSC-exosomes promoted axonal growth, whereas attenuation of argonaut 2 protein, one of the primary microRNA (miRNA) machinery proteins, in MSC-exosomes abolished their effect on axonal growth. Both neuronal cell bodies and axons internalized MSC-exosomes, which was blocked by botulinum neurotoxins (BoNTs) that cleave proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Moreover, tailored MSC-exosomes carrying elevated miR-17-92 cluster further enhanced axonal growth compared to native MSC-exosomes. Quantitative RT-PCR and Western blot analysis showed that the tailored MSC-exosomes increased levels of individual members of this cluster and activated the PTEN/mTOR signaling pathway in recipient neurons, respectively. Together, our data demonstrate that native MSC-exosomes promote axonal growth while the tailored MSC-exosomes can further boost this effect and that tailored exosomes can deliver their selective cargo miRNAs into and activate their target signals in recipient neurons. Neuronal internalization of MSC-exosomes is mediated by the SNARE complex. This study reveals molecular mechanisms that contribute to MSC-exosome-promoted axonal growth, which provides a potential therapeutic strategy to enhance axonal growth