GCSH antisense regulation determines breast cancer cells' viability.

Since it is known that cancer cells exhibit a preference for increased glycine consumption, the respective glycine metabolizing enzymes are in focus of many research projects. However, no cancer associated studies are available for the Glycine Cleavage System Protein H (GCSH) to date. Our initial analysis revealed a GCSH-overexpression of the protein-coding transcript variant 1 (Tv1) in breast cancer cells and tissue. Furthermore, a shorter (391 bp) transcript variant (Tv*) was amplified with an increased expression in healthy breast cells and a decreased expression in breast cancer samples. The Tv1/Tv* transcript ratio is 1.0 in healthy cells on average, and between 5-10 in breast cancer cells. Thus, a GCSH-equilibrium at the transcript level is likely conceivable for optimal glycine degradation. A possible regulative role of Tv* was proven by Tv1-Tv*-RNA-binding and overexpression studies which consequently led to serious physiological alterations: decreased metabolic activity, release of the lactate dehydrogenase, increased extracellular acidification, and finally necrosis as a result of impaired plasma membranes. In contrast, Tv1-overexpression led to an additional increase in cellular vitality of the tumor cells, primarily due to the acceleration of the mitochondrial glycine decarboxylation activity. Ultimately, we provide the first evidence of a sensitive GCSH-antisense regulation which determines cancerous cell viability.

First evidence of SGPL1 expression in the cell membrane silencing the extracellular S1P siren in mammary epithelial cells.

The bioactive lipid sphingosine-1-phosphate (S1P) is a main regulator of cell survival, proliferation, motility, and platelet aggregation, and it is essential for angiogenesis and lymphocyte trafficking. In that S1P acts as a second messenger intra- and extracellularly, it might promote cancer progression. The main cause is found in the high S1P concentration in the blood, which encourage cancer cells to migrate through the endothelial barrier into the blood vessels. The irreversible degradation of S1P is solely caused by the sphingosine-1-phosphate lyase (SGPL1). SGPL1 overexpression reduces cancer cell migration and therefore silences the endogenous S1P siren, which promotes cancer cell attraction-the main reason for metastasis. Since our previous metabolomics studies revealed an increased SGPL1 activity in association with successful breast cancer cell treatment in vitro, we further investigated expression and localization of SGPL1. Expression analyses confirmed a very low SGPL1 expression in all breast cancer samples, regardless of their subtype. Additionally, we were able to prove a novel SGPL expression in the cytoplasm membrane of non-tumorigenic breast cells by fusing three independent methods. The general SGPL1 downregulation and the loss of the plasma membrane expression resulted in S1P dependent stimulation of migration in the breast cancer cell lines MCF-7 and BT-20. Not only S1P stimulated migration could be repressed by overexpressing the natural SGPL1 variant not but also more general migratory activity was significantly reduced. Here, for the first time, we report on the SGPL1 plasma membrane location in human, non-malignant breast epithelial cell lines silencing the extracellular S1P siren in vitro, and thereby regulating pivotal cellular functions. Loss of this plasma membrane distribution as well as low SGPL1 expression levels could be a potential prognostic marker and a viable target for therapy. Therefore, the precise role of SGPL1 for cancer treatment should be evaluated.

Silicon-dioxide-polyvinylpyrrolidone as a wound dressing for skin defects in a murine model.

OBJECTIVE: There is a high demand for temporary wound dressings that improve wound healing and regeneration. Silicon (as SiO2) has been shown to support the growth and collagen formation in biological systems. METHODS: A nanocomposite was made from PVP (polyvinylpyrrolidon), nano-sized silica aggregates and water and served for fabrication of a wet dressing material (SiO2-PVP gel, by cross-linking the gel) and a freeze-dried dressing material (SiO2-PVP fleece). Materials were characterized by SAXS, DSC, EDX and viscosity measurements. A 10 mm circular defect was set on both sides of the back of SKH1-hr mice (n = 40) and both dressing materials were compared with untreated controls. After 3, 6, 9, 12 and 15 days, the defect regions were explanted and evaluated by histomorphometric measurements and CD31-immunohistochemistry. RESULTS: The microstructure of the compound was composed of fiber like structures. SiO2 nano-aggregates inside the composite remained stable and embedded in a rigid amorphous PVP fraction. In animal experiments, all groups showed a non-irritated defect closure after 9 days. EDX of SiO2-PVP gel and fleeces revealed SiO2-PVP diffusion into the wound. Wound contraction was significantly enhanced after treatment with SiO2-PVP gel followed by SiO2-PVP fleece compared to controls. Re-epithelialization was increased in SiO2-PVP treated wounds and the regenerated epidermis showed a well-differentiated layer structure compared to untreated controls. CONCLUSIONS: The results indicate that silica diffuses from the dressing into the wound. Both dressings affect the wound healing. The SiO2-based wound dressing may counteract scarring and might be suitable as a temporary wound dressing.

Stem cell labeling with iron oxide nanoparticles: impact of 3D culture on cell labeling maintenance.

AIM: We aimed to analyze the suitability of nanoparticles (M4E) for safe human mesenchymal stem cell (hMSC) labeling and determined cell labeling maintenance in 2D and 3D culture. MATERIALS & METHODS: We investigated cell-particle interaction and the particles' impact on cell viability, growth and proliferation. We analyzed cell labeling maintenance in 2D and 3D culture invasively and noninvasively. RESULTS: M4E do not affect cell viability, growth and proliferation and do not cause chromosomal aberrations. Cell labeling maintenance is up to five-times higher in 3D conditions compared with 2D culture. CONCLUSION: M4E allow safe hMSC labeling and noninvasive identification. Our hMSC-loaded, 3D tissue-engineered construct could serve as a graft for regenerative therapies, in which M4E-labeled hMSCs can migrate to their target.

Guidance of mesenchymal stem cells on fibronectin structured hydrogel films.

Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.

Comparative in vitro study on magnetic iron oxide nanoparticles for MRI tracking of adipose tissue-derived progenitor cells.

Magnetic resonance imaging (MRI) using measurement of the transverse relaxation time (R2*) is to be considered as a promising approach for cell tracking experiments to evaluate the fate of transplanted progenitor cells and develop successful cell therapies for tissue engineering. While the relationship between core composition of nanoparticles and their MRI properties is well studied, little is known about possible effects on progenitor cells. This in vitro study aims at comparing two magnetic iron oxide nanoparticle types, single vs. multi-core nanoparticles, regarding their physico-chemical characteristics, effects on cellular behavior of adipose tissue-derived stem cells (ASC) like differentiation and proliferation as well as their detection and quantification by means of MRI. Quantification of both nanoparticle types revealed a linear correlation between labeling concentration and R2* values. However, according to core composition, different levels of labeling concentrations were needed to achieve comparable R2* values. Cell viability was not altered for all labeling concentrations, whereas the proliferation rate increased with increasing labeling concentrations. Likewise, deposition of lipid droplets as well as matrix calcification revealed to be highly dose-dependent particularly regarding multi-core nanoparticle-labeled cells. Synthesis of cartilage matrix proteins and mRNA expression of collagen type II was also highly dependent on nanoparticle labeling. In general, the differentiation potential was decreased with increasing labeling concentrations. This in vitro study provides the proof of principle for further in vivo tracking experiments of progenitor cells using nanoparticles with different core compositions but also provides striking evidence that combined testing of biological and MRI properties is advisable as improved MRI properties of multi-core nanoparticles may result in altered cell functions.

Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors.

The control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. Because integrin receptors function as mechanotransducers, we applied drag forces to ß1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors.