Cell adhesion of PC12 cells to AGE-modified ECM proteins.

<p>AGE-modified and non-modified collagen IV and laminin (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g002" target="_blank">Fig. 2</a>) were coated on E-plates. Cell adhesion of 5×10⁵ PC12 cells was quantified by RTCA real time analysis as described. Adhesion to non-modified ( = control) substrates was set to 100% and adhesion to AGE-modified substrates was calculated in % of control. Each bar represents values of three independent experiments carried out in triplicates (*p≤0.0001).</p

Cell viability after MGO treatment.

<p>PC12 cells were incubated with PBS, 0.1 mM MGO, 0.3 mM MGO or 1 mM MGO for 4 hours. A. Micrographs of typical PC12 cells. B. Tryphan blue staining of PC12 cells. Bars represent three independent experiments carried out in quadruplicates. Cell viability of cells cultured in the presence of PBS ( = control) was set to 1 and cell viability expressed in relation to the control. C. FACS analysis of PC12 cells stained with annexin V and propidium iodide.</p

AGE-modification of PC12 cells using MGO.

<p>PC12 cells were incubated with PBS, 0.1 mM MGO, 0.3 mM MGO or 1.0 mM MGO for 4 hours. A. Washed cells were solubilized and subjected to SDS-gel electrophoresis. Proteins were blotted and detected using monoclonal CML26 antibody B&C. Permeabilized (B) and non-permeabilized (C) were analyzed by flow cytometry using monoclonal CML26 antibody.</p

Real-time analysis of neurite outgrowth of AGE-modified PC12 cells.

<p>PC12 cells were AGE-modified using 1 mM MGO for 4 h as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g004" target="_blank">Figs. 4</a> &<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g005" target="_blank">5</a>. Cells were cultured on LN-coated E-plates and neurite outgrowth was induced by application of 100 ng/ml NGF. Neurite outgrowth was continuously quantified over 48 hours by RTCA as described in Pollscheit et al. (2012). Total neurite outgrowth of non-modified control cells during 48 h was set to 100% and neurite outgrowth of AGE-modified cells was expressed in % of control. Bars represent two independent experiments carried out in quadruplicates.</p

Formation of advanced glycation endproducts (AGE).

<p>Formation of advanced glycation endproducts (AGE).</p

AGE modification of ECM proteins using MGO.

<p>20 µg collagen IV (Col. IV) or laminin (LN) were spotted on a nitrocellulose membrane. One half of the membrane was incubated with 1 mM MGO for 4 hours. AGE formation was detected by dot blot analysis using the monoclonal CML26 antibody.</p

Comparative data about wild type C57Bl/6 <i>GNE</i>+/+ and heterozygous C57Bl/6 <i>GNE</i>+/− mice.

<p>±SD; range in parentheses; n.s., not significant. Data are mean</p><p><i>GNE</i>^{+/+ and} C57Bl/6 <i>GNE</i>^+/− at 24 weeks and at 80 weeks. There was no statistical difference between the parameters in C57Bl/6 </p

Treadmill exercise in the cohort of the wild type C57Bl/6 <i>GNE</i>^+/+ (black) and the C57Bl/6 <i>GNE</i>^+/− (grey)

<p>(<b>A</b>). Electron microscopic findings of 80-week old female mice: depicts subsarcolemmal nucleus and perinuclear vacuole containing abundant dense granular material (unlikely to be lipofuscin) in the wild type mouse. Adjacent to the large vacuole are round structures (arrowhead) of sarcoplasmatic tubules, which are confined by double membranes and contain dark granules and in the centre a corpuscule of medium densitiy (gastrocnemius muscle at 16.700×magnification) (<b>B</b>). The perinuclear vacuole is surrounded by a single membrane in the heterozygous mouse and displays similar features. Note the two rounded structures in the vicinity containing either membranous structures or dense, granular material (arrowhead) (quadriceps muscle at 16.700×magnification) (<b>C</b>). Quantification of sialylation from membrane bound sialic acid in in anterior tibial, gastrocnemic, and quadriceps femoral muscle in wild type C57Bl/6 <i>GNE</i>^+/+ (black bars) and the C57Bl/6 <i>GNE</i>^+/− (gray bars). Values represent means ± 1 SD of three independent experiments (<b>D</b>). Relative sialic acid concentrations in different muscles (anterior tibial, gastrocnemic and quadriceps femoral muscle). Sialic acid concentration after 180 days was set to 100% and all other values were expressed in percent of this value. Note that maximal sialic acid concentration was reached after 180 days and no further increase of sialic acid concentration was observed after that time (<b>E</b>).</p

Sialic Acid Metabolic Engineering: A Potential Strategy for the Neuroblastoma Therapy

<div><p>Background</p><p>Sialic acids (Sia) represent negative-charged terminal sugars on most glycoproteins and glycolipids on the cell surface of vertebrates. Aberrant expression of tumor associated sialylated carbohydrate epitopes significantly increases during onset of cancer. Since Sia contribute towards cell migration ( =  metastasis) and to chemo- and radiation resistance. Modulation of cellular Sia concentration and composition poses a challenge especially for neuroblastoma therapy, due to the high heterogeneity and therapeutic resistance of these cells. Here we propose that Metabolic Sia Engineering (MSE) is an effective strategy to reduce neuroblastoma progression and metastasis.</p><p>Methods</p><p>Human neuroblastoma SH-SY5Y cells were treated with synthetic Sia precursors N-propanoyl mannosamine (ManNProp) or N-pentanoyl mannosamine (ManNPent). Total and Polysialic acids (PolySia) were investigated by high performance liquid chromatography. Cell surface polySia were examined by flow-cytometry. Sia precursors treated cells were examined for the migration, invasion and sensitivity towards anticancer drugs and radiation treatment.</p><p>Results</p><p>Treatment of SH-SY5Y cells with ManNProp or ManNPent (referred as MSE) reduced their cell surface sialylation significantly. We found complete absence of polysialylation after treatment of SH-SY5Y cells with ManNPent. Loss of polysialylation results in a reduction of migration and invasion ability of these cells. Furthermore, radiation of Sia-engineered cells completely abolished their migration. In addition, MSE increases the cytotoxicity of anti-cancer drugs, such as 5-fluorouracil or cisplatin.</p><p>Conclusions</p><p>Metabolic Sia Engineering (MSE) of neuroblastoma cells using modified Sia precursors reduces their sialylation, metastatic potential and increases their sensitivity towards radiation or chemotherapeutics. Therefore, MSE may serve as an effective method to treat neuroblastoma.</p></div

Cytotoxicity assay with 5-Fluorouracil.

<p>0.05×10⁶ SHSY5Y cells were seeded in the RTCA E plate and cultured in the presence or absence of 10 mM ManNProp or ManNPent followed by treatment with 5-FU. A. Cytotoxicity induced by 5-FU towards the engineered cells was measured. B. ManNProp and ManNPent treated cells displayed significant increase in cytotoxicity at 10-fold reduced 5-Fluorouracil concentration compared to untreated cells. ** P<0.0001 for ManNProp and ManNPent treatment until 25 µM 5-Flurouracil.</p