Glycation is regulated by isoflavones

The effect of soy isoflavones on the Maillard reaction (MR) was investigated. Model systems composed of the soy protein glycinin (10 mg mL -1) and fructose (40 mg mL-1) under basic pH (∼12) conditions were employed for testing the anti-glycative effect of the major antioxidant soy isoflavones (genistin and genistein at 10 μg mL-1) and a soy isoflavone-rich extract. The contents of total phenols (TPCs) and total flavonoids (TFCs) of the isoflavone-rich extract were determined. Glycinin was pre-incubated with isoflavones for 1 h and 16 h at 60 °C prior to MR. The progress of MR was estimated by analysis of free amino groups by OPA assay; carbohydrate covalently bound to the protein backbone using phenol-sulfuric acid assay, protein-bound Nε-(carboxymethyl)lysine (CML) by UPLC-MS and spectral analysis of fluorescent protein-bound AGEs. Genistin (10 μg mL-1, 23 μM) and its aglycone genistein (10 μg mL-1, 37 μM) did not prevent protein glycation (p > 0.05). The soy isoflavone-rich extract containing 2.5 mg mL-1 of TFC efficiently decreased the amount of carbohydrate bound to the protein skeleton (20%) (p 80%) (p < 0.05). The anti-glycative mechanism of isoflavones may be related to its conjugation to glycation sites of the protein structure (free amino groups), their antioxidant character and trapping of dicarbonyl intermediates. Extracts based on mixtures of isoflavones may be useful for producing glycated conjugates avoiding the substantial formation of AGEs bound to protein. © the Partner Organisations 2014

In Vivo Evolution of Tumor-Derived Endothelial Cells

The growth of a malignant tumor beyond a certain, limited size requires that it first develop an independent blood supply. In addition to providing metabolic support, this neovasculature also allows tumor cells to access the systemic circulation, thus facilitating metastatic dissemination. The neovasculature may originate either from normal blood vessels in close physical proximity to the tumor and/or from the recruitment of bone marrow-derived endothelial cell (EC) precursors. Recent studies have shown that human tumor vasculature ECs may also arise directly from tumor cells themselves and that the two populations have highly similar or identical karyotypes. We now show that, during the course of serial in vivo passage, these tumor-derived ECs (TDECs) progressively acquire more pronounced EC-like properties. These include higher-level expression of EC-specific genes and proteins, a greater capacity for EC-like behavior in vitro, and a markedly enhanced propensity to incorporate into the tumor vasculature. In addition, both vessel density and size are significantly increased in neoplasms derived from mixtures of tumor cells and serially passaged TDECs. A comparison of early- and late-passage TDECs using whole-genome single nucleotide polymorphism profiling showed the latter cells to have apparently evolved by a process of clonal expansion of a population with a distinct pattern of interstitial chromosomal gains and losses affecting a relatively small number of genes. The majority of these have established roles in vascular development, tumor suppression or epithelial-mesenchymal transition. These studies provide direct evidence that TDECs have a strong evolutionary capacity as a result of their inherent genomic instability. Consequently such cells might be capable of escaping anti-angiogenic cancer therapies by generating resistant populations

The Phenotypic Radiation Resistance of CD44+/CD24−or low Breast Cancer Cells Is Mediated through the Enhanced Activation of ATM Signaling

Cancer initiating cells (CIC) are stem-like cells. CIC may contribute not only to the initiation of cancer but also to cancer recurrence because of the resistance of CIC both to chemotherapy and radiation therapy. From the MCF-7 and MDA-MB231 breast cancer cell lines and primary culture of patient breast cancer cells, we isolated by flow cytometry a CIC subset of cells with the CD44+/CD24−or low phenotype. The CD44+/CD24−or low subset showed increased sphere formation and resistance to radiation compared to the non- CD44+/CD24−or low subset. The increased radiation resistance was not dependent on the result of altered non-homologous end joining (NHEJ) DNA repair activity as both NHEJ activity and expression of the various proteins involved in NHEJ were not significantly different between the CD44+/CD24−or low and non- CD44+/CD24−or low subsets. However, activation of ATM signaling was significantly increased in CD44+/CD24−or low cells compared to non- CD44+/CD24−or low cells in both from breast cancer cell lines and primary human breast cancer cells. Application of an ATM inhibitor effectively decreased the radiation resistance of CD44+/CD24−or low subset, suggesting that targeting ATM signaling may provide a new tool to eradicate stem-like CIC and abolish the radiation resistance of breast cancer

Eya1 acts as a critical regulator for specifying the metanephric mesenchyme

AbstractAlthough it is well established that the Gdnf-Ret signal transduction pathway initiates metanephric induction, no single regulator has yet been identified to specify the metanephric mesenchyme or blastema within the intermediate mesoderm, the earliest step of metanephric kidney development and the molecular mechanisms controlling Gdnf expression are essentially unknown. Previous studies have shown that a loss of Eya1 function leads to renal agenesis that is a likely result of failure of metanephric induction. The studies presented here demonstrate that Eya1 specifies the metanephric blastema within the intermediate mesoderm at the caudal end of the nephrogenic cord. In contrast to its specific roles in metanephric development, Eya1 appears dispensable for the formation of nephric duct and mesonephric tubules. Using a combination of null and hypomorphic Eya1 mutants, we now demonstrated that approximately 20% of normal Eya1 protein level is sufficient for establishing the metanephric blastema and inducing the ureteric bud formation but not for its normal branching. Using Eya1, Gdnf, Six1 and Pax2 mutant mice, we show that Eya1 probably functions at the top of the genetic hierarchy controlling kidney organogenesis and it acts in combination with Six1 and Pax2 to regulate Gdnf expression during UB outgrowth and branching. These findings uncover an essential function for Eya1 as a critical determination factor in acquiring metanephric fate within the intermediate mesoderm and as a key regulator of Gdnf expression during ureteric induction and branching morphogenesis

Overexpression of receptor of advanced glycation end products hypersensitizes cells for amyloid beta peptide-induced cell death

Hadding A, Kaltschmidt B, Kaltschmidt C. Overexpression of receptor of advanced glycation end products hypersensitizes cells for amyloid beta peptide-induced cell death. Biochim Biophys Acta. 2004;1691(1):67-72

Permanently blocked stem cells derived from breast cancer cell lines

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs

EC-specific marker expression increases in serially-passaged H460-derived TDECs.

<p>(A) Cell surface staining for hCD31. TDECs from the indicated serial passages were isolated and the cell surface expression of the EC-specific marker hCD31 was then assessed by flow cytometry. (B) AcLDL uptake, E-lectin binding and anti-human vWF (hvWF) and anti-human ESAM (hESAM) immuno-staining were assessed in early and late TDECs using confocal microscopy (16). HUVECs were included as positive controls. Nuclei were stained with DAPI (blue). H&E staining under light microscopy was used to compare the morphologies of H460 tumor cells, TDECs, and HUVECs. Images were obtained at 20−40× magnification. (C) Expression of CD31 and vWF assessed by immuno-blotting. Early- and late-passage TDECs were examined by immuno-blotting for hCD31 and hvWF, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037138#pone.0037138-Sajithlal1" target="_blank">[16]</a>. All lanes in the hCD31 blot were from the same X-ray film. β-actin levels were measured on the same samples and served as a loading control. (D) qRT-PCR was used to assess mRNA expression for the EC markers ESAM, CD31, and VWF in late passage TDECs relative to early passage TDECs. Values shown represent the average of at least triplicate reactions and <i>p</i> values were from a standard Student’s <i>t</i> test.</p

Summary of genomic alterations in late passage H460 TDECs.

<p>The nucleotide coordinates of genomic alterations in SP5 and SP6 TDECs were determined by the first and last SNPs that differed from those of early passage TDECs and the precise locations were obtained using the Ensembl Genome Browser 57 database.</p