The fluorescent dye Cy3 and galactose derivatives are covalently assembled with different ratios on the surfaces of magnetic nanoparticles (MNPs) to produce multifunctional HepG2 cancer cell–targeting agents and the effect of ligand spatial orientation on the MNP surface is investigated on targeting specificity. By using a mixture of bis‐N‐hydroxysuccinimide ester (a bifunctional linker) and OSu‐activated Cy3 (w/w = 30:1), stable and quantifiable fluorescent MNPs (Cy3@MNPs) are synthesized that could be subsequently loaded with galactosyl ligands. A mono‐antennary and two different tri‐antennary galactosyl ligands are individually immobilized on Cy3@MNPs, and the uptake efficiencies of the resulting galactosyl Cy3@MNPs by HepG2 and HeLa cells are investigated using confocal microscopy. The confocal images show that galactosyl Cy3@MNPs are sprayed over cytoplasm of the HepG2 cells, indicating that the MNP uptake occurs via receptor‐mediated endocytosis that is followed by release from endosomes. The results also reveal that the ligand spatial orientation affects the efficiency of the receptor‐mediated endocytosis and one of the tri‐antennary galactosyl ligands shows the best uptake efficiency owing to the optimal spatial presentation of the galactosyl moieties. Overall, it is shown that the MNP is a good ligand carrier and that, when pre‐assembled, the multivalent ligand structure enhances the interactions between the surface ligands of the MNPs and receptors of HepG2 cells. Additionally, the galactosyl Cy3@MNPs are not cytotoxic, indicating that they may potentially be used for in vivo applications.
Angiogenesis is a highly organized process controlled by a series of molecular events. While much effort has been devoted to identifying angiogenic factors and their reciprocal receptors, far less information is available on the molecular mechanisms underlying directed endothelial cell migration. To search for novel proteins that participate in this process, we used the serial analysis of gene expression (SAGE) transcript profiling approach to identify genes that are selectively expressed in endothelial cells (ECs). Two EC SAGE libraries were constructed from human umbilical vein and artery ECs to enable data-mining against other non-ECs. A novel endothelial protein, Thrombospondin Type I Domain Containing 7A (THSD7A), with preferential expression in placenta vasculature and in human umbilical vein endothelial cells (HUVECs) was identified and targeted for further characterization. Overexpression of a THSD7A carboxyl-terminal fragment in HUVECs inhibited cell migration and disrupted tube formation, while suppression of THSD7A expression enhanced HUVEC migration and tube formation. Immunohistological analysis revealed that THSD7A was expressed at the leading edge of migrating HUVECs, and it co-localized with alpha(V)beta(3) integrin and paxillin. This distribution was dispersed from focal adhesions after disruption of the actin cytoskeleton, suggesting the involvement of THSD7A in cytoskeletal organization. Our results show that THSD7A is a novel placenta endothelial protein that mediates EC migration and tube formation, and they highlight its potential as a new target for anti-angiogenic therapy.
SummaryThe events that prime pluripotent cells for differentiation are not well understood. Inhibitor of DNA binding/differentiation (Id) proteins, which are inhibitors of basic helix-loop-helix (bHLH) transcription factor activity, contribute to pluripotency by blocking sequential transitions toward differentiation. Using yeast-two-hybrid screens, we have identified Id-regulated transcription factors that are expressed in embryonic stem cells (ESCs). One of these, Tcf15, is also expressed in the embryonic day 4.5 embryo and is specifically associated with a novel subpopulation of primed ESCs. An Id-resistant form of Tcf15 rapidly downregulates Nanog and accelerates somatic lineage commitment. We propose that because Tcf15 can be held in an inactive state through Id activity, it may prime pluripotent cells for entry to somatic lineages upon downregulation of Id. We also find that Tcf15 expression is dependent on fibroblast growth factor (FGF) signaling, providing an explanation for how FGF can prime for differentiation without driving cells out of the pluripotent state.
Aberrant expression of cyclin D1, frequently observed in human malignant disorders, has been linked to the control of G(1)→S cell cycle phase transition and development and progression in carcinogenesis. Cyclin D1 level changes are partially controlled by GSK-3β-dependent phosphorylation at threonine-286 (Thr286), which targets cyclin D1 for ubiquitination and proteolytic degradation. In our continuing studies on the mechanism of prostate cancer prevention by resveratrol, focusing on the role of its recently discovered target protein, quinone reductase 2 (NQO2), we generated NQO2 knockdown CWR22Rv1 using short hairpin RNA (shRNA)-mediated gene silencing approach. We found that, compared with cells expressing NQO2 (shRNA08), NQO2 knockdown cells (shRNA25) displayed slower proliferation and G(1) phase cell accumulation. Immunoblot analyses revealed a significant decrease in phosphorylation of retinoblastoma Rb and cyclin D1 in shRNA25 compared with shRNA08. Moreover, shRNA25 cells showed a 37% decrease in chymotrypsin-like proteasome activity. An increase in AKT activity was also observed in shRNA25, supported by a ∼1.5-fold elevation in phosphorylation and ∼50% reduction/deactivation of GSK-3α/β at Ser21/9, which were accompanied by a decrease in phosphorylation of cyclin D1 at T286. NQO2 knockdown cells also showed attenuation of resveratrol-induced downregulation of cyclin D1. Our results indicate a hitherto unreported role of NQO2 in the control of AKT/GSK-3β/cyclin D1 and highlight the involvement of NQO2 in degradation of cyclin D1, as part of mechanism of chemoprevention by resveratrol.
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