Silencing of PTK7 in Colon Cancer Cells: Caspase-10-Dependent Apoptosis via Mitochondrial Pathway

Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference (siRNA)-mediated gene silencing. Following transfection, the siRNA successfully suppressed PTK7 mRNA and protein expression. Knocking down of PTK7 in HCT 116 cells inhibited cell proliferation compared to control groups and induced apoptosis. Furthermore, this apoptosis was characterized by decreased mitochondrial membrane potential and activation of caspase-9 and -10. Addition of a caspase-10 inhibitor totally blocked this apoptosis, suggesting that caspase-10 may play a critical role in PTK7-knockdown-induced apoptosis, downstream of mitochondria. These observations may indicate a role for PTK7 in cell proliferation and cell apoptosis and may provide a potential therapeutic pathway for the treatment of a variety of cancers

Microbead-Based Platform for Multiplex Detection of DNA and Protein

We present a novel microbead-based detection platform as a simple and universal strategy for simultaneous determination of multiple biomolecules. This platform is composed of streptavidin coated uniform-sized polystyrene microbeads, dye and biotin-labeled ssDNA or aptamer probes, and quencher-labeled complementary sequences. By this method, upon target binding to the probes, quencher strand dissociation is triggered, which results in fluorescence reactivation of the microbead linked probes. The fluorescence variation is readily monitored by flow cytometry and with a high sensitivity. Explicitly, this microbead-based detection platform shows a high sensitivity for target DNA with a detection limit as low as 0.20 nM, alongside good selectivity from one-base mismatched DNA. This novel platform also shows good selectivity and high sensitivity for protein detection when aptamer is used as a probe. The detection limit for lysozyme is as low as 8.56 nM. Moreover, simultaneous detection of multiple targets has been achieved via incorporating different dye-labeled probes on the microbeads concurrently. We have also applied this developed strategy to the detection of target DNA in human serum. This strategy can be easily extended to other targets through simple probe and quencher variation

A DNA Aptameric Ligand of Human Transferrin Receptor Generated by Cell-SELEX

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics

Carbon Dots Based Dual-Emission Silica Nanoparticles as a Ratiometric Nanosensor for Cu²⁺

A simple and effective strategy for designing ratiometric fluorescent nanosensor has been described in this work. A carbon dots (CDs) based dual-emission nanosensor for Cu²⁺ detection was prepared by coating CDs on the surface of Rhodamine B-doped silica nanoparticles. The fluorescent CDs were synthesized using <i>N</i>-(β-aminoethyl)-γ-aminopropyl methyldimethoxysilane (AEAPMS) as the main raw material, so that the residual ethylenediamine groups and methoxysilane groups on the surface of CDs can serve as the Cu²⁺ recognition sites and the silylation reaction groups. The obtained nanosensor showed characteristic fluorescence emissions of Rhodamine B (red) and CDs (blue) under a single excitation wavelength. Upon binding to Cu²⁺, only the fluorescence of CDs was quenched, resulting in the ratiometric fluorescence response of the dual-emission silica nanoparticles. This ratiometric nanosensor exhibited good selectivity to Cu²⁺ over other substances, such as metal ions, amino acids, proteins, and vitamin C. The ratio of <i>F</i>₄₆₇/<i>F</i>₅₈₅ linearly decreased with the increasing of Cu²⁺ concentration in the range of 0 to 3 × 10^–6 M, a detection limit as low as 35.2 nM was achieved. Additionally, this nanosensor was successfully applied for the ratiometric fluorescence imaging of Cu²⁺ in cells and determination of Cu²⁺ in real tap water

Comparative Study of the Chemical Constituents and Bioactivities of the Extracts from Fruits, Leaves and Root Barks of <i>Lycium barbarum</i>

The fruits, leaves and root barks of L. barbarum plant are widely used as functional foods and as ingredients in traditional Chinese prescriptions and patent medicines. They are considered to have different pharmacological activities and health benefits because of their diverse constituents. Here, the chemical constituents of the extracts from fruits, leaves and root barks of L. barbarum were compared by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HR-MS). A total of 131 compounds were identified and seven of them were quantified. Among them, 98, 28 and 35 constituents were detected in fruits, leaves and root barks respectively. Dicaffeoylspermidine/spermine derivatives were the most detected compounds (74/131); among them, dicaffeoylspermine isomers and propionyl-dicaffeoylspermidine were found in root barks in very large amounts (e.g., kukoamine B = 10.90 mg/g dry powder); dicaffeoyl-spermidine isomers were detected in fruits/leaves in a high amount, and many of their glycosylated derivatives were mainly detected in fruits. In addition, six saponins from L. barbarum fruits were reported for the first time, and 5,6-dihydrosolasonine was reported for the first time in plants. The activity assays showed that the root bark extract possessed the strongest antioxidative activity and cytotoxicity, which was presumed due to the large amount of dicaffeoylspermine/spermidines in root barks. Fourteen potential bioactive components from fruits were identified by a target cell-based screening method. These results will help to understand the different biological activities of these three parts of L. barbarum plant and will benefit the discovery of new functional components

Dicyanomethylene Substituted Benzothiazole Squaraines: The Efficiency of Photodynamic Therapy In Vitro and In Vivo

The lack of ideal photosensitizers limits the clinical application of photodynamic therapy (PDT). Here we report the PDT efficiency of dicyanomethylene substituted benzothiazole squaraine derivatives. This class of squaraine derivatives possess strong absorption and long excitation and emission wavelengths (ex/em, 685/720 nm). They show negligible dark toxicity, but can generate singlet oxygen under irradiation resulting in the apoptosis and necrosis of cells (phototoxicity). Changing the side chains of these compounds greatly influences their albumin-binding rate, cellular uptake and their phototoxicity. One of the squaraine derivatives with two methyl butyrate side chains shows high PDT efficiency in a mouse subcutaneous xenograft model under the irradiation of a 690 nm laser. These results show the great potential of dicyanomethylene substituted benzothiazole squaraines to be the leading compound of near-infrared photosensitizers in PDT

Ratiometric Fluorescent Biosensing of Hydrogen Peroxide and Hydroxyl Radical in Living Cells with Lysozyme–Silver Nanoclusters: Lysozyme as Stabilizing Ligand and Fluorescence Signal Unit

Construction of facile ratiometric fluorescent probes which possess sensitive and selective sensing ability for bioactive small molecules is highly desirable and challenging. Herein, silver nanoclusters capped with denatured lysozyme (dLys-AgNCs) were synthesized and proved to be dual emissive. The facility of the dLys-AgNCs ratiometric probe was attributed to the finding that the lysozyme acted not only as stabilizing ligand but also as fluorescence signal unit. In the presence of Fenton reagents, the emission of dLys-AgNCs at 640 nm was quenched by ^•OH, whereas the emission at 450 was enhanced due to ^•OH-induced oxidation of tyrosine in the lysozyme. This probe could be used for highly sensitive detection of H₂O₂. The fluorescence changes of <i>F</i>₄₅₀<i>/F</i>₆₄₀ had fantastic linearity to H₂O₂ concentrations in the range of 0.8–200 μmol/L (<i>R</i>² = 0.9993), with a limit of detection (LOD) as low as 0.2 μmol/L. Additionally, this probe was also applied to H₂O₂-generated oxidase-based biosensing. As a proof-of-concept, glucose and acetylcholine chloride were detected with benefical LOD values of 0.6 μmol/L and 0.8 μmol/L, respectively. Furthermore, fluorescence confocal imaging demonstrated dLys-AgNCs had a sensitive response to fluctuation of ^•OH levels in living cells, which might have promising application in study of ^•OH-induced oxidative damage to proteins

General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs) are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes) have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s

Immunomodulatory Effects of Lycium barbarum Polysaccharide Extract and Its Uptake Behaviors at the Cellular Level

Lycium barbarum L. is a widely used functional food and medicinal herb in Asian countries. L. barbarium polysaccharides (LBP) are considered as one of the major medicinal components of L. barbarium fruit and exhibits a wide range of biological activities. Here, we investigated the immunomodulatory effects of LBP and its uptake behaviors at the cellular level. LBP was prepared by water extraction and ethanol precipitation, and divided into two fractions based on the molecular weight distribution by ultrafiltration (LBP &gt; 10 kDa and LBP &lt; 10 kDa). The physicochemical properties of LBP and LBP fractions were well characterized. The LBP &gt; 10 kDa fraction greatly enhanced the viability of macrophages RAW264.7 cells and induced cell polarization, but had weak effects to other tested tumor cell lines and normal cell line. This fraction could regulate the production of NO, TNF-&alpha;, IL-6 and ROS in RAW264.7 cells, suggesting both pro-inflammatory and anti-inflammatory effects. The dye-labeled LBP could be internalized into all tested cell lines and accumulated in lysosomes. The internalization of LBP in RAW264.7 cells is mainly through the clathrin-mediated endocytosis pathway. The Caco-2 intestinal transport experiment demonstrated that the dye labeled LBP could be transported through the Caco-2 cell monolayer (mimic intestinal epithelium) through clathrin-mediated endocytosis. These results demonstrate the immunomodulatory effects of LBP and its effective uptake by macrophages and intestine