CX-4945 inhibits TGF-β1-induced non-Smad signaling.

<p>The effect of CX-4945 on TGF-β1-induced non-Smad activation was evaluated. Briefly, after 24 h serum starvation, A549 cells were treated with TGF-β1 (5 ng/ml) alone or in combination with CX-4945 in media containing 0.1% FBS for 72 h (A) or 48 h (B). The relative, normalized ratio between phosphorylated protein and the protein itself was presented.</p

CK2 Inhibitor CX-4945 Blocks TGF-β1-Induced Epithelial-to-Mesenchymal Transition in A549 Human Lung Adenocarcinoma Cells

<div><p>Background</p><p>The epithelial-to-mesenchymal transition (EMT) is a major phenotype of cancer metastasis and invasion. As a druggable cancer target, the inhibition of protein kinase CK2 (formally named to casein kinase 2) has been suggested as a promising therapeutic strategy to treat EMT-controlled cancer metastasis. This study aimed to evaluate the effect of the CK2 inhibitor CX-4945 on the processes of cancer migration and invasion during the EMT in A549 human lung adenocarcinoma cells.</p> <p>Materials and Methods</p><p>The effect of CX-4945 on TGF-β1-induced EMT was evaluated in A549 cells treated with TGF-β1 (5 ng/ml) and CX-4945. The effect of CX-4945 on TGF-β1-induced cadherin switch and activation of key signaling molecules involved in Smad, non-Smad, Wnt and focal adhesion signaling pathways were investigated by Western blot analysis, immunocytochemistry and reporter assay. Additionally, the effect of CX-4945 on TGF-β1-induced migration and invasion was investigated by wound healing assay, Boyden chamber assay, gelatin zymography, and the quantitative real-time PCR.</p> <p>Results</p><p>CX-4945 inhibits the TGF-β1-induced cadherin switch and the activation of key signaling molecules involved in Smad (Smad2/3, Twist and Snail), non-Smad (Akt and Erk), Wnt (β-catenin) and focal adhesion signaling pathways (FAK, Src and paxillin) that cooperatively regulate the overall process of EMT. As a result, CX-4945 inhibits the migration and invasion of A549 cells accompanied with the downregulation of MMP-2 and 9.</p> <p>Conclusions</p><p>Clinical evaluation of CX-4945 in humans as a single agent in solid tumors and multiple myeloma has established its promising pharmacokinetic, pharmacodynamic, and safety profiles. Beyond regression of tumor mass, CX-4945 may be advanced as a new therapy for cancer metastasis and EMT-related disorders.</p> </div

CX-4945 inhibits TGF-β1-induced Smad signaling.

<p>(A) The effect of CX-4945 on TGF-β1-induced activation of Smad and expression of Snail and Twist was evaluated using Western blot analysis. Briefly, after 24 h serum starvation, A549 cells were treated with TGF-β1 (5 ng/ml) alone or in combination with CX-4945 in media containing 0.1% FBS for 48 h. Cytosolic and Nuclear fractions were obtained as described in ‘Materials and Methods’. Actin and histone H3 were used as internal control of cytosolic and nuclear fraction, respectively. The relative, normalized ratio between p-Smad2 and actin was presented. (B) Nuclear translocation of Smad2/3 and its inhibition by CX-4945 were confirmed by immunocytochemistry. Nuclei were counterstained with Hoechst 33342. All scale bars represent 50 µm.</p

Scheme of the molecular mechanism proposed for action of CX-4945 to inhibit the TGF-β1-induced EMT.

<p>CX-4945 has the potential to inhibit the TGF-β1-induced EMT through Smad, non-Smad, Wnt, focal adhesion and MMPs/NF-κB signaling pathways.</p

CX-4945 inhibits TGF-β1-induced migration and invasion.

<p>The effect of CX-4945 on the TGF-β1-induced migration and invasion of A549 cells was evaluated using IncuCyte software (A) and Boyden chambers (B), respectively. The red and white dashed line (A) represent the wounded area and the edge of migrated cells, respectively. Values (% RWD; Relative Wound Density) represent mean ± SD of triplicate samples and reported images are representatives of triplicate experiments. The effect of CX-4945 on the TGF-β1-induced activation of MMP-2/9 was evaluated using gelatin zymography (C), while its effect on TGF-β1-induced MMP-2/9 transcription was evaluated using real-time PCR (D). The effect of CX-4945 on the TGF-β1-induced activation of molecules such as FAK, Src, and paxillin was evaluated using Western blot analysis (E). Briefly, proteins were prepared in serum-deprived A549 cells treated with TGF-β1 (5 ng/ml) alone or in combination with CX-4945 in serum-free media for 48 h. Actin was used as a loading control. The relative, normalized ratio between phosphorylated protein and the protein itself was presented.</p

CX-4945 inhibits TGF-β1-induced Wnt signaling.

<p>The effect of CX-4945 on TGF-β1-induced Wnt activation was evaluated by measuring the transcriptional activity of β-catenin (A) and its expression in cytosolic and nuclear fractions (B). For Western blot analysis, A549 cells were treated with TGF-β1 (5 ng/ml) alone or in combination with CX-4945 in media containing 0.1% FBS for 48 h. Actin was used as a loading control. (C) Nuclear translocation of β-catenin and its inhibition by CX-4945 were confirmed by immunocytochemistry. Nuclei were counterstained with Hoechst 33342. All scale bars represent 20 µm.</p

Application of the Koutecký-Levich Method to the Analysis of Steady State Voltammograms with Ultramicroelectrodes

We demonstrate a new experimental approach to measure heterogeneous electron transfer rates. We adapted the classical Koutecký-Levich model for a rotating disk electrode (RDE) to a general heterogeneous electrochemical kinetic study with ultramicroelectrodes (UMEs) even for fast redox systems, where different sizes of UMEs are used to modulate the mass transfer rate (<i>m</i>). Subsequently, a linear plot of (1/current density) vs 1/<i>m</i> at different potentials can be created from the obtained steady state voltammograms, which is analogous to the traditional Koutecký-Levich plot. A simple numerical treatment with a slope and <i>y</i>-intercept from a linear plot allows for extracting kinetic parameters. A unifying treatment is presented for the steady state quasi-reversible, irreversible, and reversible voltammograms for a simple electron transfer reaction at UMEs. This new experimental approach with submicrometer to ∼micrometer sized UMEs exceeds the mass transfer rates achieved by conventional electrochemical methods using rotating electrodes or solely tens of micrometer sized electrodes, thus enables us to study much faster heterogeneous electron transfer kinetics with simple instrumentation. The method should be particularly useful in studying particle size and structure effects

Improved Electrochemical Microsensor for the Real-Time Simultaneous Analysis of Endogenous Nitric Oxide and Carbon Monoxide Generation

An amperometric dual NO/CO microsensor was developed on the basis of a working electrode incorporating dual Pt microdisks (each diameter, 76 μm) and a Ag/AgCl reference electrode covered with a gas permeable membrane. One of the Pt disks was sequentially electrodeposited with Pt and Sn; the other Pt disk was deposited with Pt–Fe­(III) oxide nanocomposites. The first showed activity for the oxidation of both NO and CO; the second showed activity only for NO oxidation. In the copresence of NO and CO, the currents measured at each electrode, respectively, represented the concentrations of CO and NO. The sensor showed high stability during the monitoring of organ tissue for at least 2.5 h and high selectivity to NO over CO at the Pt–Fe­(III) oxide working electrode. Real-time coupled dynamic changes of NO and CO generated by a living C57 mouse kidney were monitored simultaneously and quantitatively in response to a NO synthase inhibitor (N^G-nitro-l-arginine methyl ester), for the first time. CO was found to increase and NO decreased upon addition of the inhibitor, suggesting a possible reciprocal interaction between these endogenous gases

Glucose Sensor using Liquid-Crystal Droplets Made by Microfluidics

Micrometer-sized, 4-cyno-4-pentylbiphenyl (5CB) droplets were developed for glucose detection in an aqueous medium by coating with poly­(acrylicacid-<i>b</i>-4-cynobiphenyl-4-oxyundecylacrylate) (PAA-<i>b</i>-LCP) at the 5CB/water interface and covalently immobilizing glucose oxidase (GOx) to the PAA chains. This functionalized liquid-crystal (LC) droplet detected glucose from a radial to bipolar configurational change by polarized optical microscopy under crossed polarizers at concentrations as low as 0.03 mM and response times of ∼3 min and showed the selective detection of glucose against galactose. This new and sensitive LC-droplet-based glucose biosensor has the merits of low production cost and easy detection by the naked eye and might be useful for prescreening the glucose level in the human body

Detection of the Short-Lived Cation Radical Intermediate in the Electrochemical Oxidation of <i>N</i>,<i>N</i>‑Dimethylaniline by Scanning Electrochemical Microscopy

The short-lived intermediate <i>N</i>,<i>N</i>-dimethyl­aniline (DMA) cation radical, DMA^•+, was detected during the oxidation of DMA in MeCN with 0.1 M tetra-<i>n</i>-butyl­ammonium hexafluoro­phosphate. The detection was accomplished at steady state by scanning electrochemical microscopy (SECM) with ultramicroelectrodes using the tip generation/​substrate collection mode. Cyclic voltammetry (CV) with a 2 mm Pt electrode indicates that DMA oxidation in acetonitrile is followed by a dimerization and two electrochemical reactions, which is consistent with previous results. The DMA^•+ intermediate is detected by SECM, where the DMA^•+ generated at the ca. 500 nm radius Pt tip is collected on a 5 μm radius Pt substrate when the gap between the tip and the substrate is a few hundred nanometers. Almost all of the DMA^•+ is reduced at the substrate when the gap is 200 nm or less, yielding a dimerization rate constant of 2.5 × 10⁸ M^–1·s^–1 based on a simulation. This is roughly 3 orders of magnitude larger than the value estimated by fast-scan CV. We attribute this discrepancy to the effects of double-layer capacitance charging and adsorbed species in the high scan rate CV