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 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

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 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

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

Safety analysis of elderberry extract powder.

With aging, men develop testosterone-deficiency syndrome (TDS). The development is closely associated with age-related mitochondrial dysfunction of Leydig cell and oxidative stress-induced reactive oxygen species (ROS). Testosterone-replacement therapy (TRT) is used to improve the symptoms of TDS. However, due to its various side effects, research on functional ingredients derived from natural products that do not have side effects is urgently needed. In this study, using the mitochondrial dysfunction TM3 (mouse Leydig) cells, in which testosterone biosynthesis is reduced by H2O2, we evaluated the effects of elderberry extract and monosaccharide-amino acid (fructose–leucine; FL) on mRNA and protein levels related to steroidogenesis-related enzymes steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1(CYP11A1, cytochrome P450 17A1(CYP17A1), cytochrome P450 19A1(CYP19A1, aromatase), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase(17β-HSD). We analyzed elderberry extract and extract-derived FL for changes in ROS scavenging activity and testosterone secretion. Elderberry extract and FL significantly reduced H2O2-induced intracellular ROS levels, improved testosterone secretion, and increased the mRNA and protein expression levels of steroidogenesis-related enzymes (StAR, 3b-HSD, 17b-HSD, CYP11A1, CYp17A1). However, the conversion of testosterone to estradiol was inhibited by elderberry extract and extract-derived FL, which reduced the mRNA and protein expression of CYP19A1. In conclusion, elderberry extract and FL are predicted to have value as novel functional ingredients that may contribute to the prevention of TDS by ameliorating reduced steroidogenesis.</div

Schematic representation of the effects of elderberry extract and the monosaccharide-amino acids derived from elderberry extract in the improvement of testosterone-deficiency syndrome.

EB: elderberry extract, FL: Fructose–leucine, E∙R: endoplasmic reticulum, ROS: reactive oxygen species.</p

Effects of elderberry extract and elderberry extract-derived FL on inhibition of H₂O₂ treatment-induced reactive oxygen species (ROS) production and improvement of testosterone secretion in TM3 cells.

TM3 cells were treated with EB and FL combined with H2O2 (600 μM) for 24h and analyzed for intracellular ROS (A, B) and testosterone secretion (C, D). Data are expressed as mean ± SD (n = 3). #p ###p p p p 2O2 group, analyzed via one-way ANOVA. CTL: control, EB: elderberry extract, FL: fructose–leucine.</p