KY-05009 inhibits TGF-β1-induced activation of focal adhesion and non-Smad signaling pathways.

<p>Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h. Effects of KY-05009 on TGF-β1-induced activation of focal adhesion-related and ERK and JNK MAP kinase signaling molecules was evaluated by Western blot analysis. The ERK1 and 2 (p44 and p42, respectively) or JNK1 and 2 (p46 and p54, respectively) isoforms could be separated and differentially evaluated by gel electrophoresis, according to their molecular weight differences. The expression of p-FAK, p-Src, p-Paxillin, p-ERK1/2, and p-JNK2/1 was normalized by endogenous FAK, Src, Paxillin, ERK1/2, and JNK2/1, respectively. Actin was used as a loading control. Reported results are representatives of triplicate experiments.</p

KY-05009 inhibits TGF-β1-mediated modulation of EMT markers.

<p>The effect of KY-05009 on TGF-β1-mediated modulation of EMT markers was evaluated by (A) Western blot analysis and (B) immunofluorescence microscopy. Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h. Actin was used as a loading control. Nuclei were counterstained with Hoechst 33342, and all scale bars represent 20 µm. The expressions of E-cadherin and vimentin were represented by the relative intensity of green fluorescence (n = 30), and reported images are representatives of triplicate experiments. ^##<i>p</i><0.01,* <i>p</i><0.05 (versus ‘the group treated with TGF-β1 only’).</p

A Novel Aminothiazole KY-05009 with Potential to Inhibit Traf2- and Nck-Interacting Kinase (TNIK) Attenuates TGF-β1-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Adenocarcinoma A549 Cells

<div><p>Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (<i>K</i>_i, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.</p></div

KY-05009 inhibits TGF-β1-induced migration and invasion.

<p>The effect of KY-05009 on TGF-β1-induced migration and invasion of A549 cells was evaluated using (A) IncuCyte software and (B) Boyden chambers, respectively. The red and white dashed lines 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. Numbers of invaded cells were represented by an average number of cells per randomly selected three high-power field (HPF). Effects of KY-05009 on TGF-β1-induced expression and activation of MMP-2 and MMP-9 were measured by (C) quantitative RT-PCR and (D) gelatin zymography, respectively. (E) The effect of KY-05009 on NF-κB transcriptional activity was determined by reporter assay. ^##<i>p</i><0.01, ^###<i>p</i><0.001 (versus ‘the control’); * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 (versus ‘the group treated with TGF-β1 only’).</p

KY-05009 inhibits TGF-β1-induced Smad signaling.

<p>(A) Effects of KY-05009 on TGF-β1-activated Smad signaling. Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h, and then the levels of p-Smad2 and endogenous Smad2/3 in cytosolic and nuclear fractions were evaluated by Western blot analysis. The nuclear expression levels of Snail and Twist were also evaluated. The expression of cytosol p-Smad2 was normalized by endogenous Smad2/3 and all nucleus proteins were normalized by histone H3. (B) Immunocytochemical confirmation of KY-05009 inhibition of TGF-β1-activated p-Smad2. Nuclei were counterstained with Hoechst 33342. All scale bars represent 50 µm. The expression of p-Smad2 was represented by the relative intensity of green fluorescence (n = 30), and reported images are representatives of triplicate experiments. ^##<i>p</i><0.01 (versus ‘the control’), ** <i>p</i><0.01 (versus ‘the group treated with TGF-β1 only’).</p

Binding mode and <i>K</i>_i of KY-05009 for TNIK.

<p>(A) Chemical structure of KY-05009. (B) Binding mode of KY-05009 for TNIK. KY-05009 has two H-bond interactions with Cys108 (red dotted lines) in the hinge region, and CH/π interactions with Val31, Gly111, and Leu160. The yellow ball represents the CH/π interactions among Val31, ligand, and Gly111. (C) The binding constant, <i>K</i>_i, of KY-05009 for TNIK was determined using an ATP competition assay.</p

KY-05009 inhibits TGF-β1-induced Wnt signaling.

<p>(A) Effects of KY-05009 on cell viability and TCF4 transcriptional activity. Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h, and then cell viability and TOPflash luciferase activity were measured. FOPflash-normalized TOPflash luciferase activity was represented to the relative TCF/LEF luciferase activity. The expression of TNIK and β-catenin in cytosolic and nuclear fractions (B), and the protein levels of TCF4-interacting proteins c were measured by Western blot analysis and immunoprecipitation assay, respectively. Actin, histone H3, and IgG were used as loading controls. The expression of cytosol and nucleus proteins was normalized by actin and histone H3, respectively. Reported results are representatives of triplicate experiments. ^###<i>p</i><0.001 (versus ‘the control’); ** <i>p</i><0.01, *** <i>p</i><0.001 (versus ‘the group treated with TGF-β1 only’).</p

Inflammatory Bone Resorption and Antiosteosarcoma Potentials of Zinc Ion Sustained Release ZnO Chips: Friend or Foe?

Multifunctional zinc oxide (ZnO) has been generated as nanoparticles or nanorods and applied to various medical purposes since it exhibits several biological actions including anticancer activity. Especially, due to antibacterial activity and effects on bone regeneration, ZnO is widely used in implants and scaffolds in the orthopedic and dental fields. However, concerns over side effects have been raised recently in the clinical use of ZnO, and it is necessary to assess the safety of ZnO regarding its inflammatory potential in the bone environment. This made us hypothesize that the inflammatory activity of zinc ions released from ZnO NPs could be harmful to induce bone resorption but that their cytotoxicity would be beneficial to kill osteosarcoma. To clarify this hypothesis, in the present work, the effects of ZnO on bone matrix and abnormal bone environments were investigated quantitatively using ZnO chips, filter paper, or glass slides coated with thin films of ZnO grown via atomic layer deposition (ALD). ALD-grown ZnO thin films exhibit thickness with atomic precision, which enables the quantitative analysis of the effects of ZnO. <i>In vivo</i> application of ZnO chips to mouse calvarial bone induced bone resorption, presumably due to the activation of osteoclasts by zinc ion-induced TNF-α release. However, <i>in vitro</i> application of ZnO chips to osteosarcoma cells induced caspase-dependent apoptosis and oxidative stress. Taken together, the results showed two sides of ZnO as our hypothesis. Therefore, careful design and multiple evaluations for the safety and efficacy of ZnO materials are necessary for its successful clinical application

Effect of praeruptorin A on RANKL-induced osteoclast differentiation.

<p>(A) Chemical structure of praeruptorin A. (B) BMMs were pretreated with vehicle (0.1% DMSO) or praeruptorin A for 2 h and then incubated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) for 4 days. Multinucleated cells were fixed, permeabilized, and stained with TRAP solution. Mature TRAP-positive multinucleated osteoclasts (MNCs) were photographed under a light microscope. TRAP-positive MNCs (nuclear number >3) were counted (C), and TRAP activity of osteoclasts was measured (D). (E) The effect of praeruptorin A on the viability of BMMs was evaluated by CCK-8 assay. *, <i>P</i><0.05; **, <i>P</i><0.01; ***<i>P</i><0.001.</p

Anti-Osteoclastogenic Activity of Praeruptorin A via Inhibition of p38/Akt-c-Fos-NFATc1 Signaling and PLCγ-Independent Ca²⁺ Oscillation

<div><p>Background</p><p>A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of <i>Peucedanum praeruptorum</i> Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet.</p><p>Materials and Methods</p><p>The effect of praeruptorin A on the differentiation of bone marrow–derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca²⁺ levels were also measured.</p><p>Results</p><p>Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca²⁺ oscillation without any changes in the phosphorylation of PLCγ.</p><p>Conclusion</p><p>Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca²⁺ oscillation.</p></div