Pengaruh Ekstrak Jahe Merah (Zingiber Officinale Roscoe Var. Rubrum) Terhadap Kadar Mda Serum Tikus Setelah Terpapar Asap Rokok

Latar Belakang: Asap rokok merupakan salah satu sumber radikal bebas. Kadar radikal bebas yang tinggi dapat menyebabkan terjadinya kondisi stres oksidatif dan memicu terjadinya peroksidasi lipid pada membran sel yang akan menghasilkan Malondialdehyde (MDA). Sebenarnya, tubuh mempunyai sejumlah enzim dan zat yang dapat menetralkan radikal bebas yang disebut antioksidan. Kadar radikal bebas yang tinggi dapat menyebabkan antioksidan endogen tidak mampu untuk menetralisir. Kemampuan jahe sebagai antioksidan alami juga tidak terlepas dari kadar komponen fenolik total yang terkandung di dalamnya sehingga memiliki efek protektif yang tinggi dalam menangkal stres oksidatif. Tujuan: Penelitian ini bertujuan untuk mengetahui pengaruh pemberian ekstrak jahe merah terhadap kadar MDA serum tikus setelah terpapar asap rokok. Metode: Penelitian ini merupakan penelitian true experimental dengan desain pre-post test only control group design pada tikus. Perlakuan yang diberikan yaitu dengan pemberian ekstrak jahe merah (Zingiber officinale var. Rubrum) dan pemberian paparan asap rokok pada tikus, sedangkan keluarannya (outcome) adalah kadar MDA serum tikus. Hasil: Pemberian paparan asap rokok pada kelompok 2 meningkatkan kadar MDA serum dibandingkan dengan kelompok 1. Kelompok 3 yang diberikan ekstrak jahe merah 200 mg/kgBB/hari tidak efektif menurunkan kadar MDA serum. Pemberian ekstrak jahe merah menurunkan kadar MDA serum setelah diberikan paparan asap rokok (K4) jika dibandingkan dengan kelompok yang diberikan jahe merah tanpa diberikan paparan asap rokok (K3), penurunan tersebut tidak bermakna secara statistik. Kelompok 4 meningkatkan kadar MDA serum tikus dibandingkan dengan kelompok 2. Kesimpulan: Tidak terdapat pengaruh yang bermakna dari pemberian jahe merah (Zingiber officinale var. Rubrum) terhadap kadar MDA darah tikus setelah terpapar asap rokok

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 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

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

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 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

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-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

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