• Biomolecules & Therapeutics
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• v.31 no.2
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• pp.200-209
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• 2023

Patients with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) amplification or sensitive mutations initially respond to the tyrosine kinase inhibitor gefitinib, however, the treatment becomes less effective over time by resistance mechanism including mesenchymal-epithelial transition (MET) overexpression. A therapeutic strategy targeting MET and EGFR may be a means to overcoming resistance to gefitinib. In the present study, we found that picropodophyllotoxin (PPT), derived from the roots of Podophyllum hexandrum, inhibited both EGFR and MET in NSCLC cells. The antitumor efficacy of PPT in gefitinib-resistant NSCLC cells (HCC827GR), was confirmed by suppression of cell proliferation and anchorage-independent colony growth. In the targeting of EGFR and MET, PPT bound with EGFR and MET, ex vivo, and blocked both kinases activity. The binding sites between PPT and EGFR or MET in the computational docking model were predicted at Gly772/Met769 and Arg1086/Tyr1230 of each ATP-binding pocket, respectively. PPT treatment of HCC827GR cells increased the number of annexin V-positive and subG1 cells. PPT also caused G2/M cell-cycle arrest together with related protein regulation. The inhibition of EGFR and MET by PPT treatment led to decreases in the phosphorylation of the downstream-proteins, AKT and ERK. In addition, PPT induced reactive oxygen species (ROS) production and GRP78, CHOP, DR5, and DR4 expression, mitochondrial dysfunction, and regulated involving signal-proteins. Taken together, PPT alleviated gefitinib-resistant NSCLC cell growth and induced apoptosis by reducing EGFR and MET activity. Therefore, our results suggest that PPT can be a promising therapeutic agent for gefitinib-resistant NSCLC.

• Journal of Veterinary Science
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• v.24 no.2
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• pp.26.1-26.11
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• 2023

Background: Angiotensin-converting enzyme inhibitor (ACEi) inhibits the catalysis of angiotensin I to angiotensin II and the degradation of substance P (SP) and bradykinin (BK). While the possible relationship between ACEi and SP in nociceptive mice was recently suggested, the effect of ACEi on signal transduction in astrocytes remains unclear. Objectives: This study examined whether ACE inhibition with captopril or enalapril modulates the levels of SP and BK in primary cultured astrocytes and whether this change modulates PKC isoforms (PKCα, PKCβI, and PKCε) expression in cultured astrocytes. Methods: Immunocytochemistry and Western blot analysis were performed to examine the changes in the levels of SP and BK and the expression of the PKC isoforms in primary cultured astrocytes, respectively. Results: The treatment of captopril or enalapril increased the immunoreactivity of SP and BK significantly in glial fibrillary acidic protein-positive cultured astrocytes. These increases were suppressed by a pretreatment with an angiotensin-converting enzyme. In addition, treatment with captopril increased the expression of the PKCβI isoform in cultured astrocytes, while there were no changes in the expression of the PKCα and PKCε isoforms after the captopril treatment. The captopril-induced increased expression of the PKCβI isoform was inhibited by a pretreatment with the neurokinin-1 receptor antagonist, L-733,060, the BK B1 receptor antagonist, R 715, or the BK B₂ receptor antagonist, HOE 140. Conclusions: These results suggest that ACE inhibition with captopril or enalapril increases the levels of SP and BK in cultured astrocytes and that the activation of SP and BK receptors mediates the captopril-induced increase in the expression of the PKCβI isoform.

• Korean Journal of Plant Resources
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• v.36 no.3
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• pp.207-218
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• 2023

Hepatocellular carcinoma (HCC) has a poor prognosis and high metastasis and recurrence rates. Although extracts of Alnus hirsuta (Turcz. ex Spach) Rupr. (AH) have been demonstrated to possess potential anti-inflammatory and anti-cancer activities, the underlying mechanism of AH in HCC treatment remains to be elucidated. We investigated the effects and potential mechanisms of AH on migration and invasion of Hep3B cells. Within the non-cytotoxic concentration range, AH significantly inhibited motility and invasiveness of Hep3B cells in a concentration-dependent manner. Inhibitory effects of AH on cell invasiveness are associated with tightening of tight junctions (TJs), as demonstrated by an increase in transepithelial electrical resistance. Immunoblotting indicated that AH decreased levels of claudins, which form major components of TJs and play key roles in the control and selectivity of paracellular transport. Furthermore, AH inhibited the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9 and simultaneously increased the levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/AKT pathway in Hep3B cells. Therefore, AH inhibits migration and invasion of Hep3B cells by inhibiting the activity of MMPs and tightening TJs through suppression of claudin expression, possibly by suppressing the PI3K/AKT signaling pathway.

• Molecules and Cells
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• v.46 no.6
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• pp.387-398
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• 2023

Microtubule acetylation has been proposed as a marker of highly heterogeneous and aggressive triple-negative breast cancer (TNBC). The novel microtubule acetylation inhibitors GM-90257 and GM-90631 (GM compounds) cause TNBC cancer cell death but the underlying mechanisms are currently unknown. In this study, we demonstrated that GM compounds function as anti-TNBC agents through activation of the JNK/AP-1 pathway. RNA-seq and biochemical analyses of GM compound-treated cells revealed that c-Jun N-terminal kinase (JNK) and members of its downstream signaling pathway are potential targets for GM compounds. Mechanistically, JNK activation by GM compounds induced an increase in c-Jun phosphorylation and c-Fos protein levels, thereby activating the activator protein-1 (AP-1) transcription factor. Notably, direct suppression of JNK with a pharmacological inhibitor alleviated Bcl2 reduction and cell death caused by GM compounds. TNBC cell death and mitotic arrest were induced by GM compounds through AP-1 activation in vitro. These results were reproduced in vivo, validating the significance of microtubule acetylation/JNK/AP-1 axis activation in the anti-cancer activity of GM compounds. Moreover, GM compounds significantly attenuated tumor growth, metastasis, and cancer-related death in mice, demonstrating strong potential as therapeutic agents for TNBC.

• Korean Journal of Exercise Nutrition
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• v.23 no.3
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• pp.13-21
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• 2019

[Purpose] Chronic stress is a precipitating factor for depression, whereas exercise is beneficial for both the mood and cognitive process. The current study demonstrates the anti-depressive effects of regular exercise and the mechanisms linked to hippocampal neurogenesis. [Methods] Mice were subjected to 14 consecutive days of restraint, followed by 3 weeks of treadmill running, and were then subjected to behavioral tests that included the forced swimming and Y-maze tests. Protein levels were assessed using western blot analysis and newborn cells were detected using 5-bromo-2'-deoxyuridine (BrdU). [Results] Three weeks of treadmill running ameliorated the behavioral depression caused by 14 days of continuous restraint stress. The exercise regimen enhanced BrdU-labeled cells and class III β-tubulin levels in the hippocampal dentate gyrus, as well as those of thioredoxin-1 (TRX-1) and synaptosomal β2-adrenergic receptors (β2-AR) under stress. In vitro experiments involving treatment with recombinant human TRX-1 (rhTRX-1) augmented the levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2), nuclear β-catenin, and proliferating cell nuclear antigens, which were previously inhibited by U0216 and FH535 (inhibitors of ERK1/2 and β-catenin/T cell factor-mediated transcription, respectively). The hippocampal neurogenesis elicited by a 7-day exercise regimen was abolished by a selective inhibitor of β2-AR, butoxamine. [Conclusion] These results suggest that TRX-1-mediated hippocampal neurogenesis by β2-AR function is a potential mechanism underlying the psychotropic effect of exercise.

• BMB Reports
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• v.57 no.2
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• pp.92-97
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• 2024

Elevated blood glucose is associated with an increased risk of atherosclerosis. Data from the current study showed that glucosamine (GlcN), a normal glucose metabolite of the hexosamine biosynthetic pathway (HBP), promoted lipid accumulation in RAW264.7 macrophage cells. Oleic acid- and lipopolysaccharide (LPS)-induced lipid accumulation was further enhanced by GlcN in RAW264.7 cells, although there was no a significant change in the rate of fatty acid uptake. GlcN increased acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), scavenger receptor class A, liver X receptor, and sterol regulatory element-binding protein-1c (SREBP-1c) mRNA expression, and; conversely, suppressed ATP-binding cassette transporter A1 (ABCA-1) and ABCG-1 expression. Additionally, GlcN promoted O-GlcNAcylation of nuclear SREBP-1 but did not affect its DNA binding activity. GlcN stimulated phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Rapamycin, a mTOR-specific inhibitor, suppressed GlcN-induced lipid accumulation in RAW264.7 cells. The GlcN-mediated increase in ACC and FAS mRNA was suppressed, while the decrease in ABCA-1 and ABCG-1 by GlcN was not significantly altered by rapamycin. Together, our results highlight the importance of the mTOR signaling pathway in GlcN-induced macrophage lipid accumulation and further support a potential link between mTOR and HBP signaling in lipogenesis.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.1
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• pp.49-57
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• 2024

While arterial tone is generally determined by the phosphorylation of Ser¹⁹ in myosin light chain (p-MLC2), Thr¹⁸/Ser¹⁹ diphosphorylation of MLC2 (pp-MLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-β and MYPT1 levels were higher in MA than in PA. Interestingly, the level of pp-MLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 µM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 µM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1073-1081
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• 2024

Obesity is spawned by an inequality between the portion of energy consumed and the quantity of energy expended. Disease entities such as cardiovascular disease, arteriosclerosis, hypertension, and cancer, which are correlated with obesity, influence society and the economy. Suppression of adipogenesis, the process of white adipocyte generation, remains a promising approach for treating obesity. Oil Red O staining was used to differentiate 3T3-L1 cells for screening 20 distinct Lactobacillus species. Among these, Lactobacillus acidophilus DS0079, referred to as YBS1, was selected for further study. YBS1 therapy decreased 3T3-L1 cell development. Triglyceride accumulation and mRNA expression of the primary adipogenic marker, peroxisome proliferator-activated receptor gamma (PPARγ), including its downstream target genes, adipocyte fatty acid binding protein 4 and adiponectin, were almost eliminated. YBS1 inhibited adipocyte differentiation at the early stage (days 0-2), but no significant difference was noted between the mid-stage (days 2-4) and late-stage (days 4-6) development. YBS1 stimulated the activation of p38 mitogen-activated protein kinase (p38 MAPK) during the early stages of adipogenesis; however, this effect was eliminated by the SB203580 inhibitor. The data showed that YBS1 administration inhibited the initial development of adipocytes via stimulation of the p38 MAPK signaling pathway, which in turn controlled PPARγ expression. In summary, YBS1 has potential efficacy as an anti-obesity supplement and requires further exploration.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.2
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• pp.125-132
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• 2006

Objective: In the previous study, we complied the differentially expressed genes during early folliculogenesis. Objective of the present study was to identify downstream target genes of transcription factors (TFs) using bioinformatics for selecting the target TFs among the gene lists for further functional analysis. Materials & Methods: By using bioinformatics tools, constituent domains were identified from database searches using Gene Ontology, MGI, and Entrez Gene. Downstream target proteins/genes of each TF were identified from database searches using TF database ($TRANSFAC^{(R)}$ 6.0) and eukaryotic promoter database (EPD). Results: DNA binding and trans-activation domains of all TFs listed previously were identified, and the list of downstream target proteins/genes was obtained from searches of TF database and promoter database. Based on the known function of identified downstream genes and the domains, 3 (HNF4, PPARg, and TBX2) out of 26 TFs were selected for further functional analysis. The genes of wee1-like protein kinase and p21WAF1 (cdk inhibitor) were identified as potential downstream target genes of HNF4 and TBX2, respectively. PPARg, through protein-protein interaction with other protein partners, acts as a transcription regulator of genes of EGFR, p21WAF1, cycD1, p53, and VEGF. Among the selected 3 TFs, further study is in progress for HNF4 and TBX2, since wee1-like protein kinase and cdk inhibitor may involved in regulating maturation promoting factor (MPF) activity during early folliculogenesis. Conclusions: Approach used in the present study, in silico analysis of downstream target genes, was useful for analyzing list of TFs obtained from high-throughput cDNA microarray study. To verify its binding and functions of the selected TFs in early folliculogenesis, EMSA and further relevant characterizations are under investigation.

• Journal of Life Science
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• v.29 no.4
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• pp.410-420
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• 2019

Citrus unshiu peel extracts possess a variety of beneficial effects, and studies on their anticancer activity have been reported. However, the exact mechanisms underlying this activity remain unclear. In the current study, the apoptotic effect of ethanol extract of C. unshiu peel (EECU) on human breast adenocarcinoma MDA-MB-231 cells and related mechanisms were investigated. The results showed that the survival rate of MDA-MB-231 cells treated with EECU was significantly inhibited in a concentration-dependent manner, which was associated with the induction of apoptosis. EECU-induced apoptosis was associated with the activation of caspase-8 and caspase-9, which initiate extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3, a representative effect caspase. EECU suppressed the expression of the inhibitor of apoptosis family of proteins, leading to an increased Bax/Bcl-2 ratio and proteolytic degradation of poly (ADP-ribose) polymerase. EECU also enhanced the loss of the mitochondrial membrane potential and cytochrome c release from the mitochondria to the cytosol, along with truncation of Bid. In addition, EECU activated AMP-activated protein kinase (AMPK), and compound C, an AMPK inhibitor, significantly weakened EECU-induced apoptosis and cell viability reduction. Furthermore, EECU promoted the generation of reactive oxygen species (ROS), which acted as upstream signals for AMPK activation as pretreatment of cells, with the antioxidant N-acetyl cysteine reversing both EECU-induced AMPK activation and apoptosis. Collectively, these findings suggest that EECU inhibits MDA-MB-231 adenocarcinoma cell proliferation by activating intrinsic and extrinsic apoptotic pathways, which was mediated through ROS/AMPK-dependent pathways.