• Biomolecules & Therapeutics
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• 제32권1호
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• pp.94-103
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• 2024

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in the liver, and there is a global increase in its incidence owing to changes in lifestyle and diet. Recent findings suggest that p53 is involved in the development of non-alcoholic fatty liver disease; however, the association between p53 expression and the disease remains unclear. Doxorubicin, an anticancer agent, increases the expression of p53. Therefore, this study aimed to investigate the role of doxorubicin-induced p53 upregulation in free fatty acid (FFA)-induced intracellular lipid accumulation. HepG2 cells were pretreated with 0.5 ㎍/mL of doxorubicin for 12 h, followed by treatment with FFA (0.5 mM) for 24 h to induce steatosis. Doxorubicin pretreatment upregulated p53 expression and downregulated the expression of endoplasmic reticulum stress- and lipid synthesis-associated genes in the FFA -treated HepG2 cells. Additionally, doxorubicin treatment upregulated the expression of AMP-activated protein kinase, a key modulator of lipid metabolism. Notably, siRNA-targeted p53 knockdown reversed the effects of doxorubicin in HepG2 cells. Moreover, doxorubicin treatment suppressed FFA -induced lipid accumulation in HepG2 spheroids. Conclusively, these results suggest that doxorubicin possesses potential application for the regulation of lipid metabolism by enhance the expression of p53 an in vitro NAFLD model.

• Biomolecules & Therapeutics
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• 제21권2호
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• pp.107-113
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• 2013

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.

• Biomolecules & Therapeutics
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• 제23권1호
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• pp.45-52
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• 2015

To explore the anti-allergic and anti-inflammatory effects of extracts of Petasites genus, we studied the effects of s-petasin, a major sesquiterpene from Petasites formosanus (a butterbur species) on asthma and peritonitis models. In an ovalbumin-induced mouse asthma model, s-petasin significantly inhibited the accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar fluids. S-petasin inhibited the antigen-induced degranulation of ${\beta}$-hexosamidase but did not inhibit intracellular $Ca^{2+}$ increase in RBL-2H3 mast cells. S-petasin inhibited the LPS induction of iNOS at the RNA and protein levels in mouse peritoneal macrophages. Furthermore, s-petasin inhibited the production of NO (the product of iNOS) in a concentration-dependent manner in the macrophages. Furthermore, in an LPS-induced mouse model of peritonitis, s-petasin significantly inhibited the accumulation of polymorpho nuclear and mononuclear leukocytes in peritoneal cavity. This study shows that s-petasin in Petasites genus has therapeutic effects on allergic and inflammatory diseases, such as, asthma and peritonitis through degranulation inhibition in mast cells, suppression of iNOS induction and production of NO in macrophages, and suppression of inflammatory cell accumulation.

• Molecules and Cells
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• 제38권6호
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• pp.528-534
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• 2015

Hypoxia-inducible factor (HIF) is a key regulator of tumor growth and angiogenesis. Recent studies have shown that, BIX01294, a G9a histone methyltransferase (HMT)-specific inhibitor, induces apoptosis and inhibits the proliferation, migration, and invasion of cancer cells. However, not many studies have investigated whether inhibition of G9a HMT can modulate HIF-$1{\alpha}$ stability and angiogenesis. Here, we show that BIX01294 dose-dependently decreases levels of HIF-$1{\alpha}$ in HepG2 human hepatocellular carcinoma cells. The half-life of HIF-$1{\alpha}$, expression of proline hydroxylase 2 (PHD2), hydroxylated HIF-$1{\alpha}$ and von Hippel-Lindau protein (pVHL) under hypoxic conditions were decreased by BIX01294. The mRNA expression and secretion of vascular endothelial growth factor (VEGF) were also significantly reduced by BIX01294 under hypoxic conditions in HepG2 cells. BIX01294 remarkably decreased angiogenic activity induced by VEGF in vitro, ex vivo, and in vivo, as demonstrated by assays using human umbilical vein endothelial cells (HUVECs), mouse aortic rings, and chick chorioallantoic membranes (CAMs), respectively. Furthermore, BIX01294 suppressed VEGF-induced matrix metalloproteinase 2 (MMP2) activity and inhibited VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), focal adhesion kinase (FAK), and paxillin in HUVECs. In addition, BIX01294 inhibited VEGF-induced formation of actin cytoskeletal stress fibers. In conclusion, we demonstrated that BIX01294 inhibits HIF-$1{\alpha}$ stability and VEGF-induced angiogenesis through the VEGFR-2 signaling pathway and actin cytoskeletal remodeling, indicating a promising approach for developing novel therapeutics to stop tumor progression.

• Biomolecules & Therapeutics
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• 제24권4호
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• pp.410-417
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• 2016

Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance ${\alpha}7$ nicotinic acetylcholine receptor (${\alpha}7$ nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of ${\alpha}7$ nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current ($I_{ACh}$) in Xenopus oocytes expressing the ${\alpha}7$ nAChR. $I_{ACh}$ was measured with a two-electrode voltage clamp technique. In oocytes injected with ${\alpha}7$ nAChR copy RNA, quercetin enhanced $I_{ACh}$, whereas quercetin glycosides inhibited $I_{ACh}$. Quercetin glycosides mediated an inhibition of $I_{ACh}$, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of $I_{ACh}$ inhibition by quercetin glycosides was Rutin${\geq}$Rham1>Rham2. Quercetin glycosides-mediated $I_{ACh}$ enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated $I_{ACh}$ inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated ${\alpha}7$ nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the ${\alpha}7$ nAChR in a differential manner.

• Biomolecules & Therapeutics
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• 제24권3호
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• pp.252-259
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• 2016

Neuropathic pain is a complex state showing increased pain response with dysfunctional inhibitory neurotransmission. The TREK family, one of the two pore domain $K^+$ (K2P) channel subgroups were focused among various mechanisms of neuropathic pain. These channels influence neuronal excitability and are thought to be related in mechano/thermosensation. However, only a little is known about the expression and role of TREK-1 and TREK-2, in neuropathic pain. It is performed to know whether TREK-1 and/or 2 are positively related in dorsal root ganglion (DRG) of a mouse neuropathic pain model, the chronic constriction injury (CCI) model. Following this purpose, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot analyses were performed using mouse DRG of CCI model and compared to the sham surgery group. Immunofluorescence staining of isolectin-B4 (IB4) and TREK were performed. Electrophysiological recordings of single channel currents were analyzed to obtain the information about the channel. Interactions with known TREK activators were tested to confirm the expression. While both TREK-1 and TREK-2 mRNA were significantly overexpressed in DRG of CCI mice, only TREK-1 showed significant increase (~9 fold) in western blot analysis. The TREK-1-like channel recorded in DRG neurons of the CCI mouse showed similar current-voltage relationship and conductance to TREK-1. It was easily activated by low pH solution (pH 6.3), negative pressure, and riluzole. Immunofluorescence images showed the expression of TREK-1 was stronger compared to TREK-2 on IB4 positive neurons. These results suggest that modulation of the TREK-1 channel may have beneficial analgesic effects in neuropathic pain patients.

• Biomolecules & Therapeutics
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• 제10권2호
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• pp.71-77
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• 2002

Heme oxygenase-1 (HO-1) is the inducible from of the rate-limiting enzyme of heme degradation; it regulates the cellular contents of heme. HO-1 is up-regulated by various stimuli including oxidative stress so that it is thought to participate in general cellular defense mechanisms against oxidative stress in mammalian cells. To investigate the role of the cAMP-dependent protein kinase A (PKA) signaling pathway on nitrogen oxidative stress-induced HO-1 gene expression, RAW 264.7 cell cultures were treated with sodium nitroprusside (SNP). SNP increased the expression of HO-1 mRNA and protein, time- and concentration-dependently. Treatment with H89, PKA inhibitor, but not LY83583, guanylate cyclase inhibitor, significantly diminished the HO-1 expression by SNP, indicating that cAMP plays a crucial role in the induction of HO-1. Incubation with cAMP-elevating agents, such as forskolin or isoproterenol resulted in up-regulation of the expression of HO-1. Forskolin-induced expression of HO-1 was inhibited by H89. Furthermore, propranolol, $\beta$-adrenoceptor blocker, inhibited the isoproterenol-induced HO-1 expression, supporting the importance of cAMP in the induction of HO-1 expression. Higenamine-S, but not higenamineR, enhanced the HO-1 expression induced by SNP. Furthermore, cellular toxicity induced by hydrogen peroxide was attenuated by the presence of SNP, which was further increased by the presence of ZnPPIX, HO-1 inhibitor. Collectively, these results strongly suggest that up-regulation of HO-1 expression in RAW 264.7 cells involves PKA signal pathway.

• The Korean Journal of Physiology and Pharmacology
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• 제15권4호
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• pp.195-201
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• 2011

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as ${\alpha}3{\beta}4$, ${\alpha}7$ and ${\alpha}9{\alpha}10$. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current ($I_{ACh}$) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited $I_{ACh}$. Pre-treatment of quercetin further inhibited $I_{ACh}$ in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of $I_{ACh}$ by quercetin was reversible and concentration-dependent. The $IC_{50}$ of quercetin was $18.9{\pm}1.2{\mu}M$ in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of $I_{ACh}$ by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.

• Biomolecules & Therapeutics
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• 제24권6호
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• pp.595-603
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• 2016

(E)-3-Phenyl-1-(2-pyrrolyl)-2-propenone (PPP) is a pyrrole derivative of chalcone, in which the B-ring of chalcone linked to ${\beta}$-carbon is replaced by pyrrole group. While pyrrole has been studied for possible Src inhibition activity, chalcone, especially the substituents on the B-ring, has shown pharmaceutical, anti-inflammatory, and anti-oxidant properties via inhibition of NF-${\kappa}B$ activity. Our study is aimed to investigate whether this novel synthetic compound retains or enhances the pharmaceutically beneficial activities from the both structures. For this purpose, inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 cells were analyzed. Nitric oxide (NO) production, inducible NO synthase (iNOS) and tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) mRNA expression, and the intracellular inflammatory signaling cascade were measured. Interestingly, PPP strongly inhibited NO release in a dose-dependent manner. To further investigate this anti-inflammatory activity, we identified molecular pathways by immunoblot analyses of nuclear fractions and whole cell lysates prepared from LPS-stimulated RAW264.7 cells with or without PPP pretreatment. The nuclear levels of p50, c-Jun, and c-Fos were significantly inhibited when cells were exposed to PPP. Moreover, according to the luciferase reporter gene assay after cotransfection with either TRIF or MyD88 in HEK293 cells, NF-${\kappa}B$-mediated luciferase activity dose-dependently diminished. Additionally, it was confirmed that PPP dampens the upstream signaling cascade of NF-${\kappa}B$ and AP-1 activation. Thus, PPP inhibited Syk, Src, and TAK1 activities induced by LPS or induced by overexpression of these genes. Therefore, our results suggest that PPP displays anti-inflammatory activity via inhibition of Syk, Src, and TAK1 activity, which may be developed as a novel anti-inflammatory drug.

• Biomolecules & Therapeutics
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• 제25권4호
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• pp.417-426
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• 2017

4-O-methylhonokiol, a neolignan compound from Magnolia Officinalis, has been reported to have various biological activities including hair growth promoting effect. However, although transforming growth factor-${\beta}$ (TGF-${\beta}$) signal pathway has an essential role in the regression induction of hair growth, the effect of 4-O-methylhonokiol on the TGF-${\beta}$ signal pathway has not yet been elucidated. We thus examined the effect of 4-O-methylhonokiol on TGF-${\beta}$-induced canonical and noncanonical pathways in HaCaT human keratinocytes. When HaCaT cells were pretreated with 4-O-methylhonokiol, TGF-${\beta}1$-induced G1/G0 phase arrest and TGF-${\beta}1$-induced p21 expression were decreased. Moreover, 4-O-methylhonokiol inhibited nuclear translocation of Smad2/3, Smad4 and Sp1 in TGF-${\beta}1$-induced canonical pathway. We observed that ERK phosphorylation by TGF-${\beta}1$ was significantly attenuated by treatment with 4-O-methylhonokiol. 4-O-methylhonokiol inhibited TGF-${\beta}1$-induced reactive oxygen species (ROS) production and reduced the increase of NADPH oxidase 4 (NOX4) mRNA level in TGF-${\beta}1$-induced noncanonical pathway. These results indicate that 4-O-methylhonokiol could inhibit TGF-${\beta}1$-induced cell cycle arrest through inhibition of canonical and noncanonical pathways in human keratinocyte HaCaT cell and that 4-O-methylhonokiol might have protective action on TGF-${\beta}1$-induced cell cycle arrest.