• Biomolecules & Therapeutics
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• v.15 no.3
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• pp.150-155
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• 2007

In the present study, we have tested the effect of dioleoyl phosphatidic acid (PA) on intracellular $Ca_{2+}$ concentration ($[Ca^{2+}]_{i}$) in two human colon cancer cell lines (HCT116 and HT29). PA and lysophosphatidic acid (LPA), a bioactive lysolipid, increased $[Ca^{2+}]_{i}$ in both HCT116 and HT29 cell lines. Increases of $[Ca^{2+}]_{i}$ by PA and LPA were more robust in HCT116 cells than in HT29 cells. A specific inhibitor of phospholipase C (U73122), however, was not inhibitory to the cell responses. Pertussis toxin, a specific inhibitor of $G_{i/o}$ type G proteins, however, had an inhibitory effect on the responses except for an LPA-induced one in HT29 cells. Ruthenium red, an inhibitor of the ryanodine receptor, was not inhibitory on the responses, however, 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor, completely inhibited both lipid-induced $Ca^{2+}$ increases in both cell types. Furthermore, by using Ki16425 and VPC32183, two structurally dissimilar specific antagonists for $LPA_{1}/LPA_{3}$ receptors, an involvement of endogenous LPA receptors in the $Ca^{2+}$ responses was observed. Ki16425 completely inhibited the responses but the susceptibility to VPC32183 was different to PA and LPA in the two cell types. Expression levels of five LPA receptors in the HCT116 and HT29 cells were also assessed. Our data support the notion that PA could increase $[Ca^{2+}]_{i}$ in human colon cancer cells, probably via endogenous LPA receptors, G proteins and $IP_{3}$ receptors, thereby suggesting a role of PA as an intercellular lipid mediator.

• Biomolecules & Therapeutics
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• v.22 no.3
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• pp.184-192
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• 2014

${\beta}$-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of ${\beta}$-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. ${\beta}$-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in ${\beta}$-lapachone-treated AGS cells. Treatment with ${\beta}$-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor-mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase-3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD-fmk, significantly abolished ${\beta}$-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by ${\beta}$-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased ${\beta}$-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of ${\beta}$-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to ${\beta}$-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

• Biomolecules & Therapeutics
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• v.22 no.3
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• pp.223-231
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• 2014

In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its $IC_{50}$ value was $175{\mu}g/ml$. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated $[CA^{2+}]_i$ mobilization and thromboxane $A_2$ ($TXA_2$) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated $[CA^{2+}]_i$ level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor ($IP_3R$) phosphorylation. These results suggest that the inhibition of $[CA^{2+}]_i$ mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of $IP_3R$. CE-WIB801C suppressed $TXA_2$ production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and $TXA_2$ synthase (TXAS). These results suggest that the inhibition of $TXA_2$ production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent $CA^{2+}$-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

• Biomolecules & Therapeutics
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• v.21 no.3
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• pp.222-228
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• 2013

Although the role of ${\alpha}$-synuclein aggregation on Parkinson's disease is relatively well known, the physiological role and the regulatory mechanism governing the expression of ${\alpha}$-synuclein are unclear yet. We recently reported that ${\alpha}$-synuclein is expressed and secreted from cultured astrocytes. In this study, we investigated the effect of valproic acid (VPA), which has been suggested to provide neuroprotection by increasing ${\alpha}$-synuclein in neuron, on ${\alpha}$-synuclein expression in rat primary astrocytes. VPA concentration-dependently increased the protein expression level of ${\alpha}$-synuclein in cultured rat primary astrocytes with concomitant increase in mRNA expression level. Likewise, the level of secreted ${\alpha}$-synuclein was also increased by VPA. VPA increased the phosphorylation of Erk1/2 and JNK and pretreatment of a JNK inhibitor SP600125 prevented the VPA-induced increase in ${\alpha}$-synuclein. Whether the increased ${\alpha}$-synuclein in astrocytes is involved in the reported neuroprotective effects of VPA awaits further investigation.

• Biomolecules & Therapeutics
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• v.26 no.2
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• pp.146-156
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• 2018

Spermidine is a naturally occurring polyamine compound that has recently emerged with anti-aging properties and suppresses inflammation and oxidation. However, its mechanisms of action on anti-inflammatory and antioxidant effects have not been fully elucidated. In this study, the potential of spermidine for reducing pro-inflammatory and oxidative effects in lipopolysaccharide (LPS)-stimulated macrophages and zebrafish was explored. Our data indicate that spermidine significantly inhibited the production of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$), and cytokines including tumor necrosis $factor-{\alpha}$ and $interleukin-1{\beta}$ in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects of spermidine accompanied by a marked suppression in their regulatory gene expression at the transcription levels. Spermidine also attenuated the nuclear translocation of $NF-{\kappa}B$ p65 subunit and reduced LPS-induced intracellular accumulation of reactive oxygen species (ROS) in RAW 264.7 macrophages. Moreover, spermidine prevented the LPS-induced NO production and ROS accumulation in zebrafish larvae and was found to be associated with a diminished recruitment of neutrophils and macrophages. Although more work is needed to fully understand the critical role of spermidine on the inhibition of inflammation-associated migration of immune cells, our findings clearly demonstrate that spermidine may be a potential therapeutic intervention for the treatment of inflammatory and oxidative disorders.

• Biomolecules & Therapeutics
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• v.26 no.2
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• pp.121-129
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• 2018

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and apoptosis play critical roles in the pathogenesis of atherosclerosis. Thioredoxin-1 (Trx) is an antioxidant that potently protects various cells from oxidative stress-induced cell death. However, the protective effect of Trx on ox-LDL-induced macrophage foam cell formation and apoptosis has not been studied. This study aims to investigate the effect of recombinant human Trx (rhTrx) on ox-LDL-stimulated RAW264.7 macrophages and elucidate the possible mechanisms. RhTrx significantly inhibited ox-LDL-induced cholesterol accumulation and apoptosis in RAW264.7 macrophages. RhTrx also suppressed the ox-LDL-induced overproduction of lectin-like oxidized LDL receptor (LOX-1), Bax and activated caspase-3, but it increased the expression of Bcl-2. In addition, rhTrx markedly inhibited the ox-LDL-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). Furthermore, anisomycin (a p38 MAPK activator) abolished the protective effect of rhTrx on ox-LDL-stimulated RAW264.7 cells, and SB203580 (a p38 MAPK inhibitor) exerted a similar effect as rhTrx. Collectively, these findings indicate that rhTrx suppresses ox-LDL-stimulated foam cell formation and macrophage apoptosis by inhibiting ROS generation, p38 MAPK activation and LOX-1 expression. Therefore, we propose that rhTrx has therapeutic potential in the prevention and treatment of atherosclerosis.

• Biomolecules & Therapeutics
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• v.26 no.5
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• pp.512-519
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• 2018

Phthalates widely used in the manufacture of plastics have deeply penetrated into our everyday lives. Recently, a concern over the toxicity of phthalates on thyroid, has been raised but in most of cases, the doses employed were unrealistically high. To investigate the effects of phthalates on thyroid, we investigated the effects of the repeated oral exposure to low to high doses (0.3, 3, 30 and 150 mg/kg) di-2-ethylhexylphthalate (DEHP) from weaning to maturity for 90 days in juvenile rats on the thyroid. The histological examination revealed that DEHP significantly induced hyperplasia in the thyroid from the doses of 30 mg/kg, which was confirmed with Ki67 staining. In line with this finding, increased mRNA expression of thyrotropin releasing hormone (Trh) was observed in the thyroid of female at 0.3 mg/kg and 150 mg/kg as determined by RNAseq analysis. Moreover, significantly increased expression of parathyroid hormone (Pth) in the female at 0.3 mg/kg, and thyroglobulin (Tg) and thyroid hormone responsive (Thrsp) in the male at 0.3 mg/kg were noted in the blood, of which changes were substantially attenuated at 150 m/kg, alluding the meaningful effects of low dose DEHP on the thyroid hormone regulation. Urinary excretion of mono-2-ethylhexyl-phthalate (MEHP), a major metabolite of DEHP was determined to be 4.10 and 12.26 ppb in male, 6.65 and 324 ppb in female at 0.3 and 30 mg/kg DEHP, respectively, which fell within reported human urine levels. Collectively, these results suggest a potential adverse effects of low dose phthalates on the thyroid.

• Biomolecules & Therapeutics
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• v.26 no.5
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• pp.439-445
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• 2018

T-type calcium channels are low voltage-activated calcium channels that evoke small and transient calcium currents. Recently, T-type calcium channels have been implicated in neurodevelopmental disorders such as autism spectrum disorder and neural tube defects. However, their function during embryonic development is largely unknown. Here, we investigated the function and expression of T-type calcium channels in embryonic neural progenitor cells (NPCs). First, we compared the expression of T-type calcium channel subtypes (CaV3.1, 3.2, and 3.3) in NPCs and differentiated neural cells (neurons and astrocytes). We detected all subtypes in neurons but not in astrocytes. In NPCs, CaV3.1 was the dominant subtype, whereas CaV3.2 was weakly expressed, and CaV3.3 was not detected. Next, we determined CaV3.1 expression levels in the cortex during early brain development. Expression levels of CaV3.1 in the embryonic period were transiently decreased during the perinatal period and increased at postnatal day 11. We then pharmacologically blocked T-type calcium channels to determine the effects in neuronal cells. The blockade of T-type calcium channels reduced cell viability, and induced apoptotic cell death in NPCs but not in differentiated astrocytes. Furthermore, blocking T-type calcium channels rapidly reduced AKT-phosphorylation (Ser473) and $GSK3{\beta}$-phosphorylation (Ser9). Our results suggest that T-type calcium channels play essential roles in maintaining NPC viability, and T-type calcium channel blockers are toxic to embryonic neural cells, and may potentially be responsible for neurodevelopmental disorders.

• Biomolecules & Therapeutics
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• v.26 no.5
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• pp.487-493
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• 2018

Cluster of differentiation 44 (CD44), a cell surface receptor for hyaluronic acid (HA), is involved in aggressive cancer phenotypes. Herein, we investigated the role of the CD44 standard isoform (CD44s) in hypoxia-inducible $factor-1{\alpha}$ ($HIF-1{\alpha}$) regulation using MCF7 overexpressing CD44s (pCD44s-MCF7). When pCD44s-MCF7 was incubated under hypoxia, levels of $HIF-1{\alpha}$, vascular endothelial growth factor, and the $HIF-1{\alpha}$ response element-derived luciferase activity were significantly increased compared to those in the control MCF7. Incubation of pCD44s-MCF7 cells with HA further increased $HIF-1{\alpha}$ accumulation, and the silencing of CD44s attenuated $HIF-1{\alpha}$ elevation, which verifies the role of CD44s in $HIF-1{\alpha}$ regulation. In addition, the levels of phosphorylated extracellular signal-regulated kinase (ERK) was higher in hypoxic pCD44s-MCF7 cells, and $HIF-1{\alpha}$ accumulation was diminished by the pharmacological inhibitors of ERK. CD44s-mediated $HIF-1{\alpha}$ augmentation resulted in two functional outcomes. First, pCD44s-MCF7 cells showed facilitated cell motility under hypoxia via the upregulation of proteins associated with epithelial-mesenchymal transition, such as SNAIL1 and ZEB1. Second, pCD44s-MCF7 cells exhibited higher levels of glycolytic proteins, such as glucose transporter-1, and produced higher levels of lactate under hypoxa. As a consequence of the enhanced glycolytic adaptation to hypoxia, pCD44s-MCF7 cells exhibited a higher rate of cell survival under hypoxia than that of the control MCF7, and glucose deprivation abolished these differential responses of the two cell lines. Taken together, these results suggest that CD44s activates hypoxia-inducible $HIF-1{\alpha}$ signaling via ERK pathway, and the $CD44s-ERK-HIF-1{\alpha}$ pathway is involved in facilitated cancer cell viability and motility under hypoxic conditions.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.159-165
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• 2007

For the development of basic genetic materials for specific and effective therapeutic approach to suppress multiplication of hepatitis C virus (HCV), HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was developed. The ribozyme targeted most effectively to +382 nucleotide (nt) site of HCV IRES RNA. The allosteric ribozyme was designed to be composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nt of HCV IRES. Noticeably, we employed in vitro selection technology to identify the most appropriate communication module sequence which can induce ribozyme activity depending on the US5B protein. We demonstrated that the ribozyme was nonfunctional either in the absence of any proteins or in the presence of control bovine serum albumin. In sharp contrast, the allosteric ribozyme can induce activity of cleavage reaction with HCV IRES RNA in the presence of the HCV NS5B protein. This allosteric ribozyme can be used as lead compound for specific and effective anti-HCV agent, tool for highthroughput screening to isolate lead chemicals for HCV therapeutics, and ligand for biosensor system for HCV diagnosis.