• The Korean Journal of Physiology and Pharmacology
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• v.11 no.2
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• pp.37-43
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• 2007

Adenosine has been reported to provide cytoprotection in the central nervous systems as well as myocardium by activating cell surface adenosine receptors. However, the exact target and mechanism of its action still remain controversial. The present study was performed to examine whether adenosine has a protective effect against $A{\beta}$-induced injury in neuroglial cells. The astrocyte-derived human neuroglioma cell line, A172 cells, and $A{\beta}_{25{\sim}35}$ were employed to produce an experimental $A{\beta}$-induced glial cell injury model. Adenosine significantly prevented $A{\beta}$-induced apoptotic cell death. Studies using various nucleotide receptor agonists and antagonists suggested that the protection was mediated by $A_1$ receptors. Adenosine attenuated $A{\beta}$-induced impairment in mitochondrial functional integrity as estimated by cellular ATP level and MTT reduction ability. In addition, adenosine prevented $A{\beta}$-induced mitochondrial permeability transition, release of cytochrome c into cytosol and subsequent activation of caspase-9. The protective effect of adenosine disappeared when cells were pretreated with 5-hydroxydecanoate, a selective blocker of the mitochondrial ATP-sensitive $K^+$ channel. In conclusion, therefore we suggest that adenosine exerts protective effect against $A{\beta}$-induced cell death of A172 cells, and that the underlying mechanism of the protection may be attributed to preservation of mitochonarial functional integrity through opening of the mitochondrial ATP-sensitive $K^+$ channels.

• Journal of Pharmacopuncture
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• v.15 no.3
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• pp.31-38
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• 2012

Sophorae Radix (SR) plays a role in a number of physiologic and pharmacologic functions in many organs. Objective: The aim of this study was to clarify the potential role for transient receptor potential melastatin 7 (TRPM7) channels in SR-inhibited growth and survival of AGS and MCF-7 cells, the most common human gastric and breast adenocarcinoma cell lines. Methods: The AGS and the MCF-7 cells were treated with varying concentrations of SR. Analyses of the caspase-3 and - 9 activity, the mitochondrial depolarization and the poly (ADPribose) polymerase (PARP) cleavage were conducted to determine if AGS and MCF-7 cell death occured by apoptosis. TRPM7 channel blockers ($Gd^{3+}$ or 2-APB) and small interfering RNA (siRNA) were used in this study to confirm the role of TRPM7 channels. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in AGS and MCF-7 cell growth and survival. Results: The addition of SR to a culture medium inhibited AGS and MCF-7 cell growth and survival. Experimental results showed that the caspase-3 and -9 activity, the mitochondrial depolarization, and the degree of PARP cleavage was increased. TRPM7 channel blockade, either by $Gd^{3+}$ or 2-APB or by suppressing TRPM7 expression with small interfering RNA, blocked the SR-induced inhibition of cell growth and survival. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SR-induced cell death. Conclusions: These findings indicate that SR inhibits the growth and survival of gastric and breast cancer cells due to a blockade of the TRPM7 channel activity. Therefore, TRPM7 channels may play an important role in the survival of patients with gastric and breast cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7995-8001
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• 2014

Interferon-gamma (IFN-${\gamma}$) has been used to treat various malignant tumors. However, the molecular mechanisms underlying the direct anti-proliferative activity of IFN-${\gamma}$ are poorly understood. In the present study, we examined the in vitro antitumor activity of IFN-${\gamma}$ on two human non-small-cell lung carcinoma (NSCLC) cell lines, H322M and H226. Our findings indicated that IFN-${\gamma}$ treatment caused a time-dependent reduction in cell viability and induced apoptosis through a FADD-mediated caspase-8/tBid/mitochondria-dependent pathway in both cell lines. Notably, we also postulated that IFN-${\gamma}$ increased indoleamine 2,3-dioxygenase (IDO) expression and enzymatic activity in H322M and H226 cells. In addition, inhibition of IDO activity by the IDO inhibitor 1-MT or tryptophan significantly reduced IFN-${\gamma}$-induced apoptosis and death receptor 5 (DR5) expression, which suggests that IDO enzymatic activity plays an important role in the anti-NSCLC cancer effect of IFN-${\gamma}$. These results provide new mechanistic insights into interferon-${\gamma}$ antitumor activity and further support IFN-${\gamma}$ as a potential therapeutic adjuvant for the treatment of NCSLC.

• BMB Reports
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• v.34 no.6
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• pp.537-543
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• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• Molecules and Cells
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• v.40 no.10
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• pp.752-760
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• 2017

A2B adenosine receptor (A2BAR) is known to be the regulator of bone homeostasis, but its regulatory mechanisms in osteoclast formation are less well-defined. Here, we demonstrate the effect of A2BAR stimulation on osteoclast differentiation and activity by RANKL. A2BAR was expressed in bone marrow-derived monocyte/macrophage (BMM) and RANKL increased A2BAR expression during osteoclastogenesis. A2BAR stimulation with its specific agonist BAY 60-6583 was sufficient to inhibit the activation of ERK1/2, p38 MAP kinases and $NF-{\kappa}B$ by RANKL as well as it abrogated cell-cell fusion in the late stage of osteoclast differentiation. Stimulation of A2BAR suppressed the expression of osteoclast marker genes, such as c-Fos, TRAP, Cathepsin-K and NFATc1, induced by RANKL, and transcriptional activity of NFATc1 was also inhibited by stimulation of A2BAR. A2BAR stimulation caused a notable reduction in the expression of Atp6v0d2 and DC-STAMP related to cell-cell fusion of osteoclasts. Especially, a decrease in bone resorption activity through suppression of actin ring formation by A2BAR stimulation was observed. Taken together, these results suggest that A2BAR stimulation inhibits the activation of ERK1/2, p38 and $NF-{\kappa}B$ by RANKL, which suppresses the induction of osteoclast marker genes, thus contributing to the decrease in osteoclast cell-cell fusion and bone resorption activity.

• YAKHAK HOEJI
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• v.45 no.6
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• pp.694-700
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• 2001

Synthetic pyrethroids are analysis of a natural chemical moiety, pyrethrin derived from the pyrethrum plant Chrysanthemum. The natural pyrethrin structure has been modified to be highly lipophilic and photostable, creating an effective pesticide and resulting in an increased presence in the environment. Worldwide, they are commonly used insecticides against ticks, mites, mosquitoes, and as treatment for human head lice and scabies. Therefore, human exposure to their compounds in extensive. Several studies on the effects of pyrethroids on thyroid hormone regulation, estrogen and androgen function have been reported and yet little has been done try assess their potential hormonal activities. Among humans, a pyrethroid compound was suggested to be the causal agent for gynecomastia in a group of Haitian men. The reports suggest that some pyrethroid compounds are capable of disrupting endocrine function. Therefore, we examined estrogenic/antiestrogenic potential of three pyrethroid insecticides, that is permethrin, allethrin and fenvalerate in human breast cancer cell and action mechanism mediated by the estrogen receptor. Fenvalerate showed weak estrogenic activity but aallethrin and permethrin showed no effect. In combination with high levels (10$^{-10}$ M, 10$^{-11}$ M) of 17$\beta$-estradiol and three synthetic pyrethroids inhibited cert proliferations in MCF7-BUS cell by 17$\beta$-estradiol. Whereas, fenvalerate increased cell proliferative activity at lower level of estradiol (10$^{-12}$ M, 10$^{-13}$ M). The relative affinities to the estrogen receptor were observed by allethrin and permethrin treatment, but not by fenvalerate. These results indicated that some of pyrethroid insecticides may modulate estrogen functions in human breast cancer cell. The action mechanisms of estrogen receptor mediated antiestrogenicity by allethrin and permethrin were postulated.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.3
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• pp.179-187
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• 2011

Regulation of B cell receptor (BCR)-induced $Ca^{2+}$ signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to ${\alpha}$-IgM antibody (0.5~48 hr), the initial transient decrease in BCR-induced $[Ca^{2+}]_i$ was followed by spontaneous recovery to control level within 24 hr. The recovery of $Ca^{2+}$ signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of $PLC{\gamma}2$ and $IP_3R-3$. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced $Ca^{2+}$ signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of $PLC{\gamma}2$ and $IP_3R-3$. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of $Ca^{2+}$ signaling. In contrast to immature WEHI-231 cells, identical long-term ${\alpha}$-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced $[Ca^{2+}]_i$, regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced $Ca^{2+}$ signaling.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.1
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• pp.61-73
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• 2023

Esophageal squamous cell carcinoma (ESCC) is a kind of malignant tumor with high incidence and mortality in the digestive system. The aim of this study is to explore the function of lnc-ABCA12-3 in the development of ESCC and its unique mechanisms. RT-PCR was applied to detect gene transcription levels in tissues or cell lines like TE-1, EC9706, and HEEC cells. Western blot was conducted to identify protein expression levels of mitochondrial apoptosis and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway. CCK-8 and EdU assays were carried out to measure cell proliferation, and cell apoptosis was examined by flow cytometry. ELISA was used for checking the changes in glycolysis-related indicators. Lnc-ABCA12-3 was highly expressed in ESCC tissues and cells, which preferred it to be a candidate target. The TE-1 and EC9706 cells proliferation and glycolysis were obviously inhibited with the downregulation of lnc-ABCA12-3, while apoptosis was promoted. TLR4 activator could largely reverse the apoptosis acceleration and relieved the proliferation and glycolysis suppression caused by lnc-ABCA12-3 downregulation. Moreover, the effect of lnc-ABCA12-3 on ESCC cells was actualized by activating the TLR4/NF-κB signaling pathway under the mediation of exosome. Taken together, the lnc-ABCA12-3 could promote the proliferation and glycolysis of ESCC, while repressing its apoptosis probably by regulating the TLR4/NF-κB signaling pathway under the mediation of exosome.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.45-54
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• 1999

In the present study we have analyzed the characteristics and distribution of the mu-opioid receptor(MOR) by raising anti-peptide antisera to the C-terminal peptide of MOR. The antisera against MOR was produced in New Zealand White rabbit against 15 residue corresponding to amino acids, 384-398 of the cloned rat MOR. The antigenic peptide was synthesized using an Applied Biosystems 432 solid-phase peptide synthesizer. The specificity and identification of the antisera were tested by analysis of transfected cells, epitope mapping and immunohistochemical method. COS-7 cells electroporated with MOR cDNA were used to evaluate the characteristics and subcellular distribution of MOR. MOR immunoreactivity was prodominent in the plasmalemma and subcellular compartments such as endoplasmic reticulum, Golgi apparatus and vesicle like structure. Furthermore, both tissue sections and transfected cell lines could be immunostained with these antisera and the immunoreactivity was abolished when anti-MOR sera were preincubated with the peptide against which they were raised. Based on epitope mapping analysis, all antisera appeared to have a similar epitope, which was determined to be within the last amino acid, 391-398. Moreover, immunohistochemistry showed that MOR immunoreactivity was observed in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus and the superficial laminae of the dorsal horn. These stained spinal cord and brain areas showed the mirrored pattern observed in auto radiographic studies of mu-opioid binding as well as a pattern similar to that seen by is situ hybridization for MOR. Thus, several lines of evidence support the conclusion that the antisera produced in the present study most likely recognize mu-opioid receptor. These results suggest that MOR antisera may be utilized as useful tool to analyze the physiological and pharmacological studies for mu-opioid receptor in the future.

• Archives of Pharmacal Research
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• v.10 no.4
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• pp.212-218
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• 1987

Intact brain cell aggregates were dissociated from adult rat brains without cerebellum using a sieving technique. This proparation was used to elucidate the binding characteristics of agonist to muscarinic acetylcholine receptors (mAchR) in brain. Incubation of cells with carbamylcholine (carbachol) was shown agonist-induced receptor down-regulation depending on the concentration of agonist, not depending on the incubation time. This effect of carbachol was due to a reduction in the maximal binding capacity ($B_{max}$) to the mAchR without decreasing the affinity of the remaining receptors in incubation at 37.deg.C but was not apparent inincubation at $15^{\circ}}C$In addition, it was abolished when the receptors were blocked by atropine. The decline in ($^3H$)N-methylscopolamine (($^3H$)NMS) binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that 'true' receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist ($^3H$) quinuclidinyl benzilate (($^3H$) QNB) insted of the hydrophilic ligand ($^3H$)NMS, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggested that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded.