• International Journal of Oral Biology
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• 제44권2호
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• pp.50-54
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• 2019

Melatonin is a neurotransmitter that modulates various physiological phenomena including regulation and maintenance of the circadian rhythm. Nicotinic acetylcholine receptors (nAChRs) play an important role in oral functions including orofacial muscle contraction, salivary secretion, and tooth development. However, knowledge regarding physiological crosstalk between melatonin and nAChRs is limited. In the present study, the melatonin-mediated modulation of nAChR functions using bovine adrenal chromaffin cells, a representative model for the study of nAChRs, was investigated. Melatonin inhibited the nicotinic agonist dimethylphenylpiperazinium (DMPP) iodide-induced cytosolic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increase and norepinephrine secretion in a concentration-dependent manner. The inhibitory effect of melatonin on the DMPP-induced $[Ca^{2+}]_i$ increase was observed when the melatonin treatment was performed simultaneously with DMPP. The results indicate that melatonin inhibits nAChR functions in both peripheral and central nervous systems.

• The Korean Journal of Physiology and Pharmacology
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• 제25권3호
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• pp.189-195
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• 2021

Fluoxetine (FLX), a selective serotonin reuptake inhibitor antidepressant, exhibits various other mechanisms of action in numerous cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The aim of this study was to determine the off-target effects of the anti-depressant drug FLX, on the human ovarian granulosa tumor COV434 cells stimulated by forskolin (FSK), by measuring the real-time kinetics of intracellular cyclic AMP (cAMP), ATP level, cytoplasmic calcium ([Ca2+]cyt) and survival of COV434 cells. We show that incubating COV434 cells with FLX (between 0.6 and 10 μM) induces a decrease in intracellular cAMP response to FSK, a drop in ATP content and stimulates cytoplasmic Ca2+ accumulation in COV434 cells. Only the highest concentrations of FLX (5-10 μM) diminished cell viability. The present report is the first to identify an action mechanism of FLX in human tumor ovarian cells COV434 cells and thus opening the way to potential use of fluoxetine as a complementary tool, in granulosa tumor treatments.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1835-1842
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• 2020

Ergosterol, an essential constituent of membrane lipids of yeast, is distributed in both the cell membrane and intracellular endomembrane components such as vacuoles. Honokiol, a major polyphenol isolated from Magnolia officinalis, has been shown to inhibit the growth of Candida albicans. Here, we assessed the effect of honokiol on ergosterol biosynthesis and vacuole function in C. albicans. Honokiol could decrease the ergosterol content and upregulate the expression of genes related with the ergosterol biosynthesis pathway. The exogenous supply of ergosterol attenuated the toxicity of honokiol against C. albicans. Honokiol treatment could induce cytosolic acidification by blocking the activity of the plasma membrane Pma1p H+-ATPase. Furthermore, honokiol caused abnormalities in vacuole morphology and function. Concomitant ergosterol feeding to some extent restored the vacuolar morphology and the function of acidification in cells treated by honokiol. Honokiol also disrupted the intracellular calcium homeostasis. Amiodarone attenuated the antifungal effects of honokiol against C. albicans, probably due to the activation of the calcineurin signaling pathway which is involved in honokiol tolerance. In conclusion, this study demonstrated that honokiol could inhibit ergosterol biosynthesis and decrease Pma 1p H+-ATPase activity, which resulted in the abnormal pH in vacuole and cytosol.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8503-8512
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• 2016

The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline, WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of WP631 and DOX suggest that this bisanthracycline can be an effective alternative in ovarian cancer treatment.

• 한국식품위생안전성학회지
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• 제22권3호
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• pp.223-230
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• 2007

저자들은 녹차 카테킨(GTC)이 강한 항 혈전 작용을 나타내며, 이는 항 혈소판 활성에 의한 것임을 보고한 바 있다. 본 연구에서는, 8가지 녹차 카테킨 성분들의 항 혈소판 활성을 비교하였다. 실험결과, 갈레이트(gallate) 구조를 갖는 카테킨들(EGCG, GCG, ECG, CG)은 콜라겐$(5{\mu}g/ml)$으로 유도한 토끼 혈소판 응집능을 강하게 억제하였으며, 50% 억제농도$(IC_{50})$는 각각 79.8, 63.0, 168.2, $67.3{\mu}M$이었다. 또한 EGCG, GCG, CG는 아라키돈산(AA, $100{\mu}M$)으로 유도한 토끼 혈소판 응집능을 억제하였고, 50% 억제농도$(IC_{50})$는 각각 98.9, 200.0, $174.3{\mu}M$이었다. 반면에, 갈레이트 구조를 가지지 않는 카테킨들은 혈소판 응집능 억제 효과가 매우 약했다. 이 결과는 항 혈소판 활성에서 카테킨들의 C-3 위치의 갈레이트 구조의 존재가 매우 중요하다는 것과 카테킨들과 B-ring 갈레이트 구조의 존재 또한 녹차카테킨의 항 혈소판 활성에 중요한 작용한다는 것을 의미한다. 그리고, EGCG는 농도 의존적으로 세포내 칼슘 생성과 아리키돈산의 생성을 억제시켰는데, 이는 혈소판 응집능의 억제와 일치하였다. 반면에, EGC는 세포 내 칼슘 및 다른 혈소판 활성 기전에 아무런 영향이 없었다. 이들 결과는 EGCG의 항 혈소판 활성은 C-ring에서 carbon 3 자리의 에스테르화 된 갈레이트 구조의 존재에 의해서 강화된 항 혈소판 작용으로, 아라키돈산 생성과 세포 내 칼슘 생성을 억제하는 효과에 기인한 것이라 사료된다.

• 대한약리학회지
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• 제26권1호
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• pp.55-66
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• 1990

안정 상태 및 활성화된 중성 백혈구에서 ATP는 superoxide 라디칼 생성을 자극하였으나 adenosine은 약간 억제하였다. ATP에 대한 활성화된 중성 백혈구의 반응이 안정상태의 중성 백혈구에서보다 크게 나타났다. 칼슘이 제거된 반응액에서 superoxide 라디칼 생성에 대한 adenosine의 억제효과가 관찰되었으나 ATP는 영향을 주지않았다. Superoxide 라디칼 생성에 대한 ATP의 자극 효과는 adenosine에 의하여 용량에 따라 억제되었다. ATP와 adenosine은 NADPH oxidase 활성도에 영향을 주지 않았다. ATP 또는 adenosine에 의한 superoxide 라디칼 생성의 변경은 다른 triphosphate nucleotide나 nucleoside에 의한 것보다 현저하였다. 활성화된 중성 백혈구에서 ATP와 ADP는 칼슘이온의 흡수를 더 자극하였고 세포질 유리 칼슘농도를 증가시켰으나, adenosine은 칼슘이온의 이동을 억제하였다. APT에 의한 세포질 유리 칼슘이온 농도의 증가는 verapamil에 의하여 효과적으로, tetrodotoxin에 의하여 약간 억제되었다. ATP에 노출된 활성화된 중성 백혈구에서의 superoxide 라디칼 생성에 대한 verapamil 과 tetrodotoxin의 억제 효과는 ATP의 영향이 없는 활성화된 중성 백혈구에서보다 크게 나타났다. Tetraethylammonium chloride는 superoxide 생성에 뚜렷한 영향을 미치지 못했다. CCCP, 2,4-dinitrophenol, diphenylhydantoin과 procaine은 활성화된 중성 백혈구에서 superoxide 라디칼의 생성을 억제하였다. 이들 가운데 CCCP만이 ATP의 자극 효과를 억제하였다. ATP는 활성화된 중성 백혈구에서의 sulfhydryl기의 손실을 더 자극하였으나 adenosin의 영향은 관찰되지 않았다. 이상의 결과로부터 중성 백혈구의 기능적 반응은 부분적으로 purine에 의하여 조절될 것으로 시사되었다. ATP와 adenosine은 칼슘 흡수와 그리고 아마도 세포막 인산화 반응 및 용해성 sulfhydryl기의 산화에 대한 영향을 통하여 활성화된 중성 백혈구의 반응을 더 변경 시킬 수 있을 것으로 추정된다.

• Journal of Applied Biological Chemistry
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• 제66권
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• pp.236-243
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• 2023

혈소판은 1차 및 2차 지혈에서 근본적인 역할을 하는 세포지만 혈소판의 과도한 활성화는 혈전증을 유발할 수 있다. 따라서 혈소판 응집의 적절한 조절은 혈전증 매개 질환을 예방하는 데 중요하다. 최근 곤충소재의 개발이 주목을 받고 있다. 다양한 곤충 자원 중 고영양 기능성 식품원으로는 쌍별귀뚜라미(Gryllus bimaculatus)와 같은 곤충류가 있다. 쌍별귀뚜라미 는 고단백 및 불포화지방산을 함유하고 있으며 2015년 9월 식품의약품안전처로부터 식품원료로 등록되었다. 본 연구에서는 쌍별귀뚜라미 에탄올 추출물(G. bimaculatus extract)이 혈소판 응집, 세포 내 Ca²⁺ 조절, thromboxane A₂ 생산 및 glycoprotein IIb/IIIa (integrin αIIb/β3) 활성화를 억제하는지 여부를 확인하고. 1, 4, 5-triphosphate receptor type I, extracellular signal-regulated kinase, cytosolic phospholipase A₂, mitogen-activated protein kinases p38, vasodilator-stimulated phosphoprotein, phosphatidylinositol-3 kinase, Akt, glycogen synthase kinase-3α/β 및 SYK 같은 신호 분자를 조절할 수 있는지 여부를 조사했다. 우리는 쌍별귀뚜라미 추출물이 혈소판 관련 혈전증 및 심혈관 질환을 예방할 수 있는 잠재적인 치료 약물로 가치가 있음을 규명하였다.

• Archives of Pharmacal Research
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• 제25권6호
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• pp.879-884
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• 2002

Previous study showed that an amidrazonophenylalanine derivative, LB30057, which has high water solubility, inhibited the catalytic activity of thrombin potently by interaction with the active site of thrombin. In the current investigation, we examined whether LB30057 inhibited platelet aggregation and vascular relaxation induced by thrombin. Treatment with LB30057 to plateletrich plasma (PRP) isolated from human blood resulted in a concentration-dependent inhibition of thrombin-induced aggregation. Values for $IC_{50}$ and $IC_{100}$ were $54{\pm}4$ nM and $96{\pm}3$ nM, respectively. This inhibition was agonist (thrombin) specific, since $IC_{50}$ values for collagen and ADP were \much greater than those for thrombin. In addition, concentration-dependent inhibitory effects were observed on the serotonin secretion induced by thrombin in PRP. Consistent with these findings, thrombin-induced increase in cytosolic calcium levels was inhibited in a concentration-dependent manner. When LB30057 was treated with aortic rings isolated from rats, LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular relaxation. All these results suggest that LB30057 is a potent inhibitor of platelet aggregation and blood vessel relaxation induced by thrombin.

• 한국약용작물학회지
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• 제13권5호
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• pp.219-226
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• 2005

Sanguisorbae radix (SR) from Sanguisorba officinalis L. (Losaceae) is widely used in Korea and China due to its various pharmacological activity. The present study aims to investigate the effect of the methanol extract of SR on amyloid ${\beta}$ Protein(25-35) $(A{\beta}\;(25-35))$, a synthetic 25-35 amyloid peptide, -induced neurotoxicity using cultured rat cortical neurons. SR, over a concentration range of $10-50\;{\mu}g/ml$, inhibited the $A{\beta}$ (25-35) $(10\;{\mu}M)-induced$ neuronal cell death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. Pretreatment of SR $(50\;{\mu}g/ml)$ inhibited $10\;{\mu}M\;A{\beta}$ (25-35)-induced} elevation of cytosolic calcium concentration $([Ca^{2+}]c)$, which was measured by a fluorescent dye, fluo-4 AM. SR $(10\;and\;50\;{\mu}g/ml)$ inhibited glutamate release into medium induced by $10\;{\mu}M\;A{\beta}(25-35)$, which was measured by HPLC, and generation of reactive oxygen species. These results suggest that SR prevents $A{\beta}$ (25-35)-induced neuronal cell damage in vitro.

• Molecules and Cells
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• 제23권1호
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• pp.11-16
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• 2007

N,N-dimethyl-D-erythro-sphingosine (DMS) is an N-methyl derivative of sphingosine and an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In the present study, we examined the effects of DMS on intracellular $Ca^{2+}$ concentration, pH, and glutamate uptake in human 1321N1 astrocytes. DMS increased intracellular $Ca^{2+}$ concentration and cytosolic pH in a concentration-dependent manner. Pretreatment of the cells with the $G_{i/o}$ protein inhibitor PTX and the PLC inhibitor U73122 had no obvious effect. However, removal of extracellular $Ca^{2+}$ with the $Ca^{2+}$ chelator EGTA or depletion of intracellular $Ca^{2+}$ stores with thapsigargin impeded the DMS-induced increase of intracellular $Ca^{2+}$ concentration. Pretreatment of cells with $NH_4Cl$ or monensin reduced the DMS-induced $Ca^{2+}$ increase. However, inhibition of the DMS-induced $Ca^{2+}$ increase with BAPTA did not influence the DMS-induced pH increase. DMS also inhibited glutamate uptake by the 1321N1 astrocytes in a concentration-dependent manner. It also increased intracellular $Ca^{2+}$ and pH in PC12 neuronal cells. Our observations on the effects of DMS on 1321N1 astrocytes and PC12 neuronal cells point to a physiological role of DMS in the brain.