• Title/Summary/Keyword: Apoptosis assay

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Protective Effects of Potassium Ion on Rotenone-Induced Apoptosis in Neuronal (Neuro 2A) Cells

  • Park, Ji-Hwan;Kim, Yun-Ha;Moon, Seong-Keun;Kim, Tae-Young;Kim, Jong-Moon
    • Journal of Korean Neurosurgical Society
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    • v.38 no.6
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    • pp.456-464
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    • 2005
  • Objective : The authors investigated whether rotenone induces cellular death also in non-dopaminergic neurons and high concentration of potassium ion can show protective effect for non-dopaminergic neuron in case of rotenone-induced cytotoxicity. Methods : Neuro 2A cells was treated with rotenone, and their survival as well as cell death mechanism was estimated using 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium[MTT] assay, Lactate dehydrogenase[LDH] release assay, fluorescence microscopy, and agarose gel electrophoresis. The changes in rotenone-treated cells was also studied after co-treatment of 50mM KCl. And the protective effect of KCl was evaluated by mitochondrial membrane potential assay and compared with the effects of various antioxidants. Results : Neuro 2A cells treated with rotenone underwent apoptotic death showing chromosome condensation and fragmentation as well as DNA laddering. Co-incubation of neuro 2A cells with 50mM KCl prevented it from the cytotoxicity induced by rotenone. Intracellular accumulation of reactive oxygen species[ROS] resulting by rotenone were significantly reduced by 50mM KCl. Potassium exhibited significantly similar potency compared to the antioxidants. Conclusion : The present findings showed that potassium attenuated rotenone-induced cytotoxicity, intracellular accumulation of ROS, and fragmentation of DNA in Neuro 2A cells. These findings suggest the therapeutic potential of potassium ion in neuronal apoptosis, but the practical application of high concentration of potassium ion remains to be settled.

Alpha-lipoic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced cell injury by inhibiting autophagy and apoptosis

  • Kang, Kyeong-Rok;Kim, Jae-Sung;Kim, Tae-Hyeon;Seo, Jeong-Yeon;Lim, HyangI;Park, Jong-Hyun;Yang, Kwang Yeol;Yu, Sun-Kyoung;Kim, Heung-Joong;Kim, Chun Sung;Chun, Hong Sung;Lee, Dong-Seol;Park, Joo-Cheol;Kim, Do Kyung
    • International Journal of Oral Biology
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    • v.46 no.1
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    • pp.15-22
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    • 2021
  • Alpha-lipoic acid (ALA) is a naturally occurring antioxidant and has been previously used to treat diabetes and cardiovascular disease. However, the autophagy effects of ALA against oxidative stress-induced dopaminergic neuronal cell injury remain unclear. The aim of this study was to investigate the role of ALA in autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cell line. We examined SH-SY5Y phenotypes using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (cell viability/proliferation), 4′,6-diamidino-2-phenylindole dihydrochloride nuclear staining, Live/Dead cell assay, cellular reactive oxygen species (ROS) assay, immunoblotting, and immunocytochemistry. Our data showed ALA attenuated hydrogen peroxide (H2O2)-induced ROS generation and cell death. ALA effectively suppressed Bax up-regulation and Bcl-2 and Bcl-xL down-regulation. Furthermore, ALA increased the expression of the antioxidant enzyme, heme oxygenase-1. Moreover, the expression of Beclin-1 and LC-3 autophagy biomarkers was decreased by ALA in our cell model. Combined, these data suggest ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.

The anti-tumor efficacy of 20(S)-protopanaxadiol, an active metabolite of ginseng, according to fasting on hepatocellular carcinoma

  • Li, Wenzhen;Wang, Yifan;Zhou, Xinbo;Pan, Xiaohong;Lu, Junhong;Sun, Hongliu;Xie, Zeping;Chen, Shayan;Gao, Xue
    • Journal of Ginseng Research
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    • v.46 no.1
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    • pp.167-174
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    • 2022
  • Background: 20(S)-protopanaxadiol (20(S)-PPD), one of the main active metabolites of ginseng, performs a broad spectrum of anti-tumor effects. Our aims are to search out new strategies to enhance anti-tumor effects of natural products, including 20(S)-PPD. In recent years, fasting has been shown to be multi-functional on tumor progression. Here, the effects of fasting combined with 20(S)-PPD on hepatocellular carcinoma growth, apoptosis, migration, invasion and cell cycle were explored. Methods: CCK-8 assay, trypan blue dye exclusion test, imagings photographed by HoloMonitorTM M4, transwell assay and flow cytometry assay were performed for functional analyses on cell proliferation, morphology, migration, invasion, apoptosis, necrosis and cell cycle. The expressions of genes on protein levels were tested by western blot. Tumor-bearing mice were used to evaluate the effects of intermittent fasting combined with 20(S)-PPD. Results: We firstly confirmed that fasting-mimicking increased the anti-proliferation effect of 20(S)-PPD in human HepG2 cells in vitro. In fasting-mimicking culturing medium, the apoptosis and necrosis induced by 20(S)-PPD increased and more cells were arrested at G0-G1 phase. Meanwhile, invasion and migration of cells were decreased by down-regulating the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in fasting-mimicking medium. Furthermore, the in vivo study confirmed that intermittent fasting enhanced the tumor growth inhibition of 20(S)-PPD in H22 tumor-bearing mice without obvious side effects. Conclusion: Fasting significantly sensitized HCC cells to 20(S)-PPD in vivo and in vitro. These data indicated that dietary restriction can be one of the potential strategies of chinese medicine or its active metabolites against hepatocellular carcinoma.

Anti-Cancer Effect of Ursolic Acid in Melanoma Cell A375SM and A375P (Ursolic acid의 악성 흑색종 세포주 A375SM과 A375P에서의 항암효능)

  • Woo, Joong-Seok;Kim, Na-Won;Lee, Jin-Gyu;Kim, Jae-Hyuk;Lim, Da-Young;Kang, Shin-Woo;Kim, Sung-Hyun;Yoo, Eun-Seon;Lee, Jae-Han;Han, So-Hee;Park, Young-Seok;Kim, Byeong-Soo;Kim, Sang-Ki;Park, Byung-Kwon;Jung, Ji-Youn
    • Journal of Food Hygiene and Safety
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    • v.34 no.2
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    • pp.183-190
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    • 2019
  • Ursolic acid is recognized for various effects such as anti-cancer, antioxidant, and anti-inflammatory activity. In this study, we confirmed the anti-cancer effect of ursolic acid on human melanoma cancer cells, A375SM and A375P. Survival rate of the melanoma cells was confirmed by MTT assay and the proliferation rate was confirmed by wound healing assay. The rate of apoptotic bodies was confirmed by DAPI staining, and apoptosis rate was confirmed by flow cytometry. The induction of apoptosis protein was examined by western blotting according to the concentration of ursolic acid in melanoma cells. The survival and proliferation rates of melanoma cells were decreased according to the treatment concentrations of ursolic acid. DAPI staining showed that chromosomal condensation of melanoma cells was increased with increasing concentrations of ursolic acid, and increased apoptosis rate of melanoma cells by ursolic acid was confirmed by flow cytometry. We also confirmed by western blotting that cleaved-PARP and Bax were increased and Bcl-2 was decreased at $12{\mu}M$ concentration of uricolic acid in melanoma cells. This study was carried out at low concentrations of ursolic acid, 0 to $20{\mu}M$, and analyzed 24 h after treatment. As a result of this study, it is thought that ursolic acid has the anti-cancer effect through the regulation of apoptosis-related proteins in melanoma cells A375SM and A375P.

High Glucose Induces Apoptosis through Caspase-3 Dependent Pathway in Human Retinal Endothelial Cell Line (인간망막 내피세포주에서 고농도 포도당이 caspase-3 경로를 통해 세포자연사 유도)

  • Seo, Eun-Sun;Chae, Soo-Chul;Kho, Eun-Gyeong;Lee, Jong-Bin
    • Korean Journal of Environmental Biology
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    • v.27 no.1
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    • pp.66-72
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    • 2009
  • Diabetic Retinopathy (DR) is a leading cause of blindness among adults in the western countries. Hyperglycemia is a condition, that induces apoptotic cell death in a variety of cell types in diabetes, but the mechanism remains unclear. The aim of the study is to understand the effects of high Glucose on Human Retinal Endothelial Cells. Retinal endothelial cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 5, 25 and 50 mM Glucose, incubated for 24, 36 and 48 hours in humidified 5 % CO$_2$ incubator at 37$^{\circ}C$. Human Retinal Endothelial Cell Line (HREC) were characterized for morphology with different treatment by phase contrast microscopic analysis. Number of dead and viable cells was counted by trypan blue exclusion and supported by MTT assay. The intracellular Hydrogen peroxide (H$_2$O$_2$), a Reactive Oxygen Species (ROS) generation in high glucose conditions was assessed by FOX II assay and apoptosis by caspase-3 assay. The high glucose treated cells undergoing DNA fragmentation was witnessed by Agarose gel electrophoresis. We found that the cells incubated with 25 and 50 mM glucose containing medium for 48 hours altered the morphology of the cell, induced apoptosis and DNA fragmentation. The dead cell number were high in 25 and 50 mM when compared to the cells incubated with 5 mM glucose for 24, 36, and 48 hours. Also, the H$_2$O$_2$ levels and the activity of caspase-3 were increased in high glucose treated cells. Conclusions/interpretation: Our results demonstrated that elevated glucose induces apoptosis in cultured HREC. The hyperglycemia-induced increase in apoptosis may be dependent on caspase activation. The association between ROS generation and caspase-3 activation on high glucose treated cells is yet to be investigated.

Obovatol Inhibits Inflammation Mediator Generation and Colon Carcinoma SW620, HCT116 Cell Growth Through Induction of Apoptotic Cell Death Via Inactivation of $NF-{\kappa}B$ (Obovatol의 염증매개 생성 억제와 세포자멸사를 통한 Colon Carcinoma SW620, HCT116 세포의 세포증식에 대한 영향)

  • Jung, In-Mo;Song, Ho-Sueb
    • Journal of Acupuncture Research
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    • v.25 no.2
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    • pp.75-89
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    • 2008
  • 목적 : 후박(厚朴)(Magnolia obovata)에서 추출한 낮은 농도의 obovatol 약침액의 RAW264.7 세포에서 LPS로 유발된 염증, $TNF-{\alpha}$로 유발된 human colon carcinoma SW620 및 HCT116 세포의 세포증식에 대한 영향과 그 기전을 살펴보고자 하였다. 방법 : RAW264.7 세포에서 LPS로 염증을 유발하고 낮은 농도의 obovatol 약침액을 처리한 후 cell viability, NO 생성량, iNOS와 COX-2의 발현, $NF-{\kappa}B$활성, 전사능력을 관찰하기 위해 WST-1 assay, NO determination assay, western blot analysis, EMSA, luciferase activity assay를 시행하였고, HCT116, SW620 세포에 $TNF-{\alpha}$로 증식을 유도하고 낮은 농도의 obovatol 약침액을 처리한 후 cell growth, apoptosis 및 apoptosis와 연관된 $NF-{\kappa}B$의 활성 변화를 관찰하기 위해 WST-1, Cell morphogy test, DAPI staining and TUNEL assay, EMSA, luciferase activity assay를 시행하였다. 결과 : 1. RAW264.7 세포에서 낮은 농도의 obovatol 약침액 처리는 $NF-{\kappa}B$의 활성 및 전사능력을 낮추고 iNOS와 COX-2의 발현과 NO 생성을 감소시켜 LPS로 유발된 염증을 억제하였다. 2. HCT116, SW620 세포에서 낮은 농도의 obovatol 약침액 처리는 $NF-{\kappa}B$의 활성을 낮추어 세포자멸사를 촉진함으로써 $TNF-{\alpha}$로 유발된 암세포의 성장을 억제하였다. 결론 : 이상의 결과는 낮은 농도의 obovatol 약침액이 항염 및 인간 전립선암세포주인 SW620, HCT116에 대한 증식억제 효과가 있음을 입증한 것이며, 향후 이를 바탕으로 한 생체 연구에서의 긍정적인 결과는 obovatol 약침액이 만성염증성 질환 및 대장암의 예방과 치료에 대한 효과적인 치료제 개발에 초석이 될 것으로 기대된다.

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Obovatol Extracted from Magnolia Obovata Inhibits Inflammation Mediator Generation and Prostate Carcinoma PC-3, LNCap Cell Growth Through Induction of Apoptotic Cell Death via Inactivation of $NF-{\kappa}B$ (후박에서 추출한 Obovatol의 염증매개 생성의 억제와 세포자멸사를 통한 Human Prostate Carcinoma PC-3 및 LNCap 세포의 세포증식에 대한 영향)

  • Kim, Goon-Joong;Song, Ho-Sueb
    • Journal of Acupuncture Research
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    • v.24 no.2
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    • pp.15-29
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    • 2007
  • 목적 : 후박(厚朴)(Magnolia Obovata)에서 추출한 낮은 농도의 Obovatol 약침액의 RAW264.7 세포에서 LPS로 유발된 염증,$TNF-{\alpha}$로 유발된 human Prostate carcinoma PC-3 및 LNCap 세포의 세포증식에 대한 영향과 그 기전을 살펴보고자 하였다. 방법 : RAW264.7 세포에서 LPS로 염증을 유발하고 낮은 농도의 Obovatol 약침액을 처리한 후 cell viability, NO 생성량,iNOS와 COX-2의 발현, $NF-{\kappa}B$활성,전사능력을 관찰하기 위해 MTT assay, NO determination assay, western blot analysis, EMSA, luciferase activity assay를 시행하였고,LNCap, PC-3 세포에 $TNF-{\alpha}$로 증식을 유도하고 낮은 농도의 Obovatol 약침액을 처리한 후 cell growth, apoptosis 및 apoptosis와 연관된 $NF-{\kappa}B$의 활성 변화를 관찰하기 위해 WST-1, Cell morphogy test, DAPI staining and TUNEL assay, EMSA, luciferase activity assay를 시행하였다. 결과 : 1. RAW264.7 세포에서 낮은 농도의 Obovatol 약침액 처리는 $NF-{\kappa}B$의 활성 및 전사능력을 낮추고 iNOS와 COX-2의 발현과 NO 생성을 감소시켜 LPS로 유발된 염증을 억제하였다. 2. LNCap, PC-3 세포에서 낮은 농도의 Obovatol 약침액 처리는 $NF-{\kappa}B$의 활성을 낮추어 세포자별사를 촉진함으로써 $TNF-{\alpha}$로 유발된 암세포의 성장을 억제하였다. 결론 : 이상의 결과는 낮은 농도의 Obovatol 약침액이 항염 및 인간 전립선암세포주인 PC-3, LNCap에 대한 증식억제 효과가 있음을 입증한 것이며,향후 이를 바탕으로한 생체 연구에서의 긍정적인 결과는 Obovatol 약침액이 만성염증성 질환 및 전립선암의 예방과 치료에 대한 효과적인 치료제 개발에 초석이 될 것으로 기대된다.

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Induction of Apoptosis in Human Cancer Cells with Extracts of Taraxacum coreanum, Youngia sonchifolia and Ixeris dentate (흰민들레, 고들빼기, 씀바귀 추출물의 암세포 증식 억제 효과)

  • Shin, Seong-Ah;Lee, Hae-Nim;Choo, Gang-Sik;Kim, Hyeong-Jin;Park, Byung-Kwon;Kim, Byeong-Soo;Jung, Ji-Youn
    • Journal of Food Hygiene and Safety
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    • v.31 no.1
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    • pp.51-58
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    • 2016
  • This research examined the effects of Compositae extract on the inhibition of proliferation and apoptosis in human breast and human gastric cancer cells. Compositae extracts which is used in the experiment are Taraxacum coreanum (TC), Youngia sonchifolia (YS) and Ixeris dentata (ID). The proliferation of SK-BR-3, MDA-MB-231 and AGS cells were investigated by MTT assay. ID and YS extracts inhibited proliferation of SK-BR-3, MDA-MB-231 and AGS cells in a dose-dependent manner, but TC have barely affected. In addition, the most effective extract was ID. To assess the apoptosis of ID extract, the nuclei of human cancer cells were stained with DAPI solution respectively. Chromatin condensation, indicated apoptosis, was increased in a dose-dependent manner. We investigated change of ID extract-induced apoptosis proteins on human cancer cells by western blot analysis. The level of Bcl-2 decreased, whereas the level of Bax, cleaved-PARP increased in dose-dependent manner compared with non-treatment. Also Bax/Bcl-2 ratio, which is used in clinical indicator of apoptosis, was increased at ID extract treatment group compared with non-treatment. Moreover the Bax/Bcl-2 ratio of MDA-MB-231 cell was significantly increased as against SK-BR-3, AGS cells. These results indicated that ID extract have anti-proliferation effect better than YS or TC, and induced apoptosis in human breast cancer MDA-MB-231 cell better than human breast cancer SK-BR-3 cell, human gastric cancer. Even if further research is needed, ID can be developed as a chemopreventive or therapeutic agent of breast cancer.

Dihydroaustrasulfone alcohol induces apoptosis in nasopharyngeal cancer cells by inducing reactive oxygen species-dependent inactivation of the PI3K/AKT pathway

  • Kok-Tong Tan;Yu-Hung Shih;Jiny Yin Gong;Xiang Zhang;Chiung-Yao Huang;Jui-Hsin Su;Jyh-Horng Sheu;Chi-Chen Lin
    • The Korean Journal of Physiology and Pharmacology
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    • v.27 no.4
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    • pp.383-398
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    • 2023
  • Dihydroaustrasulfone alcohol (DA), the synthetic precursor of a natural compound (austrasulfone) isolated from the coral species Cladiella australis, has shown cytotoxic effects against cancer cells. However, it is unknown whether DA has antitumor effects on nasopharyngeal carcinoma (NPC). In this study, we determined the antitumor effects of DA and investigated its mechanism of action on human NPC cells. The MTT assay was used to determine the cytotoxic effect of DA. Subsequently, apoptosis and reactive oxygen species (ROS) analyses were performed by using flow cytometry. Apoptotic and PI3K/AKT pathway-related protein expression was determined using Western blotting. We found that DA significantly reduced the viability of NPC-39 cells and determined that apoptosis was involved in DA-induced cell death. The activity of caspase-9, caspase-8, caspase-3, and PARP induced by DA suggested caspase-mediated apoptosis in DA-treated NPC-39 cells. Apoptosis-associated proteins (DR4, DR5, FAS) in extrinsic pathways were also elevated by DA. The enhanced expression of proapoptotic Bax and decreased expression of antiapoptotic BCL-2 suggested that DA mediated mitochondrial apoptosis. DA reduced the expression of pPI3K and p-AKT in NPC-39 cells. DA also reduced apoptosis after introducing an active AKT cDNA, indicating that DA could block the PI3K/AKT pathway from being activated. DA increased intracellular ROS, but N-acetylcysteine (NAC), a ROS scavenger, reduced DA-induced cytotoxicity. NAC also reversed the chances in pPI3K/AKT expression and reduced DA-induced apoptosis. These findings suggest that ROS-mediates DA-induced apoptosis and PI3K/AKT signaling inactivation in human NPC cells.

Study on the Protective Effect of EGCG Against the Cytotoxicity Induced by Topical Anesthetic Proparacaine Hydrochloride (점안마취제 성분인 Proparacaine Hydrochloride의 세포독성에 대한 Epigallocatechin-Gallate의 효과)

  • Seo, Eun-Sun
    • Journal of Korean Ophthalmic Optics Society
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    • v.18 no.4
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    • pp.525-531
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    • 2013
  • Purpose: To identify the apoptosis caused by Proparacaine hydrochloride (PPC), a topical anesthesia, applied to conjunctival cell lines and determine whether pigallocatechin-gallate (EGCG), has protective effects on. Methods: The conjunctival cell lines were treated with 0.5% of Alcaine$^{(R)}$, 0.5% of PPC and 0.01% of Benzalkonium chloride (BAC) for 15 minutes, respectively in order to investigate the effects of topical anesthesia on cells, and followed by cultured for 12 and 24 hours. The recovery effects were investigated by measuring level of cellular proliferation inhibiting using MTT assay and LDH assay. The conjunctival cell lines were pre-treated with EGCG $10{\mu}M$ for 3 hrs and post-treated with 0.5% PPC for 15 mins in order to investigate whether EGCG has protective effects, flow cytometry were performed in order to observe apoptosis. Results: A result of the additional culture of 12 and 24 hours and again immediately after the treatment for 15 minutes 0.5% of Alcaine$^{(R)}$, 0.5% of PPC, the 0.01% of BAC, cell viability was not increased in all groups (p<0.05). The cell viabilities were higher than in cells 3 hours post-treated with $10{\mu}M$ of EGCG and pre-treated PPC 0.5% (68.2%), compared to cells ($32.2{\pm}2.0%$) treated only with 0.5% of PPC. PPC 0.5% also induced apoptosis in the treated group was reduced by the addition of EGCG. Conclusions: It is considered that the EGCG has cell protective effects when it is added to PPC, a topical anesthesia, by improving cell viability and inhibiting apoptosis.