• 생명과학회지
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• 제23권7호
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• pp.926-932
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• 2013

글루타민과 혈청은 세포의 생존과 증식에 기본적으로 요구되지만, 그들의 양적 변화에 따른 내피세포 반응에 관한 신호전달 관련 연구는 거의 이루어지지 않았다. 본 연구에서는 인체 재대정맥 내피세포(human umbilical vein endothelial cells, HUVECs)의 증식에 미치는 글루타민과 혈청의 결핍에 관한 영향을 조사하였다. 본 연구의 결과에 의하면 글루타민 및 혈청이 결핍된 조건에서 배양된 HUVECs의 증식 억제는 apoptosis 유발과 연관성이 있었음을 DAPI staining에 의한 핵의 형태 변화와 유세포 분석을 통하여 확인하였다. 비록 혈청이 결핍된 조건보다 글루타민 결핍에 의한 apoptosis 유발 정도가 더 높게 나타났으나, 두 현상에 의한 apoptosis의 유발은 anti-apoptotic Bcl-2 및 Bcl-xL의 발현 저하와 pro-apoptotic Bax의 발현 증가, IAP family 단백질의 발현 감소, caspase의 활성 증가에 따른 PARP 단백질의 단편화와 연관성이 있었다. 또한 이러한 조건에서 HUVECs의 Bid 발현의 감소 또는 tBid 발현의 증가 현상이 관찰되어, 글루타민 또는 혈청 결핍에 의한 HUVECs의 apoptosis 유발은 세포막 수용체 및 미토콘드리아 활성 경로를 동시에 통하여 이루어지고 있음을 알 수 있었다. 그러나 글루타민과 혈청이 동시에 결핍된 조건에서 배양된 HUVECs의 증식 억제 현상은 각각의 조건에 비하여 증가되었으나 apoptosis는 유발되지 않았다.

• Nutrition Research and Practice
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• 제12권2호
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• pp.93-100
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• 2018

BACKGROUND/OBJECTIVE: Oxidative stress plays a key role in neuronal cell damage, which is associated with neurodegenerative disease. The aim of present study was to investigate the neuroprotective effects of perilla oil (PO) and its active component, alpha-linolenic acid (ALA), against hydrogen peroxide $(H_2O_2)$-induced oxidative stress in SH-SY5Y neuronal cells. MATERIALS/METHODS: The SH-SY5Y human neuroblastoma cells exposed to $250{\mu}M$ $H_2O_2$ for 24 h were treated with different concentrations of PO (25, 125, 250 and $500{\mu}g/mL$) and its major fatty acid, ALA (1, 2.5, 5 and $25{\mu}g/mL$). We examined the effects of PO and ALA on $H_2O_2$-induced cell viability, lactate dehydrogenase (LDH) release, and nuclear condensation. Moreover, we determined whether PO and ALA regulated the apoptosis-related protein expressions, such as cleaved-poly ADP ribose polymerase (PARP), cleaved caspase-9 and -3, BCL-2 and BAX. RESULTS: Treatment of $H_2O_2$ resulted in decreased cell viability, increased LDH release, and increase in the nuclei condensation as indicated by Hoechst 33342 staining. However, PO and ALA treatment significantly attenuated the neuronal cell death, indicating that PO and ALA potently blocked the $H_2O_2$-induced neuronal apoptosis. Furthermore, cleaved-PARP, cleaved caspase-9 and -3 activations were significantly decreased in the presence of PO and ALA, and the $H_2O_2$-induced up-regulated BAX/BCL-2 ratio was blocked after treatment with PO and ALA. CONCLUSIONS: PO and its main fatty acid, ALA, exerted the protective activity from neuronal oxidative stress induced by $H_2O_2$. They regulated apoptotic pathway in neuronal cell death by alleviation of BAX/BCL-2 ratio, and down-regulation of cleaved-PARP and cleaved caspase-9 and -3. Although further studies are required to verify the protective mechanisms of PO and ALA from neuronal damage, PO and ALA are the promising agent against oxidative stress-induced apoptotic neuronal cell death.

• 한국식품영양과학회지
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• 제46권7호
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• pp.886-890
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• 2017

상황버섯균사체 쌀의 뇌세포 보호 효과를 분석하여 상황버섯균사체 쌀에 대한 기초자료를 제공하고, 이를 이용하여 뇌 질환의 예방과 관련된 기능성 식품으로의 개발가능성을 찾고자 하였다. 상황버섯균사체 쌀 추출물을 처리하여 마우스 해마세포주 HT22의 세포보호 효과를 알아보기 위해 MTS assay를 이용한 세포생존율과 western blot을 이용한 세포사멸 단백질의 발현을 관찰하였다. 상황버섯균사체 쌀 추출물의 단일 독성을 측정한 결과 상황버섯균사체 쌀 추출물 처리군은 102.68% 이상의 생존율을 보여 신경세포의 사멸에 영향을 주지 않았다. 또한, 상황버섯균사체 쌀 추출물과 $H_2O_2$를 함께 처리하였을 때 $H_2O_2$로 자극된 세포는 63.80%의 세포생존율을 나타내었고, 상황버섯균사체 쌀 추출물과 $H_2O_2$가 함께 처리된 세포는 76.85%, 92.46%, 95.57%로 농도 의존적으로 세포생존율이 증가하였다. HT22 cell에 $H_2O_2$와 상황버섯균사체 쌀 추출물을 처리하여 apoptosis 유도 단백질을 측정한 결과, pro-apoptotic protein인 Bax는 발현이 억제되었고, anti-apoptotic protein인 Bcl-2는 발현이 증가함을 확인하였다. 또한, caspase-3와 PARP의 발현을 억제하여 상황버섯균사체 쌀 추출물이 $H_2O_2$로 유도되는 apoptosis를 억제하는 것을 확인할 수 있었다.

• The Korean Journal of Physiology and Pharmacology
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• 제10권3호
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• pp.111-121
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• 2006

Neuroprotective properties of lithium were evaluated by using in vivo NMDA excitotoxicity model. Systemic injection of NMDA to young mice induced neuronal apoptosis mediated by both TNFR-l and Fas ligand, and long-term lithium treatment showed noticeable neuroprotection against NMDA-induced excitotoxicity: NMDA-damaged neurons expressed several apoptosis-related gene products such as TNFR-l, Fas ligand, and caspase-3, and these gene expressions were not found in the brain of mice chronically treated with lithium. Therefore, it is highly likely that the protection offered by chronic lithium treatment occurred at far upstream of caspase activation, since the chronic lithium treatment increased the expression of Bcl-2, an important antiapoptotic gene known to act upstream of caspase cascade. Timm's histochemistry indicated the complete blockade of the NMDA insults by the treatment. There was no indication of axonal regeneration, which follows synaptic degeneration induced by neuronal damage. Furthermore, this study reports for the first time that TNFR-l and Fas ligand are involved in neuroprotective effects of lithium in NMDA-induced neuronal apoptosis.

• 대한약침학회지
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• 제3권1호
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• pp.1-19
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability and apoptosis were analyzed using MTT assay, tryphan blue assay, $[^3H]$thymidine release assay, flow cytometric analysis, and activity of caspase-3 protease activity assay. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, $[^3H]$thymidine release assay, and flow cytomet1 ric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and Bcl-$X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.22-22
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• 2003

This study was conducted to determine effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and bovine serum albumin (BSA) on blastocoel formation, cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. Embryos were collected from 2-cell or late 4-cell diploid parthenotes that activated with electro pulse, and in vitro cultured in the NCSU 23 medium supplemented without or with 0.1% PVA, 10% FBS or 0.4% BSA for day 7. The morphological analysis of apoptosis in embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expressions of Bcl-xL, Bak and P53 in blastocyst stage parthenotes and in vivo-derived blastocysts were determined using semiquantitative RT-PCR. The addition of 0.4% BSA to the culture medium enhanced the development of 2- or late 4-cell stage parthenotes to the blastocysts stage (P < 0.01) while FBS decreased the incidence of blastocoel formation. FBS also reduced cell numbers of blastocysts developed from both 2- (P < 0.001) and late 4-cell (P < 0.05) embryos and increased percentage of apoptosis in the blastocysts (P < 0.001). The relative abundance of Bcl-xL mRNA in diploid parthenotes cultured from 2-cell stage in the presence of BSA is similar with that in in vivo derived embryos, but is significantly higher than in parthenotes cultured with FBS, PVA or none protein supplement control. Bak mRNA showed a significant increase at the blastocyst stage in FBS supplement medium. This result suggests that apoptosis related gene expression is significantly affected by protein supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

• 한국식품영양과학회지
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• 제41권11호
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• pp.1487-1492
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• 2012

본 연구는 치콘 추출물의 항산화 효능을 확인하고자 물과 에탄올로 추출하였다. 치콘에 포함된 총 폴리페놀 함량을 측정한 결과, 에탄올 추출물($37.3{\pm}5.2$ mg/GAE/g extract)과 열수 추출물($36.3{\pm}1.0$ mg/GAE/g extract)이 유사한 총 폴리페놀 함량을 포함하고 있었으며, 총 플라보노이드 함량은 물 추출물이 $47.0{\pm}3.8$ mg CE/g extract 그리고 에탄올 추출물이 $53.9{\pm}5.1$ mg CE/g extract를 나타내었다. ABTS를 이용한 라디칼 소거활성, FRAP를 이용한 환원력을 통한 항산화 활성을 측정한 결과에서도 치콘 추출물이 항산화 효과를 나타내었다. 한편, 세포 독성을 살펴보기 위하여 신경세포를 이용하여 MTT assay를 수행한 결과, 세포의 생존율은 1.0 mg/mL의 농도 이상에서는 생존에 영향을 미치지 않았고 신경세포 보호효능 실험에서는 2.5 mM의 $H_2O_2$로 유발시킨 산화적 손상에 대해 농도 의존적인 신경세포 보호 효과가 있었으며, 항산화 효소 활성을 SOD와 CAT로 분석한 결과 SOD는 0.5 mg/mL 농도에서 95% 이상의 활성과 CAT는 손상그룹에 비해 2배 이상의 활성을 각각 확인하였다. 또한 치콘 추출물이 세포사와 관련이 있는 단백질인 Bax와 Bcl-2의 발현을 조절하는 것을 확인하였다. 이와 같은 결과는 치콘 추출물이 산화적 손상의 억제를 통해 세포를 보호하는 효과가 있는 것으로 사료된다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10407-10412
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• 2015

Background: ${\beta}$-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of ${\beta}$-elemene against glioma cells remains unclear. In the present study, we assessed effects of ${\beta}$-elemene on human glioma cells and explored the underlying mechanism. Materials and Methods: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of ${\beta}$-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of ${\beta}$-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and ${\beta}$-elemne treatment group. Results: With increase in the concentration of ${\beta}$-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability ($IC_{50}$) was $48.5{\mu}g/mL$ for 24h. ${\beta}$-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with ${\beta}$-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by ${\beta}$-elemene in a time and does-dependent manner. Conclusions: Our results indicate that ${\beta}$-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.

• 생명과학회지
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• 제18권11호
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• pp.1499-1506
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• 2008

식용 및 약용으로 이용되는 산초(Zanthoxylum schinifolium)의 줄기로부터 항암활성 성분을 분리하기 위하여, 산초 줄기를 유기용매로 추출하고 각 추출물의 인체 급성백혈병 암세포에 대한 독성 및 세포자살 유도 활성을 조사하였다. Methanol (SS-7), methylene chloride (SS-8), ethyl acetate (SS-9), n-butanol (SS-10)로 추출한 각 시료와 유기용매 추출 후 잔여분획 (SL-14)의 세포 독성을 인체 급성백혈병 Jurkat T 세포주를 대상으로 조사한 결과, 암세포에 대한 세포독성이 주로 methylene chloride 추출분획인 (SS-8)에서 확인되었다. Methylene chloride 추출물 (SS-8)의 Jurkat T 세포주에 대한 세포독성의 기전은 mitochondria로부터cytochrome c 방출, caspase-9 및 caspase-3의 활성화, PARP 분해, internucleosomal DNA fragmentation 등의 일련의 생화학적 반응을 수반하며, 항 세포자살단백질인 Bcl-xL단백질의 과발현에 의해 억제되는 세포자살 기전임을 확인하였다. FADD가 disruption된 Jurkat T cell clone I2.1 ($FADD^{-/-}$) 및 caspase-8가 결핍된 Jurkat T cell clone I9.2 (caspase-$8^{-/-}$)와 함께 the wild-type Jurkat T cell clone A3에 미치는 SS-8의 세포독성작용을 비교 분석한 결과, wild-type Jurkat A3, FADD-deficient Jurkat clone I2.1및 caspase-8-deficient Jurkat clone I9.2 모두는 SS-8의 세포독성에 대해 유사한 정도의 감수성을 나타내었다. 이는 SS-8에 의해 유도되는 apoptosis에 있어서, Fas/FasL system이 관계되지 않음을 시사한다. 한편, SS-8를 GC-MS 분석하여, 9,12-octadecanoic acid (18.62%), 2,4-dihydro-5-methyl-4-(1-methylethylidene)-2-(4-nitrophenyl)- 3H-pyrazol-3-one (14.97%), hexadecanoic acid (14.23%), (z,z)-6,9-pentadecadien-1-ol (13.73%), 5,6-dimethoxy- 2-methyl benzofuran (10.95%), 그리고 4-methoxy-2-methylcinnamic acid (5.38%) 등을 포함한 16가지의 구성 성분과 그 조성비를 확인하였다. 이상의 연구결과는 산초 줄기에 함유된 항암 활성에 대한 규명과 이해를 증진시킨다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권16호
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• pp.7103-7110
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• 2015

The present study was conducted to investigate the effect of ${\gamma}$-radiation alone or combined with a cytotoxic drug, simvastatin, on viability and cell cycling of a myeloma cell line. P3NS1 myeloma cells were treated with the selected dose of simvastatin ($0.1{\mu}M/l$) 24 hours prior to ${\gamma}$-irradiation (0.25, 0.5 and 1Gy). The cell viability, induction of apoptosis, cell death, cell cycling, generation of ROS, and expression of P53, Bax, Bcl2, caspase3, PARP1 and Fas genes were estimated. The results indicated that simvastatin ($0.1{\mu}M/l$) treatment for 24 hours prior to ${\gamma}$-irradiation increased cell death to 37.5% as compared to 4.81% by radiation (0.5Gy) alone. It was found that simvastatin treatment before irradiation caused arrest of cells in G0/G1 and G2/M phases as assessed using flow cytometry. Interestingly, simvastatin treatment of P3NS1 cells increased the intracellular ROS production and decreased antioxidant enzyme activity with increased P53, Bax and Caspase3 gene expression while that of Bcl2 was decreased. Consequently, our results indicated that pre-treatment with simvastatin increased radio sensitivity of myeloma tumor cells in addition to apoptotic effects through an intrinsic mitochondrial pathway.