• 한국수정란이식학회지
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• 제27권2호
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Asian-Australasian Journal of Animal Sciences
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• 제27권5호
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• pp.635-647
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• 2014

Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; $44h+10{\mu}M$ rapamycin/24 h, $47.52{\pm}5.68$) or control oocytes (44 h IVM; $42.14{\pm}4.40$) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, $22.04{\pm}5.68$) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

• BMB Reports
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• 제45권9호
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• pp.532-537
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• 2012

P18, a member of the INK4 family of cyclin-dependent kinase inhibitors, is a tumor suppressor protein and plays a key cell survival role in a variety of human cancers. Under pathophysiological conditions, the INK4 group proteins participate in novel biological functions associated with neuronal diseases and oxidative stress. Parkinson's disease (PD) is characterized by loss of dopaminergic neurons, and oxidative stress is important in its pathogenesis. Therefore, we examined the effects of PEP-1-p18 on oxidative stress-induced SH-SY5Y cells and in a PD mouse model. The transduced PEP-1-p18 markedly inhibited 1-methyl-4-phenyl pyridinium-induced SH-SY5Y cell death by inhibiting Bax expression levels and DNA fragmentation. Additionally, PEP-1-p18 prevented dopaminergic neuronal cell death in the substantia nigra of a 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine-induced PD mouse model. These results indicate that PEP-1-p18 may be a useful therapeutic agent against various diseases and is a potential tool for treating PD.

• BMB Reports
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• 제42권7호
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• pp.444-449
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• 2009

Different neurodegenerative disorders like prion disease, is caused by protein misfolding conformers. Reverse-transfected cytosolic prion protein (PrP) and PrP expressed in the cytosol have been shown to be neurotoxic. To investigate the possible mechanism of neurotoxicity due to accumulation of PrP in cytosol, a PrP mutant lacking the signal and GPI (CytoPrP) was introduced into the SH-SY5Y cell. MTT and trypan blue assays indicated that the viability of cells expressing CytoPrP was remarkably reduced after treatment of MG-132. Obvious apoptosis phenomena were detected in the cells accumulated with CytoPrP, including loss of mitochondrial transmembrane potential, increase of caspase-3 activity, more annexin V/PI-double positive-stained cells and reduced Bcl-2 level. Moreover, DNA fragmentation and TUNEL assays also revealed clear evidences of late apoptosis in the cells accumulated CytoPrP. These data suggest that the accumulation of CytoPrP in cytoplasm may trigger cell apoptosis, in which mitochondrial relative apoptosis pathway seems to play critical role.

• 생명과학회지
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• 제25권9호
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• pp.1051-1058
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• 2015

본 연구에 평가된 감귤 돌연변이는 기존 궁천조생 품종에서 유래된 방사선 돌연변이체로 선행 연구에서 다양한 생리활성 및 기능성 성분이 함유되어 있다는 것을 보고한 이래로, 감귤 돌연변이의 항암 제제로써 활용 가능성을 검증하였다. 여러 암종(HCT116, 인간대장암세포; A375, 인간흑색종세포; Hela, 인간자궁경부암세포; MCF-7, 인간유방암세포; A549, 인간폐암세포; HepG2, 인간간암세포)을 대상으로 감귤 돌연변이의 항암 활성을 기존 궁천조생 품종(방사선 무처치군)과 비교 분석한 결과 그 효용 가치가 높게 나타났을 뿐 아니라 자연사멸 유도 메커니즘을 탐색하였을 때 감귤 돌연변이에 의해 야기된 apoptosis에 IAP family의 발현 저해와 NO^• pathway 증강에 따라 세포 고사가 촉진됨을 확인할 수 있었다. 또한 감귤 돌연변이가 정상 세포에 대한 독성을 유발하지 않는 것으로 나타나, 암세포 선택성이 높은 chemopreventive 및 chemotherapeutic agent로 개발 가능할 것으로 예상되며 이를 위해 향후 보다 정밀한 자연사멸의 분자생물학적 기전 규명이 수반되어야 할 것으로 사료된다.

• 대한미생물학회지
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• 제34권6호
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• pp.501-512
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• 1999

Gliotoxin, a fungal metabolite, is one of the epipolythiodioxopiperazine classes and has a variety of effects including immunomodulatory and apoptotic agents. This study is designed to evaluate the effect of zinc on gliotoxin-induced death of HL-60 cells. Here, we demonstrated that treatment of gliotoxin decreased cell viability in a dose and time-dependent manner. Gliotoxin-induced cell death was confirmed as apoptosis characterized by chromatin margination, fragmentation and ladder-pattern digestion of genomic DNA. Gliotoxin increased the proteolytic activities of caspase 3, 6, 8, and 9. Caspase-3 activation was further confirmed by the degradation of procaspase-3 and PARP in gliotoxin-treated HL-60 cells. Zinc compounds including $ZnCl_2$ and $ZnSO_4$ markedly inhibited gliotoxin-induced apoptosis in HL-60 cells (from 30% to 90%). Consistent with anti-apoptotic effects, zinc also suppressed the enzymatic activities of caspase-3 and -9 proteases. In addition, cleavage of both PARP and procaspase 3 in gliotoxin-treated HL-60 cells was inhibited by the addition of zinc compounds. We further demonstrated that expression of Fas ligand by gliotoxin was suppressed by zinc compounds. These data suggest that zinc may prevent gliotoxin-induced apoptosis via inhibition of Fas ligand expression as well as suppression of caspase family cysteine proteases-3 and -9 in HL-60 cells.

• 대한한방내과학회지
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• 제21권2호
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• pp.259-266
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• 2000

Objectives : The effects of aqueous extracts of Cortex ulmi pumilae (a traditional medicine for cancer treatment in oriental medicine) on the induction of apoptotic cell death were investigated in human liver origm hepatoma cell lines, HepG2. Methods : The death of HepG2 cells was markedly induced by the addition of extracts of Cortex ulmi pumilae in a dose-dependent manner. The apoptotic characteristic ladder pattern of DNA strand break was not observed in cell death of HepG2. In addition, it was not shown nucleus chromatin condensation and fragmentation under hoechst staining. However, by the using annexin V staining assay, externalizations of phosphatidylserine in HepG2 cell which were treated with Cortex ulmi pumilae extracts were detected in the early time (at 9 hr after extract treatment). Furthermore, LDH release was not detected in this early stage. Therefore, Cortex ulmi pumilae extracts-induced cell death of HepG2 cells is mediated by apoptotic death signal processes. Result : The activity of caspase 3-like proteases remained in a basal level in HepG2 cells which treated with the extract of Cordyceps sinensis. However, it was markedly increased in HepG2 cells which treated with two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) which were differently extracted (respectively, 2.3 and 3.3 fold). On a while, the phosphotransferase activities of JNK1 was markedly induced in HepG2 cells which were treated with two extracts of Cortex ulmi pumilae. On the contrary, the activation of transcriptional activator, activating protein1(AP-1) and NF-kB were severely decreased by these two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K). In addition, antioxidants (GSH and NAC) and intracellular $Ca2^+$ level regulator (Bapta/AM and Thapsigargin) did not affect Cortex ulmi pumilae extracts-induced apoptotic death of HepG2 cells. Conclusions : In conclusion, our results suggest that two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) induces the apoptotic death of human liver origin hepatoma HepG2 cells via activation of caspase 3-like proteases as well as JNK1, and inhibition of transcriptional activators, AP-1 and $NK-{\kappa}B$.

• 동의생리병리학회지
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• 제17권6호
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• pp.1383-1392
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• 2003

Apoptosis is a morphologically and biochemically district form of cell death that occurs in many different cell types in a wide variety of organisms. Albizzia julibrissin belonging the family Leguminosae has been used for the treatment of contusion, sore throat, amnesia, and insomnia in oriental traditional medicine. This study investigates whether the water extract of A. julibrissin induce apoptotic cell death in Jurkat T-acute lymphoblastic leukemia (ALL) cells. Jurkat cells were increased inhibitions of cell viability in a concentration-dependent manner by A. julibrissin. This herbal medicine also caused apoptosis as measured by cell morphology and DNA fragmentation. The capability of A. julibrissin to induce apoptosis was associated with proteolytic cleavage of specific target proteins such as poly (ADP-ribose)polymerase (PARP) and beta-catenin proteins suggesting the possible involvement of caspases. Our result showed that Bcl-2 and Bax protein levels were not changed in all A. julibrissin-treated groups compared to control group. These results suggest that A. julibrissin-mediated apoptosis is independent with Bcl-2 related signaling pathway in this cells. The purpose of the present study is also to investigate the Effect of A. julibrissin on cell cycle progression. Our results showed that G1 checkpoint related gene products (cyclin D1, cyclin dependent kinase 4, retinoblastoma, E2F1) were decreased in their protein levels in a dose-dependent manners after treatment of the extract. These results indicate that the increase of apoptotic cell death by A. julibrissin may be due to the inhibition of cell cycle progression in wild type p53-lacking Jurkat cells.

• 동의생리병리학회지
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• 제18권4호
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• pp.1055-1060
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• 2004

Apoptosis is the ability of cells to self-destruct by the activation of an intrinsic cellular suicide program when the cells are no longer needed or when they are seriously damaged. Morphologically, apoptosis is characterized by the appearance of membrane blebbing, cell shrinkage, chromatin condensation, DNA cleavage, and the fragmentation of the cell membrane-bound apoptotic bodies. Siegesbeckia glabrescens Makino (Siegesbeckiae Herba, SG) has been widely used as treatments for arthritis, and fever, as well as detoxification properties. The present studies were undertaken to evaluate if SG has an anti-apoptotic property. Cell viability was measured by XTT and tryphan blue stain. Morphological characteristic of human aortic smooth muscle cells(HASMC) were visualized with a phase-contrast microscope. SG significantly reduced HASMC, but not human umbilical vein endothelial cell(HUVEC), viability in a dose-dependent manner. Confluent untreated cells at 24hrs showed normal morphology, flat with a uniform polygonal shape. SG-treated cells (0.5㎎/㎖) at 24hrs showed apoptotic morphology. Cells became irregular with elongated lamellipodia, and exhibited condensed chromatin in nuclei with occasional endoucleation. There was an increase in the number of apoptotic cells rounding-up and being detached from the substrate. TUNEL staining of SG-treated cells showed dark-brown stains in nuclei and cytosol. Caspases are central components of the machinery responsible for apoptosis and are generally divided into two categories; the initiator caspases, which include caspases-2,-8,-9, and -10, and the effector caspases, which include caspases-3,-6, and -7. SG decreased anti-caspase-3 protein expression, which means activation of caspases-3 activity. It has been reported that there is a link between NO formation and apoptosis. NO production was accelerated by SG treatment in HASMC. L-NNA, NOS inhibitor, inhibited SG-induced apoptosis. These results, therefore, indicated that both caspases-3 and NO production are involved in apoptosis in smooth muscle cells. According to these results, SG may have a potential effect in the treatment of hypertensive atherosclerosis.

• Biomolecules & Therapeutics
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• 제28권6호
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• pp.561-568
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• 2020

We examined the anticancer effects of a novel sirtuin inhibitor, MHY2256, on HCT116 human colorectal cancer cells to investigate its underlying molecular mechanisms. MHY2256 significantly suppressed the activity of sirtuin 1 and expression levels of sirtuin 1/2 and stimulated acetylation of forkhead box O1, which is a target protein of sirtuin 1. Treatment with MHY2256 inhibited the growth of the HCT116 (TP53 wild-type), HT-29 (TP53 mutant), and DLD-1 (TP53 mutant) human colorectal cancer cell lines. In addition, MHY2256 induced G0/G1 phase arrest of the cell cycle progression, which was accompanied by the reduction of cyclin D1 and cyclin E and the decrease of cyclin-dependent kinase 2, cyclin-dependent kinase 4, cyclin-dependent kinase 6, phosphorylated retinoblastoma protein, and E2F transcription factor 1. Apoptosis induction was shown by DNA fragmentation and increase in late apoptosis, which were detected using flow cytometric analysis. MHY2256 downregulated expression levels of procaspase-8, -9, and -3 and led to subsequent poly(ADP-ribose) polymerase cleavage. MHY2256-induced apoptosis was involved in the activation of caspase-8, -9, and -3 and was prevented by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, the autophagic effects of MHY2256 were observed as cytoplasmic vacuolation, green fluorescent protein-light-chain 3 punctate dots, accumulation of acidic vesicular organelles, and upregulated expression level of light-chain 3-II. Taken together, these results suggest that MHY2256 could be a potential novel sirtuin inhibitor for the chemoprevention or treatment of colorectal cancer or both.