• Journal of Microbiology and Biotechnology
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• 제24권4호
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• pp.568-576
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• 2014

TIM-1 (also known as KIM-1 and HAVcr-1) is a type I transmembrane glycoprotein member of the TIM family that may play important roles in innate and adaptive immune responses. The overexpression of proteins associated with membrane proteins is a major obstacle to overcome in studies of membrane protein structures and functions. In this study, we successfully coupled the overexpression of the TIM-1 protein with a C-terminal enhanced green fluorescent protein (GFP) tag in Escherichia coli. To the best of our knowledge, this report is the first to describe the overexpression of human TIM-1 in E. coli. The purified TIM-1-EGFP fusion protein recognized and bound directly to apoptotic cells and did not to bind to viable cells. Furthermore, we confirmed that the interactions of TIM-1-EGFP with apoptotic cells were blocked by TIM-1-Fc fusion proteins. This fusion protein represents a readily obtainable source of biologically active TIM-1 that may prove useful in future studies of human TIM-1.

• 약학회지
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• 제58권1호
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• pp.33-39
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• 2014

The objective of this study is to evaluate the anticancer effects of the isoflavone extract from Chungkukjang in human breast cancer, MDA-MB-453 cells. For this study, MDA-MB-453 cells were treated with 12.5, 25, and $50{\mu}g$ isoflavone extract for 24, 48, and 72 hr. Cell proliferations were decreased in a time- and dose-dependent manner. Reduced cell proliferation was suspected by apoptosis or cell cycle arrest. Therefore, after treatment of $50{\mu}g$ isoflavone extract, apoptotic cells were investigated by annexin V staining. The results indicated that isoflavone extract increased the number of early apoptotic cells compared with control. Cleaved PARP was also increased. Next, we investigated the cell cycle and related proteins. The isoflavone extract leads to cell cycle arrest at the G2/M phase. Moreover isoflavone extract had influenced cell cycle relate proteins such as cyclin B1, cyclin A, and p21. These results suggest that isoflavone extract from Chungkukjang induce apoptosis and cell cycle arrest at G2/M phase via regulation of cell cycle-related proteins in MDA-MB-453 cells.

• Journal of Microbiology and Biotechnology
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• 제32권8호
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• pp.1034-1040
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• 2022

Fas-associated death domain (FADD) is an adapter molecule that bridges the interaction between receptor-interacting protein 1 (RIP1) and aspartate-specific cysteine protease-8 (caspase-8). As the primary mediator of apoptotic cell death, caspase-8 has two N-terminal death-effector domains (DEDs) and it interacts with other proteins in the DED subfamily through several conserved residues. In the tumor necrosis receptor-1 (TNFR-1)-dependent signaling pathway, apoptosis is triggered by the caspase-8/FADD complex by stimulating receptor internalization. However, the molecular mechanism of complex formation by the DED proteins remains poorly understood. Here, we found that direct DED-DED interaction between FADD and caspase-8 and the structure-based mutations (Y8D/I128A, E12A/I128A, E12R/I128A, K39A/I128A, K39D/I128A, F122A/I128A, and L123A/I128A) of caspase-8 disrupted formation of the stable DED complex with FADD. Moreover, the monomeric crystal structure of the caspase-8 DEDs (F122A/I128A) was solved at 1.7 Å. This study will provide new insight into the interaction mechanism and structural characteristics between FADD and caspase-8 DED subfamily proteins.

• Journal of Acupuncture Research
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• 제31권2호
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• pp.87-98
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• 2014

Objectives : We investigated whether snake venom toxin(SVT) from Vipera lebetina turanica sensitizes HT29 human epithelial colorectal cancer cells to tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) induced apoptosis in cancer cells. Methods : Cell viability assay was used to assess the inhibitory effect of TRAIL on cell growth of HT29 human colorectal cancer cells. And 6-diamidino-2-phenylindole(DAPI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay(TUNEL) staining assay were used to evaluate cell-apoptosis. Western blot analysis were conducted to observe apoptosis related proteins and death receptor. To assess whether the synergized inhibitory effect of SVT and TRAIL on reactive oxygen species(ROS) generation was reversed by strong anti-oxidative agent. Results : SVT with TRAIL inhibited HT29 cell growth different from TRAIL alone. Consistent with cell growth inhibition, the expression of TRAIL receptors; Expression of death receptor(DR)4 and DR5 was significantly increased and intrinsic pro-apoptotic cleaved caspase-3, -9 was subsequently increased together with increase of Bax/Bcl-2 ratio and extrinsic pro-apototic caspase-8 was also activated. In addition, the expression of anti-apoptotic survival proteins, a marker of TRAIL resistance(eg, cFLIP, survivin, X-linked inhibitor of apoptosis protein(XIAP) and Bcl-2) was suppressed by the combination treatment of SVT and TRAIL. Pretreatment with the ROS scavenger N-acetylcysteine abolished the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and expression of the intrinsic pro-apoptotic caspase-3 and-9. Conclusion : The collective results suggest that SVT facilitates TRAIL-induced apoptosis in $HT_{29}$ human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.

• Clinical and Experimental Reproductive Medicine
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• 제49권2호
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• pp.93-100
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• 2022

Objective: The impact of imatinib, a tyrosine kinase inhibitor, on ovarian follicles and several proteins related to follicular function and apoptosis was investigated in mice. Methods: Saline, cyclophosphamide (Cp; 50 or 75 mg/kg), or imatinib (7.5 or 15 mg/kg) was injected once intraperitoneally into female B6D2F1 mice (18 mice in each group). In multiple ovarian sections, the number of various types of follicles and the proportion of good-quality (G1) follicles were counted. The levels of six proteins (anti-Müllerian hormone [AMH], BCL-xL, BAX, acid sphingomyelinase [A-SMase], caspase-3, and α-smooth muscle actin [α-SMA]) within the whole ovaries were quantified using Western blots. Results: Compared to the saline group, a significant reduction of the primordial follicle count was observed in the group treated with imatinib 7.5 and 15 mg/kg, as well as in the group treated with Cp 75 mg/kg. Administration of Cp significantly decreased the proportion of G1 primordial follicles, but administration of imatinib did not. No differences in the AMH, anti-apoptotic BCLX-L, pro-apoptotic BAX, and A-SMase levels in the ovarian tissues were observed among the five groups. However, caspase-3 and α-SMA levels were significantly higher in the imatinib and Cp groups than in the saline group. Conclusion: The administration of imatinib to mice significantly reduced the primordial follicle count and increased the protein levels of caspase-3 and α-SMA. Our findings suggest that imatinib potentially exerts ovarian toxicity via apoptotic processes, similarly to Cp.

• Journal of Animal Science and Technology
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• 제64권1호
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• pp.110-122
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• 2022

The reduction of milk yield caused by heat stress in summer is the main condition restricting the economic benefits of dairy farms. To examine the impact of hyperthermia on bovine mammary epithelial (MAC-T) cells, we incubated the MAC-T cells at thermal-neutral (37℃, CON group) and hyperthermic (42℃, HS group) temperatures for 6 h. Subsequently, the cell viability and apoptotic rate of MAC-T cells, apoptosis-related genes expression, casein and amino acid transporter genes, and the expression of the apoptosis-related proteins were examined. Compared with the CON group, hyperthermia significantly decreased the cell viability (p < 0.05) and elevated the apoptotic rate (p < 0.05) of MAC-T cells. Moreover, the expression of heat shock protein (HSP)70, HSP90B1, Bcl-2-associated X protein (BAX), Caspase-9, and Caspase-3 genes was upregulated (p < 0.05). The expression of HSP70 and BAX (pro-apoptotic) proteins was upregulated (p < 0.05) while that of B-cell lymphoma (BCL)2 (antiapoptotic) protein was downregulated (p < 0.05) by hyperthermia. Decreased mRNA expression of mechanistic target of rapamycin (mTOR) signaling pathway-related genes, amino acid transporter genes (SLC7A5, SLC38A3, SLC38A2, and SLC38A9), and casein genes (CSNS1, CSN2, and CSN3) was found in the heat stress (HS) group (p < 0.05) in contrast with the CON group. These findings illustrated that hyperthermia promoted cell apoptosis and reduced the transport of amino acids into cells, which inhibited the milk proteins synthesis in MAC-T cells.

• 생명과학회지
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• 제25권1호
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• pp.93-100
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• 2015

Pachymic acid는 복령에서 분리된 lanostane-type인 triterpenoid의 일종이다. 최근 pachymic acid가 항암 및 항염증 효능과 산화적 스트레스에 대한 항산화 효능 등과 같은 약리적인 효능이 있는 것으로 밝혀지고 있으나, 그에 대한 구체적인 분자생물학적 기전 연구는 매우 미비한 실정이다. 본 연구에서는 pachymic acid의 항암활성 및 관련 기전 조사의 일환으로 T24 인체 방광암세포 모델을 이용하여 pachymic acid에 의한 apoptosis 유발 여부를 검증하였다. 본 연구의 결과에 의하면 pachymic acid는 T24 세포의 증식을 유의적으로 억제시켰으며, 이는 apoptosis 유발과 연관성이 있음을 다양한 방법으로 확인하였다. Pachymic acid에 의한 apoptosis 유도에는 pro-apoptotic 인자들의 발현 증가와 anti-apoptotic 유전자 산물들의 발현 감소가 동반되었으며, MMP의 소실과 tBid의 발현 증가가 관찰되었다. 아울러 pachymic acid는 extrinsic 및 intrinsic apoptosis 경로의 개시에 관여하는 caspase-8 및 -9의 활성뿐 만 아니라, caspase-3의 활성도 증가시킴으로서 PARP와 같은 기질 단백질의 단편화를 초래하였다. 따라서 pachymic acid는 항암활성을 지니는 천연생리활성 물질로서의 잠재력이 매우 높음을 알 수 있었다.

• 생명과학회지
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• 제26권8호
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• pp.887-894
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• 2016

개똥쑥 추출물(AAE)은 암에 효과적인 약초로 알려져 있다. Apoptosis는 프로그램화된 세포사멸로 미토콘드리아는 세포사멸 조절에 중요한 역할을 한다. 이 연구는 A549 폐암세포에서 Bcl-2 하위조절과 미토콘드리아 경로를 통한 AAE의 p53 비의존적인 세포사멸을 보여주고 있다. AAE는 p-Akt, cox-2, p53 그리고 미토콘드리아 조절 단백질을 통해 암세포의 사멸을 촉진한다. p-Akt/cox-2 단백질은 세포 증식과 생존에 중요한 역할을 한다고 알려져 있다. Bax, Bak, Bim과 같이 세포사멸을 촉진하는 Bcl-2 단백질은 미토콘드리아 외막의 투과성을 조절한다. AAE의 처리는 p-Akt, p-Mdm2, cox-2 그리고 anti-apoptotic 단백질과 같이 세포사멸을 억제하는 단백질들의 발현을 감소시키는 반면에 암 억제자인 p53과 pro-apoptotic 단백질들을 증가시킨다. Bax/Bak의 활성화는 caspase를 활성화시키기 위해 cytochrome c를 미토콘드리아에서 세포질로 방출하도록 한다. Caspase-3는 apoptosis 과정과 관련된 주요 effector caspase이다. Caspase-3는 일반적으로 pro-enzyme형태로 세포질에 존재한다. Apoptosis의 개시단계에서 caspase-3는 proteolytic cleavage에 의해 활성화되고 활성화된 caspase-3는 PARP를 분해한다. Apoptosis와 관련된 단백질들의 신호전달 사이의 상관관계를 알기 위해 Pifithrin-α (p53 inhibitor)와 Celecoxib (cox-2 inhibitor)을 처리했다. 이러한 결과를 통해 A549 폐암 세포에 AAE를 처리하였을 때 p53-independent 경로를 통해 apoptosis가 유도된다는 것을 확인하였다.

• 생명과학회지
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• 제29권3호
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• pp.318-324
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• 2019

두경부암은 전세계에서 발병률이 여섯 번째로 높은 암으로 그동안 수술적 치료를 선호하였으나 광범위한 절제에 따른 기능적 장애로 인해 항암치료에 대한 관심이 높아지고 있다. 두경부암에서 cisplatin이 가장 많이 사용되는 항암제이나 cisplatin 내성이 문제가 되고 있다. 따라서 부작용은 줄이고, 약제내성 기전에 대해 이해하여 암세포의 사멸은 증대시키는 새로운 항암제의 개발이 필요하다. Survivin은 inhibitor of apoptosis proteins (IAPs) family 중 하나로 두경부암에서 과발현되어 있다. YM155는 survivin을 억제하는 분자로 본 연구를 통해 YM155의 처리 후 두경부 암세포의 세포자멸사가 유도되며, 뇌암 세포와 신장암 세포에서도 세포자멸사가 유도됨을 확인할 수 있었다. 반면에 정상세포인 mesangial cells에는 YM155가 세포자멸사에 영향을 주지 않았다. YM155는 caspase의 활성화를 통해 세포자멸사를 촉진하며, anti-apoptotic protein인 c-FLIP, Mcl-1, survivin의 발현을 저해하는 것으로 확인되었다. YM155는 두경부 뿐만 아니라 다른 장기의 악성종양 치료법의 개발에 활용 될 수 있을 것으로 생각된다.

• Reproductive and Developmental Biology
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• 제38권4호
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• pp.165-170
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• 2014

Cows may suffer impaired ovarian function, often accompanied by reduced conception rates and increased embryonic loss. Cystic ovarian disease (COD) is one of the most frequently diagnosed gynecological findings in dairy cattle. It causes temporary infertility and is likely to affect reproduction as well as production parameters in cattle. Therefore, the purpose of this study was to determine the expression patterns of apoptosis (Bcl-2, Bax), implantation (E-cadherin) and immune related proteins (TNF-${\alpha}$, IL-10) in uterine endometrium of Hanwoo (Korean native cattle) with ovarian cyst and normal ovarian follicles. In the Western blot analysis, the expression of anti-apoptotic Bcl-2 protein was significantly higher in endometrium with normal ovarian follicles, whereas expression of pro-apoptotic Bax protein was significantly lower. Also, the expressions of E-cadherin and TNF-${\alpha}$ proteins were significantly higher in uterine endometrium with normal ovarian follicles. On the other hand, the expression of IL-10 protein was significantly lower in uterine endometrium with normal ovarian follicles. Taken together, our results provided that the expressions of apoptosis, adhesion and immune related proteins in uterine endometrium with ovarian cyst were showed the aberrant patterns, and we suggest that different expression changes of these proteins may be affect to pregnancy ability of cattle.