• 한국자원식물학회지
• /
• 제32권4호
• /
• pp.255-263
• /
• 2019

본 연구에서는 다양한 약리학적 활성을 가지는 것으로 알려진 감초 열수추출물(GRW)의 항암효능을 알아보기 위하여 인체 방광암 T24 세포에서 생존율 및 증식억제에 미치는 영향과 이와 연관된 apoptosis 유발 여부 및 관련 인자들의 발현 변화를 조사하였다. 먼저 GRW의 처리에 따른 증식억제 정도를 조사한 결과, GRW 처리 농도 의존적으로 생존율 및 증식억제 현상이 나타났으며, 핵의 형태 변화, DNA 단편화 및 apoptosis 유발에 관하여 조사한 결과 역시 GRW 처리 농도 의존적으로 증가됨을 확인할 수 있었다. 이는 GRW의 처리에 의한 암세포의 증식억제 및 형태적 변형이 암세포의 apoptosis 유발과 밀접한 관련이 있음을 시사하여 주는 것으로 사료된다. GRW 처리에 의한 apoptosis 유발에 관여하는 유전자의 탐색을 위하여 apoptosis와 연관성을 가지는 Bcl-2 family에 속하는 유전자의 발현을 조사한 결과 GRW 처리 농도 의존적으로 Bax 단백질의 발현증가와 더불어 Bcl-2 및 Bcl-xL 단백질의 발현감소가 관찰되었다(Fig. 3A). 이는 GRW에 의한 T24 세포의 apoptosis 유발에 Bcl-2 family에 속하는 유전자의 발현 조절이 중요한 역할을 하는 것으로 사료된다. 또한 GRW의 처리에 따른 MMP의 소실은 미트콘드리아 막의 교란이 유발되었음을 의미하는 것으로, 이러한 MMP 값의 변동은 Bcl-2 family 단백질의 발현 변화에 의한 것이라 추정된다. 한편 Apoptosis에 중요한 역할을 하는 것으로 알려진 caspase(-3/-8/-9)의 발현과 이들의 활성을 억제하는 IAP family (XIAP, cIAP-1, cIAP-2)의 발현에 GRW이 어떠한 영향을 미치는지를 조사한 결과, caspase-3, -8 및 -9의 활성형 단백질 발현 및 정량적 활성증가를 확인하였으며, IAP family 속한 3가지 단백질 모두 발현이 감소하는 것이 관찰되었다. 이상의 결과에서 GRW은 외인적 및 내인적 경로의 개시에 핵심적인 역할을 하는 caspase-8 및 -9의 활성을 모두 증가시켰으며, 이에 따른 caspase-3의 활성증가에 의하여 apoptosis가 유발되었음을 알 수 있었다. 이러한 두 경로의 동시 활성화에는 미트콘드리아의 기능 소실과 Bcl-2 및 IAP family의 발현 변화가 관여하고 있었으며, 특히 Bid의 발현 감소는 GRW에 의한 내인적 경로를 증폭시키는 효과로 작용했을 것이라 추정된다. 방광암의 치료에 보다 효과적인 생리활성을 갖는 물질을 발굴하고 그와 관련된 분자 및 세포수준에서의 기전을 밝히는 것이 중요하기에 본 연구의 결과는 향후 GRW로 수행될 추가 실험을 위한 기초자료로서 그 가치가 매우 높을 것으로 사료된다.

• Journal of Microbiology and Biotechnology
• /
• 제28권1호
• /
• pp.115-121
• /
• 2018

Upon sensing of microbial infections or endogenous danger signals in macrophages, inflammasome signaling plays a significant role in triggering inflammatory responses via producing interleukin (IL)-$1{\beta}$. Recent studies revealed that active caspase-1, a product of the inflammasome complex, causes maturation of inactive pro-IL-$1{\beta}$ into the active form. However, the underlying mechanism by which this leaderless cytokine is secreted into the extracellular space remains to be elucidated. In this study, we demonstrated that prolonged lipopolysaccharide (LPS) treatment to macrophages could trigger the unexpected maturation and extracellular release of IL-$1{\beta}$ through a nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3)-independent manner. Short-term treatment (less than 6 h) of LPS induced robust production of the IL-$1{\beta}$ precursor form inside cells but did not promote the maturation and secretion of IL-$1{\beta}$ in bone marrow-derived macrophages or peritoneal macrophages. Instead, prolonged LPS treatment (more than 12 h) led to a significant release of matured IL-$1{\beta}$ with no robust indication of caspase-1 activation. Intriguingly, this LPS-triggered secretion of IL-$1{\beta}$ was also observed in NLRP3-deficient macrophages. In addition, this unexpected IL-$1{\beta}$ release was only partially impaired by a caspase-1 and NLRP3 inflammasome inhibitor. Collectively, our results propose that prolonged exposure to LPS is able to drive the maturation and secretion of IL-$1{\beta}$ in an NLRP3 inflammasome-independent manner.

• Clinical and Experimental Pediatrics
• /
• 제51권2호
• /
• pp.170-180
• /
• 2008

목 적 : 몇몇 항생제가 저산소성 허혈성 뇌 손상에서 뇌 보호 효과를 가진 것으로 밝혀졌지만 아직까지 그 기전에 대해 잘 알려지지 않고 있다. 최근 아미노글루코사이드 계열의 항생제인 G418(geneticin)이 항고사에 의한 암세포 생존을 증가 시키는 것으로 알려졌으며 본 연구는 G418이 주산기 저산소성 허혈성 뇌손상에서 세포 고사를 억제함으로서 뇌 보호 효과를 나타내는지를 알아보고자 하였다. 방 법 : 임신 18일된 백서의 대뇌피질 신경세포를 배양하여 정상산소 상태군와 저산소 상태군으로 나누고, 두 군을 각각 대조군과 G418 $10{\mu}g/mL$으로 처리한 군으로 나누어서 TUNEL 분석과 caspase 3에 대한 면역조직생화학검사를 하였고, 생후 7일된 신생 백서의 좌측 총경동맥을 결찰 후 절단하고 저산소 상태를 유도하여 G418을 저산소 상태 유도 전 30분과 유도 후 30분에 복강 내로 $0.1{\mu}g/kg$ 투여하고 7일 후에 희생시켜 H&E 염색과 caspase-3에 대해 Western blot과 real-time PCR를 하였다. 결 과 : TENEL 분석상 정상산소 상태군과 저산소 상태군 모두에서 대조군보다 G418 투여군에서 통계학적으로 유의하게 고사세포가 감소되었다(P<0.01). Caspase-3에 대한 면역조직화학검사에서 저산소 손상 전에 G418을 투여한 군에서 손상 후에 투여한 군보다 Caspase-3 발현이 적게 나타났다. 저산소 손상 7일 후에 얻은 신생 백서 뇌의 육안적 관찰과 H & E 염색에서 저산소 상태군의 뇌 용적이 정상산소 상태군의 경우보다 감소된 것을 알 수 있었고, 저산소 손상 전에 G418을 투여한 군에서 손상 후에 투여한 군보다 뇌 조직의 손상이 더 적은 것을 볼 수 있었다. Bax/Bcl-2, caspase-3에 대해 Western blot과 real-time PCR 한 경우에 G418 투여한 군에서 Bax/Bcl-2 비율과 caspase-3 발현이 감소되었다. 결 론 : 주산기 저산소성 허혈성 뇌 손상에서 G418는 뇌 조직의 고사를 억제함으로서 뇌보호 효과를 나타내었다.

• Journal of Microbiology and Biotechnology
• /
• 제21권9호
• /
• pp.903-913
• /
• 2011

Vibrio parahaemolyticus, which causes gastroenteritis, wound infection, and septicemia, has two sets of type III secretion systems (TTSS), TTSS1 and TTSS2. A TTSS1-deficient vcrD1 mutant of V. parahaemolyticus showed an attenuated cytotoxicity against HEp-2 cells, and a significant reduction in mouse lethality, which were both restored by complementation with the intact vcrD1 gene. V. parahaemolyticus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in HEp-2 cells. The ability to activate p38 and ERK1/2 was significantly affected in a TTSS1-deficient vcrD1 mutant. Experiments using MAPK inhibitors showed that p38 and ERK1/2 MAPKs are involved in V. parahaemolyticus-induced death of HEp-2 cells. In addition, caspase-3 and caspase-9 were processed into active forms in V. parahaemolyticus-exposed HEp-2 cells, but activation of caspases was not essential for V. parahaemolyticus-induced death of HEp-2 cells, as shown by both annexin V staining and lactate dehydrogenase release assays. We conclude that secreted protein(s) of TTSS1 play an important role in activation of p38 and ERK1/2 in HEp-2 cells that eventually leads to cell death via a caspase-independent mechanism.

• The Korean Journal of Physiology and Pharmacology
• /
• 제26권3호
• /
• pp.165-174
• /
• 2022

As the mechanism underlying glucose metabolism regulation by oxymatrine is unclear, this study investigated the effects of oxymatrine on pyroptosis in INS-1 cells. Flow cytometry was employed to examine cell pyroptosis and reactive oxygen species (ROS) production. Cell pyroptosis was also investigated via transmission electron microscopy and lactate dehydrogenase (LDH) release. Protein levels were detected using western blotting and interleukin (IL)-1β and IL-18 secretion by enzyme-linked immunosorbent assay. The caspase-1 activity and DNA-binding activity of nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2) were also assessed. In the high glucose and high fat-treated INS-1 cells (HG + PA), the caspase-1 activity and LDH content, as well as Nod-like receptor family pyrin domain containing 3, Gsdmd-N, caspase-1, apoptosis-associated speck-like protein containing a CARD, IL-1β, and IL-18 levels were increased. Moreover, P65 protein levels increased in the nucleus but decreased in the cytoplasm. Oxymatrine attenuated these effects and suppressed high glucose and high fat-induced ROS production. The increased levels of nuclear Nrf2 and heme oxygenase-1 (HO-1) in the HG + PA cells were further elevated after oxymatrine treatment, whereas cytoplasmic Nrf2 and Keleh-like ECH-associated protein levels decreased. Additionally, the elevated transcriptional activity of p65 in HG + PA cells was reduced by oxymatrine, whereas that of Nrf2 increased. The results indicate that the inhibition of pyroptosis in INS-1 cells by oxymatrine, a key factor in its glucose metabolism regulation, involves the suppression of the NF-κB pathway and activation of the Nrf2/HO-1 pathway.

• Journal of Microbiology and Biotechnology
• /
• 제34권7호
• /
• pp.1511-1521
• /
• 2024

This study aimed to determine the function of LINC00511 in Nod-Like Receptor Pyrin Domain 3 inflammasome-mediated chondrocyte pyroptosis via the regulation of miR-9-5p and FUT 1. Chondrocyte inflammatory injury was induced by treating chondrocytes with LPS. Afterwards, the levels of IL-1β and IL-18, the expression of NLRP3, ASC, Caspase-1, and GSDMD, cell viability, and LDH activity in chondrocytes were assessed. LINC00511 expression in LPS-treated chondrocytes was detected, and LINC00511 was subsequently silenced to analyse its role in chondrocyte pyroptosis. The subcellular localization of LINC00511 was predicted and verified. Furthermore, the binding relationships between LINC00511 and miR-9-5p and between miR-9-5p and FUT1 were validated. LINC00511 regulated NLRP3 inflammasome-mediated chondrocyte pyroptosis through the miR-9-5p/FUT1 axis. LPS-treated ATDC5 cells exhibited elevated levels of inflammatory injury; increased levels of NLRP3, ASC, Caspase-1, and GSDMD; reduced cell viability; increased LDH activity; and increased LINC00511 expression, while LINC00511 silencing inhibited the NLRP3 inflammasome to restrict LPS-induced chondrocyte pyroptosis. Next, LINC00511 sponged miR-9-5p, which targeted FUT1. Silencing LINC00511 suppressed FUT1 by upregulating miR-9-5p. Additionally, downregulation of miR-9-5p or overexpression of FUT1 neutralized the suppressive effect of LINC00511 knockdown on LPS-induced chondrocyte pyroptosis. Silencing LINC00511 inhibited the NLRP3 inflammasome to quench Caspase-1-dependent chondrocyte pyroptosis in OA by promoting miR-9-5p and downregulating FUT1.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
• /
• pp.167.1-167.1
• /
• 2003

Here we show that panaxadiol, a ginseng saponin with a dammarane skeleton, induces acute apoptotic cell death in human hepatoma SK-HEP-1 cells as evidenced by analysis of DNA fragmentation, caspase activation, and changes in cell morphology. The kinetic study showed that panaxadiol-induced apoptosis is associated with depolarization of mitochondrial membrane potential and cytochrome c release. Sequential activations of caspases-depolarization of mitochondrial membrane potential and cytochrome c release. Sequential activations of caspases-9, and -3, or -7, but not of caspase 8 coincide well in a time dependent manner with mitochondrial membrane depolarization and cytochrome c release from mitochondria during apoptosis of SK-HEP-1 cells induced by treatment with panaxadiol. (omitted)

• 한국발생생물학회지:발생과생식
• /
• 제12권3호
• /
• pp.297-303
• /
• 2008

본 논문은 인공적인 단백질인 MCL-1ES BH3M에 관한 것으로 MCL-1ES BH3M를 과발현시 세포사멸을 유도한다. MCL-1L을 주형으로 재조합 PCR을 통해서 MCL-1ES BH3M를 클로닝하였다. 새롭게 클로닝한 단백질인 MCL-1ES BH3M 단백질은 안정성을 유지하기 위해서 PEST 도메인이 제거되어 있으며, 다른 BCL-2 패밀리 단백질과의 결합을 조절하기 위해서 BH3도메인의 Leu-Arg-Arg-Val-Gly-Asp-Gly 서열을 7개의 Ala 잔기로 인위적으로 돌연변이를 유도하였다. MCL-1ES BH3M를 293T 세포에서 과발현할 경우 세포사멸을 유도하였고, 항-세포사멸 단백질인 MCL-1L을 같이 과발현하더라도 세포사멸을 유도하였다. 또한, 과발현시 Caspase 9과 3를 활성화하였으며 면역염색법을 통해서 MCL-1ES BH3M 과발현시 미토콘드리아에 MCL-1ES BH3M 단백질이 부분적으로 위치하는 것을 확인하였다. 이상의 결과로 MCL-1ES BH3M는 Caspase 9과 3의 활성을 통해서 세포사멸을 유도한다. 결론적으로 본 연구는 세포사멸을 유도하는 새로운 molecule을 클로닝하였고, 이 molecule에 의한 세포사멸 기능을 확인하였다.

• Journal of Photoscience
• /
• 제9권2호
• /
• pp.225-228
• /
• 2002

Bcl-2 is a member of large bcl-2 family and protects cells from apoptosis. Using bcl-2-expressing adenovirus vector (Ad-bc1-2), we investigated the effect of bc1-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bc1-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (lx10$^{6}$ ) were transfected at Ixl0$^{8}$ PFU/ml. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on bcl-2-expression level. Following UVB irradiation caspase 8, 3, 9 activities were stimulated in NMK cells, while in bc1-2-transfected cells, only caspase 8, but not caspase 3 or 9 activities were stimulated. In order to investigate the effect of bc1-2 in vivo, topical application of Ad-bc1-2 on tape-stripped mouse skin was performed. Following the application, Bc1-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bc1-2 at 1st day following the application of lxl0$^{9}$ PFU in 200ml. The introduced Bc1-2 remained at least for 6 days. UVB irradiation (1250 J/m$^2$) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Bc1-2-transfected mice skin were resistant to UVB-induced apoptosis. Topical application of empty adenovirus vector alone had no effect on Bc1-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2000년도 춘계총회 및 학술대회
• /
• pp.123.1-123.1
• /
• 2000