• Journal of Life Science
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• 제10권2호
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• pp.21-26
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• 2000

Cytokine deprivation-induced apoptosis can abrogate by the appropriate survival factors. Because the mechanism of Interleukin (IL)-2 deprived apoptotic cell death remains unclear, we here show the apoptosis in CTLL2 cells correlates with an increase of the activity of caspase3-like protease(s). Inhibition of caspase3-like protease(s) with caspase protease inhibitors (Z-VAD, Z-EVD, and Z-LPD) blocks typical apoptotic morphological abnormalities in CTLL2 cells. Interestingly, Bcl-{TEX}$X_{L}${/TEX} protein was decreased by IL-2 deprivation in the cells. These results suggest that caspase3-like protease(s), not caspase1, plays an important role in apoptosis execution of CTLL2 cell death.

• Animal cells and systems
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• 제16권4호
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• pp.295-301
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• 2012

Interdigital tissue regression is one of the most well-known examples of embryonic programmed cell death, providing the mechanism behind separation of developing digits. Caspases have been shown to play a key part in this process, with activated caspase-3 localized between the developing digits. In caspase-3 knock-out adult mice, however, the digits are completely separated with no webbing. In other mutants with defects in the apoptotic machinery, such as Apaf1 deficient mice, interdigital tissue regression is initially inhibited but the webbing eventually disappears as alternative/additional cell death mechanisms step in. In order to investigate whether a similar temporal effect occurs after loss of caspase-3, we have used an in vitro approach to inhibit caspase-3 at specific times during digit separation. Previous limb explant culture approaches have encountered problems with proper limb development in culture, and thus a modified technique was used. The new approach enables detailed observation of the effects of caspase-3 inhibition on interdigital regression. Using these methods, we show that caspase-3 inhibition caused a delay in the loss of interdigital tissue compared with control explants, similar to that observed in Apaf1 mutant mice. Along with immunohistochemistry, active caspase-3 positive cells of the interdigital vs. digital regions were measured by flow cytometry. Notably, activated caspase-3 in vivo was found not only in the interdigital mesenchyme but also in the TUNEL negative digit region, supporting a role for caspase-3 in nonapoptotic events.

• Journal of Oral Medicine and Pain
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• 제25권3호
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• pp.265-276
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• 2000

스트레스가 타액선 조직을 변형시키고 파괴시킬 수 있다는 것은 이미 보고된 바 있다. 이는 구속스트레스 시에 관찰되는 apoptosis에 의한 것인데, 이 과정에 관여하는 caspase-3는 세포의 DNA를 분절시킴으로서 apoptosis를 일으킨다고 알려진 세포내 단백효소이다. 이에 기존에 관찰되었던 구속 스트레스에 의한 apoptosis의 형태적 변화가 apoptosis를 유도하는 caspase-3와 어떠한 시기적 상관관계를 가지고 있는지를 구명하기 위하여 본 실험을 시행하였다. 웅성 백서 (Sprague-Dawley, 8주) 를 사용하여 실험 전 기간에 걸쳐 구속스트레스를 가한 후 30분, 1시간, 3시간, 6시간, 24시간, 3일, 5일, 7일에 희생시켰다. 그 후 실험동물의 악하선을 절취하여 동결절편을 제작한 후, caspase-3에 대한 형광항체로 면역형광법을 시행하여 관찰하였다. 1. 정상대조군에서는 caspase-3가 타액선 조직 전반에서 미약하게 관찰되었다. 2. 구속스트레스 부여 30분에서 caspase-3는 강반응을 보였고, 실험기간이 경과됨에따라 점차 6시간군에서 부터는 현저히 감소하였다. 3. Caspase-3는 구속 스트레스 30분에 도관과 선포세포 모두에서 발현되었으나, 선포세포에서는 조기에 급격히 소실되었고 도관세포에서는 전 실험 기간에 걸쳐 서서히 소실되었다. 이와 같은 연구 결과에서, 세포내 caspase-3는 조직의 형태적 변화가 나타나기 이전에 발현하는 것으로 보아 caspase-3는 형태적 변화를 예견할 수 있는 진단 지표로 사용될 수 있을 것으로 사료되며 이후 임상적으로 적용하기 위한 지속적인 연구가 필요할 것으로 생각된다.

• 통합자연과학논문집
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• 제7권2호
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• pp.87-91
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• 2014

Amyloid beta ($A{\beta}$) peptide has been implicated in the pathogenesis of Alzheimer's disease and has been reported to induce apoptotic death in cell culture. Cysteine Proteases, a family of enzymes known as caspases, mediate cell death in many models of apoptosis. In the present study, we examined the caspase activity and cell death in $A{\beta}$-treated SHSY5Y cells, as an attempt to elucidate the relationship between the type of caspase and $A{\beta}$-induced cell death. $A{\beta}$ at 20 ${\mu}M$ induce activation of caspase-3, 8 and 9 activity, but not the caspase-1. Caspase-3, 8 and 9 were processed by Ab treatment, consistent with the activity assay. Inhibition of the caspase activities by the selective inhibitors, however, marginally affected the cell death induced by $A{\beta}$. Taken together, the results indicate that $A{\beta}$-induced cell death may be independent of caspase activity and rather, the enzymes might be activated as a result of the cell death.

• 생명과학회지
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• 제13권1호
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• pp.118-127
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• 2003

Phenylalanine의 구조유사체인 p-fluotophenylalanine (FPA)은 인체 급성백혈병세포주인 Jurkat T 세포의 세포자살을 유도한다. FPA에 의한 세포자살에 미치는 Bcl-2 또는 Bcl-xL의 영향을 조사하기 위해, Bcl-2 또는 Bcl-xL을 stable transfection하거나 empty vectors만을 Transfection한 Jurkat 세포를 이용하여 FPA의 세포독성과 FPA에 의한 세포내 세포자살 신호전달경로를 비교 분석하였다. Jurkt T 세포에 0.63∼3.0 mLf의 FPA를 처리하였을 때 세포의 생육도는 농도에 비례하여 감소하였다. 또한 세포자살관련 DNA fragmentation, caspase-8 activatoin, Bid cleavage, mitochondria로 부터의 cytochrome c 방출, caspase-9 및 -3 activation, PARP degradation 등이 유도되었다. 한편, FPA에 의해 유도되는 이러한 일련의 생화학적 현상들은 Bcl-2 또는 Bcl-xL의 overexpression에 의해 현저히 저해되었다. 이상의 결과들은 caspase-8 activation, Bid cleavage, mitochondnal cytochrome c 방출에 의해 활성화되는 casuase cascade 등의 현상이, Bcl-2 또는 Bcl-xL에 의해 억제됨을 나타내며 FPA에 의해 유도되는 세포자살에 필요한 과정임을 시사한다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권7호
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• pp.4095-4099
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• 2013

Oxidative stress induces apoptosis in many cellular systems including glioblastoma cells, with caspase-8 activation was regarded as a major contribution to $H_2O_2$-induced cell death. This study focused on the role of the autophagic protein p62 in $H_2O_2$-induced apoptosis in U87MG cells. Oxidative stress was applied with $H_2O_2$, and cell apoptosis and viability were measured with use of caspase inhibitors or autophagic mediators or siRNA p62, GFP-p62 and GFP-p62-UBA (del) transfection. We found that $H_2O_2$-induced U87MG cell death was correlated with caspase-8. To understand the role of p62 in MG132-induced cell death, the levels of p62/SQSTM1 or autophagy in U87MG cells were modulated with biochemical or genetic methods. The results showed that the over-expression of wild type p62/SQSTM1 significantly reduced $H_2O_2$ induced cell death, but knockdown of p62 aggravated the process. In addition, inhibition of autophagy promoted p62 and active caspase-8 increasing $H_2O_2$-induced apoptosis while induction of autophagy manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 required its C-terminus UBA domain to attenuate $H_2O_2$ cytotoxity by inhibition of caspase-8 activity. Our results indicated that p62/SQSTM1 was a potential contributor to mediate caspase-8 activation by autophagy in oxidative stress process.

• 동의생리병리학회지
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• 제19권3호
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• pp.772-778
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• 2005

The chloromethyl-2-dihydroxyphosphinyl-6,7-dimethoxy-1,2,3,4-tetrahydro- isoquinoline (CDDT) is a newly synthesized derivative from 1,2,3,4-Tetra- hydroisoquinoline (THIQ). The THIQs include potent cytotoxic agents that display a range of antitumor activities, antimicrobial activity, and other biological properties. In this study, we investigated the effect of CDDT on the cytotoxicity, induction of apoptosis in human promyelocytic leukemia cells (HL-60 cells). CDDT showed a significant cytotoxic activity in HL-60 cells ($IC_{50}$ = approximately $37\;{\mu}g/ml$) at a 24 hr incubation. Treatment of HL-60 cells with CDDT displayed several features of apoptosis, including formation of DNA ladders in agarose gel electrophoresis, morphological changes of HL-60 cells with DAPI stain. Here we observed that CDDT caused activation of caspase-3, caspase-8, and caspase-9. The most efficacious time on the activation of caspases-3 was achieved at 12 hr. Further molecular analysis demonstrated that CDDT led to cleavage of poly(ADP-ribose) polymerase (PARP), increase of hypodiploid (Sub-G1) population in the flow cytometric analysis. In conclusion, these above results indicate that CDDT dramatically suppresses HL-60 cell growth by activation of caspase-3 with caspase-8, -9 activity. These data may support a pivotal mechanism for the use of CDDT in the prevention and treatment of leukemia.

• IMMUNE NETWORK
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• 제12권5호
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• pp.213-216
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• 2012

Our previous report showed that polyacetylene compound, 1-Heptadecene-11, 13-diyne-8, 9, 10-triol (PA) from the root of Cirsium japonicum var. ussuriense has anti-inflammatory activity. In this study we investigated the role of the PA as inhibitor of caspase-1, which converts prointerleukin-$1{\beta}$ (proIL-$1{\beta}$) to active IL-$1{\beta}$ and is activated by inflammasome involved in the inflammatory process. We tested the effect of PA on the production of pro-inflammatory cytokines, IL-$1{\beta}$ in murine macrophage cell line, RAW264.7. PA inhibited lipopolysaccharide (LPS)-induced IL-$1{\beta}$ production by macrophages at a dose dependent manner. PA also suppressed the activation of caspase-1. The mRNA level of ASC (apoptosis-associated spec-like protein containing a CARD), an important adaptor protein of inflammasome, was decreased in the PA treated group. Therefore our results suggest that the anti-inflammatory effect of PA is due to inhibit the caspase-1 activation.

• Journal of Nutrition and Health
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• 제36권1호
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• pp.18-23
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• 2003

We studied the effects of hot water extract of Inonotus obliquos mushroom on the proliferation and apoptosis of the human colon adenocarcinoma, HT-29 and the human stomach adenocarcinoma, SNU-484 cell. Cells were maintained with Dulbecco's modified Eagle medium/Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum at 37$^{\circ}C$ in a humidified $CO_2$. For the cell proliferation experiments, cells were seeded in 35 mm dishes, and were treated with the various concentrations of the extract for the different time course. Apoptosis was measured by caspase-3 activity. When we incubated HT-29 cells for 24, 48, 72, and 96 hours after treatments, the cell proliferation was more suppressed with more treatment time. In case of the human stomach cancer cell, SNU484, the extract significantly decreased the cell number. Thus, the treatment of 1.5 mg/$m\ell$ extract decreased almost half of the cell number. Caspase-3 activity in HT-29 was increased by the treatment of mushroom extracts. In SNU484, caspase-3 activity tended to increase in proportion to the amounts of the extracts and the treatment of Inonotus obliquos affected the activity a lot. Therefore, Inonotus obliquos is suggested for the prevention of gastro-intestinal cancer and strongly recommended for the treatment of stomach cancer. (Korean J Nutrition 36(1) : 18~23, 2003)

• 미생물학회지
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• 제39권4호
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• pp.216-222
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• 2003

Caspase는 세포사멸 과정에서 중심적 역할을 하는 cysteine 단백분해 효소이다. 그 중 caspase-9은 활성이 없는 전장 단백질로 존재하지만 Apaf-1과 cytochrome c와 함께 apoptosome을 이루면 활성화된다. 활성 된 caspase-9은 다른 caspase들을 활성 시키는 중요한 기능을 갖는다. 본 연구에서는 대장균에서 전장 caspase-9 단백질을 정제하고자 pGEX 발현 시스템을 이용하였다. 그러나 일반적 배양 및 유도조건에서 caspase-9을 발현시키면 과다한 단백분해로 인하여 전장 caspase-9 단백질이 매우 적게 발현되었다. 이를 극복하기 위해 배양조건과 유도인자의 농도를 변화시키자 caspase-9의 과다한 단백분해 현상이 감소되고 전장 caspase-9의 발현 정도도 상당량 향상되었다. 본 연구에서 확립된 배양조건으로 caspase-9을 발현, 정제하면 70%이상의 순도를 가진 GST-caspase-9 단백질을 손쉽게 얻을 수 있다. 더불어 GST 융합 caspase-9이 대장균에서 발현될 때도 포유세포 내에서 절단되는 것과 동일한 위치인 Asp315 에서 자가 단백분해 반응이 일어난다는 것을 확인할 수 있었다. 본 연구에서는 caspase-9의 생화학적, 세포생물학적 기능연구에 필요한 전장 단백질을 다량으로 확보할 수 있는 간단하고 효과적인 정제방법을 제안한다.