• 대한치과교정학회지
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• 제33권6호
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• pp.453-463
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• 2003

본 연구는 MC3T3El 조골세포가 저산소증에 반응하여 유발될 수 있는 세포 고사조절 기전을 구명하고자 함에 목적이 있다. $2\%$ 저산소증의 조건하에서 MC3T3El 조골세포는 DNA 사다리 분절 헝성을 보였으며 형광성 염료인 Hoechst 33258로 염색된 핵 구조 형태 관찰시 시간이 지남에 따라 세포고사 현상을 관찰할 수 있었다 Pancaspase 억제제인 Z-VAD-FMK나 특정한 caspase-3 억제제인 Z-DEVD-CHO로 사전 처치하였을 경우에는 저산소증에 의한 DNA 사다리 분절형성이 농축에 비례하여 억제되었다. caspase-3류의 프로테아제(DEVDase) 활성 증가가 세포고사 중에 관찰되었으나 caspase-1 (YVADase)의 활성은 없었다. 어떤 caspase가 세포고사에 관여하는지를 확인하기 위하여 anti-caspase-3 또는 anti-caspase-6의 항체를 이용한 western blotting이 시행되었다. caspase-3의 활성산물에 해당하는 17-KDa단백질과 caspase-6의 활성산물인 20-KDa 단백질이 세포용해물에서 발생되었다. 또한 시간 경과와 더불어 caspase-6의 활동의 상징인 Lamin A의 분열을 일으켰으며, 사이토크롬 C를 cytosol로 방출하였다. 이로써 저산소증에 의한 조골세포의 고사 과정에 사이토크롬 C의 방출이 포함된 caspase의 활성이 관여한다는 것을 확인할 수 있었다.

• 대한의생명과학회지
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• 제11권3호
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• pp.281-287
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• 2005

Although actinomycin D (AMD) is known to induce apoptotic cell death to various cell lines, the mechanism of apoptosis induced by AMD is still unclear. Understanding this mechanism may improve its therapeutic efficacy. The present study has been performed to elucidate expression of Bcl-2 and Caspase-3 proteins related to apoptosis in human leukemia K-562 cells. Five different assays were performed in this study; DNA fragmentation analysis by agarose gel electrophoresis, quantitative assay of fragmented DNA, morphological assessment of apoptotic cells, quantification of apoptosis by annexin V (AV) and propidium iodide (PI) staning, and expression of Bcl-2 and Caspase-3 proteins by the western blot analysis. The number of apoptotic cells and amount of fragmented DNA in this cell line treated with AMD was increased at 6 hour. DNA ladder pattern was also appeared at 6 hour. The expression of Bcl-2 was decreased, and disappeared from 12 hours after AMD treatment. Precursor of Caspase-3 was degraded, and 20 kDa cleavage products were detected. These results suggest that AMD induced apoptosis of K-562 cells is Caspase-3-dependent fashion, and this apoptosis is related to the degradation of Bcl-2 proteins.

• 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.

• Journal of Life Science
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• 제9권1호
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• pp.9-13
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• 1999

Reduced thiols were important compounds for the maintenance of leukemia and lymphoma cell survival (and growth). In the course of examining the microenvirn-mental effects on lymphoma and leukemia cell growth, we found that cysteine suppressed apoptosis in these cells. In a present study, in order to investigate the role of cystein on the suppression of apoptotic cell death, we used CS21, P388, and L1210 cell lines. The addition of BSO, an inhibitor of glutathione synthase, induced apoptosis of these cells by blocking the cellular uptake of cysteine in CS21 cells. Although L1210 cells underwent apoptosis without thiol compounds, the addition of these compounds suppressed the apoptosis and promoted the growth or L1210 cells. When specific inhibitors of caspase3-like proteases, but not caspase1-like proteases, were activated during the L1210 cell apoptosis but the addition of thiol compounds suppressed the activation of caspase3-like proteases. These results suggest that reduced thiols including cysteine play an important role in the suppression of cell apoptosis by inhibiting the activation of caspase3-like proteases.

• 한국식품영양과학회지
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• 제37권6호
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• pp.714-720
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• 2008

본 연구에서는 동충하초(C. militaris)의 항암작용 기전 해석을 위하여 Hep3B 간암세포의 apoptosis 유발에 미치는 동충하초 열수추출물(WECM)의 영향을 조사하였으며, apoptosis 조절에 중요한 몇 가지 유전자들의 발현 및 활성 변화를 조사하였다. AECM 처리에 의한 Hep3B 세포의 증식억제는 형태적 변형을 동반한 apoptosis 유도와 연관성이 있음을 DAPI 염색을 통한 apoptotic body 출현의 증가 및 flow cytometry 분석에 의한 sub-G1 기에 속하는 세포 빈도의 증가로 확인하였다. AECM 처리에 의한 apopotosis 유도에서 Bcl-2 family에 속하는 몇 가지 유전자들의 발현은 큰 변화가 없었으나, caspase-3 및 -8의 활성이 매우 높게 증가 되었으며 이는 PARP 및 ${\beta}$-catenin 단백질의 분해와 연관성이 있었다. 또한 caspase-3 선택적 저해제인 z-DEVD-fmk로 caspase-3의 활성을 인위적으로 차단시켰을 경우, AECM에 의한 apoptois 유도 현상이 유의적으로 감소되어 AECM에 의한 Hep3B 세포의 apoptosis 유발에 caspase-3이 중요한 역할을 하고 있음을 알 수 있었다. 본 연구의 결과만으로 동충하초에 의한 간암세포의 증식억제 기전을 명확하게 제시할 수는 없으나, 이상의 결과들은 동충하초의 생화학적 항암기전 해석을 이해하는데 중요한 기초자료로서 활용될 수 있을 것으로 생각된다.

• Nutrition Research and Practice
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• 제8권2호
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• pp.132-137
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• 2014

BACKGROUND/OBJECTIVES: In this study, the apoptogenic activity and mechanisms of cell death induced by hexane extract of aged black garlic (HEABG) were investigated in human leukemic U937 cells. MATERIALS/METHODS: Cytotoxicity was evaluated by MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide) assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay. RESULTS: Exposure to HEABG was found to result in a concentration- and time-dependent growth inhibition by induction of apoptosis, which was associated with an up-regulation of death receptor 4 and Fas legend, and an increase in the ratio of Bax/Bcl-2 protein expression. Apoptosis-inducing concentrations of HEABG induced the activation of caspase-9, an initiator caspase of the mitochodrial mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. HEABG also induced apoptosis via a death receptor mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid, and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. However, pre-treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, significantly blocked the HEABG-induced apoptosis of these cells, and increased the survival rate of HEABG-treated cells, confirming that HEABG-induced apoptosis is mediated through activation of caspase cascade. CONCLUSIONS: Based on the overall results, we suggest that HEABG reduces leukemic cell growth by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways, implying its potential therapeutic value in the treatment of leukemia.

• 대한의생명과학회지
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• 제26권2호
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• pp.57-65
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• 2020

Excessive drinking of alcohol is known to be one of the main causes of various neurological diseases, such as Alzheimer's disease. Scopoletin is known to have anti-inflammatory and antioxidative properties, and to protect nerve cells. This study examined whether scopoletin inhibits the alcohol-induced apoptosis of primary hippocampal neurons, and how scopoletin regulates several factors associated with the caspase-mediated pathway. To achieve this, the cell viability and apoptosis rate of primary hippocampal neurons were measured by Cell Counting Kit-8 and flow cytometry, respectively. Apoptosis-related protein expressions (Bax, Bid, caspase-3, caspase-9, and Poly (ADP-ribose) polymerase (PARP)) were analyzed by Western blotting, and the ANOVA method was used to confirm the significance of the measured results. As a result, scopoletin inhibited the expressions of alcohol-induced apoptosis and apoptosis-related proteins in primary hippocampal neurons. These results suggest that down-regulation of Bid, Bax, and cleaved caspase-9 expression induced by scopoletin down-regulates the expression of cleaved caspase-3, inhibits the expression of cleaved PARP, and finally, inhibits mitochondrial apoptotic pathways. The study suggests that scopoletin is worth developing as a candidate for neuroprotective agent.

• 대한임상검사과학회지
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• 제38권2호
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• pp.82-86
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• 2006

Leukemia arises in hematopoietic progenitor cells and is characterized by impaired or blocked differentiation, uncontrolled proliferation and resistance to apoptosis. Molecular mechanisms underlying cellular functions by $As_2O_3$, however, have been poorly investigated. The consensus of several reports is that $As_2O_3$ induces apoptosis in leukemia cells by activating genes for apoptosis. The present study aimed to investigate the effects of $As_2O_3$ on the cell cycle and its morphological change and a relationship between the caspase-3 and $As_2O_3$-induced apoptosis. Caspase-3 is involved in $As_2O_3$-induced apoptosis in K562 cells. In this study, to address whether $As_2O_3$-induced apoptosis is mediated by caspase-3 activity, the same samples were probed with a specific antibody. The pretreatment of $25{\mu}M$ Z-VAD-fmk, a specific inhibitor of caspase, decreased $As_2O_3$-induced cytotoxicity. And $As_2O_3$ significantly increased the percentages of the cells accumulated in the G2/M phase of the cell cycle in a time- and dose-dependent manner. Chromatin condensational changes were observed with Hoechst 33258 staining after treatment of $As_2O_3$. It was shown that $As_2O_3$-induced apoptosis is controlled through caspase-3 activation. These results may provide a useful rationale for CML treatment.

• Toxicological Research
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• 제17권3호
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• pp.215-223
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• 2001

카드뮴의 주요한 표적장기이며 카드뮴이 만성 및 급성 폭로시 축적되는 가장 중요한 장기인 간의 세포독성을 Hepalclc7세포에서 caspases및 Bax단백질의 활성과 발현 그리고 미토콘드리아 세포막 전위 변화(MPT) 등을 조사하여 다음과 같은 결과를 얻었다. 1. 카드뮴은 농도의존적으로 간세포인 Hepalclc7 세포의 생존율을 감소시켰다. 2. 카드뮴을 농도별로 처리하였을 때 100 M 이상의 농도에서 세포사멸의 특징중의 하나인 DNA분절현상을 확인하였다. 3. 카드뮴 처리 후 caspase-3, caspase-8, caspase-9 의 활성변화를 조사한 결과 caspase-3,-9 pretease 활성이 시간이 경과함에 따라 증가하였다. 4. 카드뮴 처리 후 cytochrome c가 세포질내로 방출되었고 이는 caspase-9 proteas의 활성화를 유도하였다. 5. 카드뮴 처리 후 Bax가 세포질에서 미토콘드리아로 이동하여 cytochrome c의 세포질내로의 방출에 관여하였다. 6. 카드뮴 처리시 미토콘드리아 세포막 전위차의 감소를 JC-1 형광염색을 통하여 확인하였다. 이상의 결과는 카드뮴에 의한 Hepalclc7 세포사멸의 신호전달기전은 세포질내에 있는 Bax가 미토콘드리아로 이동, cytochrome c의 세포질내로의 방출, 그리고 caspase-3, 9 pretease 활성화를 의해서 매개되는 것으로 판단된다. 또한 Bax 단백질의 발현변화가 미토콘드리아의 기능변화에 기여하였으리라 사료된다.

• Molecules and Cells
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• 제26권2호
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• pp.152-157
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• 2008

Caspases play critical roles in the execution of apoptosis. Caspase-3 and caspase-7 are closely related in sequence as well as in substrate specificity. The two caspases have overlapping substrate specificities with special preference for the DEVD motif. However, they are targeted to different subcellular locations during apoptosis, implying the existence of substrates specific for one or other caspase. To identify new caspase-7 substrates, we digested cell lysates obtained from the caspase-3-deficient MCF-7 cell line with purified recombinant caspase-7, and analyzed spots that disappeared or decreased by 2-DE (we refer to this as the caspase-7 degradome). Several proteins with various cellular functions underwent caspase-7-dependent proteolysis. The substrates of capase-7 identified by the degradomic approach were rather different from those of caspase-3 (Proteomics, 4, 3429-3435, 2004). Among the candidate substrates, we confirmed that Valosin-containing protein (VCP) was cleaved by both capspase-7 and caspase-3 in vitro and during apoptosis. Cleavage occurred at both $DELD^{307}$ and $DELD^{580}$. The degradomic study yielded several candidate caspase-7 substrates and their further analysis should provide valuables clues to the functions of caspase-7 during apoptosis.