• 통합자연과학논문집
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• 제7권1호
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• pp.25-38
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• 2014

Amyloid-${\beta}$-peptide ($A{\beta}$) is important in the pathogenesis of Alzheimer's disease (AD). Calpain ($Ca^{2+}$-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in $AD/A{\beta}$ toxicity. We found that $A{\beta}$ promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the $A{\beta}$ and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIPS). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIPS degradation in the $A{\beta}$-treated and CD95-triggered cells. Increased cellular Ca2+ per se results in calpain activation but does not lead to caspase-8 activation or FLIPS degradation. The results suggest that procaspase-8 and FLIPS association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase- 8, and CD95 pathway in $AD/A{\beta}$ toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.

• 대한수의학회지
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• 제45권4호
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• pp.495-500
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• 2005

The nucleoside analogue gemcitabine (2', 2-difluorideoxycytide) is potential against a wide variety of solid tumors and considered to be one of the most active drugs in the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated the signals of gemcitabine-induced apoptosis, especially in point of caspase pathway in A549. We exposed A549 cells to gemcitabine for dose/time dependent manner and the results showed that gemcitabine induced apoptotic cell death in a time/dose-dependent manner. We also treated to gemcitabine and Z-VAD-fmk as a pan-caspase inhibitor for 24 hours. Gemcitabine alone induced 35.3% cell death, and co-treatment with gemcitabine and Z-VAD-fmk induced 15.1% apoptotic cell death. Our results demonstrated that Z-VAD-fmk as a pan-caspase did not completely block the gemcitabine-induced apoptosis. Western blotting analysis showed that gemcitabine increased caspase-3, active caspase-8, p21 and p53 protein expressions in A549. Co-treatment with Z-VAD-fmk completely blocked caspase-3 and active caspase-8 protein expressions, but did not change the level of p21 and p53 protein expressions. Our data indicate that gemcitabine induced apoptosis through caspase-dependent and -independent pathways in A549.

• Asian Pacific Journal of Cancer Prevention
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• 제16권8호
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• pp.3293-3299
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• 2015

Background: Arm protein lost in epithelial cancers, on chromosome X (ALEX) is a novel subgroup within the armadillo (ARM) family, which has one or two ARM repeat domains as opposed to more than six-thirteen repeats in the classical Armadillo family members. Materials and Methods: In the study, we explore the biological functions of ALEX1 in breast cancer cells. Overexpression of ALEX1 and silencing of ALEX1 were performed with SK-BR3 and MCF-7 cell lines. Cell proliferation and colony formation assays, along with flow cytometry, were carried out to evaluate the roles of ALEX1. Results: ALEX1 overexpression in SK-BR3 breast cancer cells inhibited proliferation and induced apoptosis. Furthermore, depletion of ALEX1 in MCF-7 breast cancer cells increased proliferation and inhibited apoptosis. Additional analyses demonstrated that the overexpression of ALEX1 activated the intrinsic apoptosis cascades through up-regulating the expression of Bax, cytosol cytochrome c, active caspase-9 and active caspase-3 and down-regulating the levels of Bcl-2 and mitochondria cytochrome c. Simultaneouly, silencing of ALEX1 inhibited intrinsic apoptosis cascades through down-regulating the expression of Bax, cytosol cytochrome c, active caspase-9, and active caspase-3 and up-regulating the level of Bcl-2 and mitochondria cytochrome c. Conclusions: Our data suggest that ALEX1 as a crucial tumor suppressor gene has been involved in cell proliferation and apoptosis in breast cancer, which may serve as a novel candidate therapeutic target.

• Asian Pacific Journal of Cancer Prevention
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• 제16권5호
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• pp.1771-1779
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• 2015

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Presently, targeted therapy via monoclonal antibodies to specific tumor-associated antigens is being continuously developed. Hep88 mAb has proven to exert tumoricidal effects on the HepG2 cell via a paraptosis-like morphology. To verify the pathway, we then demonstrated downstream up-regulation of caspase-3, caspase-8 and caspase-9, assessingmRNA expression by real-time PCR and associated enzyme activity by colorimetric assay. Active caspase-3 determination was also accomplished by flow cytometry. Active caspase-3 expression was increased by Hep88 mAb treatment in a dose-and time-dependent manner. All of the results indicated that Hep88 mAb induced programmed cell death in the HepG2 cell line from paraptosis-like to apoptosis by downstream induction of caspases. These conclusions imply that Hep88mAb might be a promising tool for the effective treatment of HCC in the future.

• 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.

• 대한한의학회지
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• 제29권4호
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• pp.171-179
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• 2008

Objective: Bufonis Venenum is the traditional Korean medicine Chan Su, which is obtained from the skin and parotid venom gland of the toads. It has been used for myocardial diseases, inflammation diseases, pain relief, cancer and others. The main components of BV are cinobufotoxin, cinobufalin, bufalin and others. Of these, bufalin, the major active ingredient of BV, has been reported to induce apoptosis and to possess anti-tumor effects. There was no report of anti-tumor screening of BV on hepatic cancer and which signaling pathway can be involved. In order to examine the effect of BV on hepatic cancer and the related signaling pathway with BV-induced apoptosis, human Hep G2 cells were used. Methods: Analysis of apoptosis was confirmed by MTT assay. BV decreased cell viability in a dose and duration dependent manner. To observe which signaling molecules will be activated by BV, phosphorylation of MAPK (p38, ERK, JNK), caspase 8 and caspase 9 were examined by Western blot analysis. Results: The phosphorylation levels of p38 started to increase at 5 min after addition of 5 ${\mu}g$/ml of BV and sustained to increase until 48 hours. The phosphorylation levels of other MAPK (ERK and JNK), caspase 8 and caspase 9 increased in a time-dependent manner. These imply that BV may activate different signaling pathways, MAPK, caspase 8 and caspase 9. These results propose that BV may induce apoptosis on Hep G2 cells through the activation of MAPK, caspase 8 and caspase 9.

• 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.

• Journal of Acupuncture Research
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• 제23권3호
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• pp.91-102
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• 2006

목적 : 이 연구에서는 FBS에 의하여 유도되는 혈관 평활근 세포 증식에 대한 봉약침액과 Melittin의 세포 고사효과의 영향 및 작용 기전을 살펴보고자 하였다. 방법 : $I{\kappa}Ba$, p-$I{\kappa}Ba$, p-ERK1/2, p-Akt, p53, Bcl-2, Bax 및 active caspase-3는 Western blotting을, $NF-{\kappa}B$는 EMSA와 immunofluorescence staining을 이용하여 측정하였다. 결과 : 1. Melittin은 $NF-{\kappa}B$ 활성에 대하여 $I{\kappa}Ba$의 인산화를 유의하게 익제하고 $I{\kappa}Ba$를 증가시켰으며, $NF-{\kappa}B$의 DNA 결합과 $NF-{\kappa}B$ p50의 핵 내 유입을 유의하게 감소시켰다. 2. Melittin은 $NF-{\kappa}B$ 활성을 증가시키는 물질인 Akt의 인산화를 유의하게 억제하였고, ERK1/2의 인산화도 억제하였다. 3. Melittin은 세포사멸 전구 단백질인 p53, Bax 및 caspase-3의 발현을 유의하게 증가시켰고, 세포사멸억제 단백질인 Bcl-2의 발현은 감소시켰다. 결론 : 이상의 결과는 $NF-{\kappa}B$ 와 Akt 활성을 억제함으로써 혈관평활근세포 증식을 억제하는 효과가 있음을 입증한 것이며, 향후 안전성 연구를 바탕으로 혈관성형술 후 재발성협착증과 동맥경화증의 치료제로 사용될 수 있을 것으로 기대된다.

• Journal of Ginseng Research
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• 제38권1호
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• pp.22-27
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• 2014

Background: Research has been conducted with regard to the development of methods for improving the pharmaceutical effect of ginseng by conversion of ginsenosides, which are the major active components of ginseng, via high temperature or high-pressure processing. Methods: The present study sought to investigate the anticancer effect of heat-processed American ginseng (HAG) in human gastric cancer AGS cells with a focus on assessing the role of apoptosis as an important mechanistic element in its anticancer actions. Results and Conclusion: HAG significantly reduced the cancer cell proliferation, and the contents of ginsenosides Rb1 and Re were markedly decreased, whereas the peaks of less-polar ginsenosides [20(S,R)-Rg3, Rk1, and Rg5] were newly detected. Based on the activity-guided fractionation of HAG, ginsenoside 20(S)-Rg3 played a key role in inducing apoptosis in human gastric cancer AGS cells, and it was generated mainly from ginsenoside Rb1. Ginsenoside 20(S)-Rg3 induced apoptosis through activation of caspase-3, caspase-8, and caspase-9, as well as regulation of Bcl-2 and Bax expression. Taken together, these findings suggest that heat-processing serves as an increase in the antitumor activity of American ginseng in AGS cells, and ginsenoside 20(S)-Rg3, the active component produced by heat-processing, induces the activation of caspase-3, caspase-8, and caspase-9, which contributes to the apoptotic cell death.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 1998년도 Advances in Ginseng Research - Proceedings of the 7th International Symposium on Ginseng -
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• pp.231-243
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• 1998

In the present study, we provide evidence that ginsenoside $Rh_2$ (G-$Rh_2$) as well as staurosporine induces apoptosis of human hepatoma SK-HEP-1 cells by caspase 3-mediated processing of $p21^{WAFI/CIPI}$ in the early stage of apoptosls. Immunoblottings showed that G-$Rh_2$ as well as statrosporine induced the processing of caspase-3 to an active form, pl7. In stable Bcl-2 transfectants however, G-$Rh_2$ induced DNA fragmentation, while staurosporine did not. In the early stage of apoptosis, $p21^{WAFI/CIPI}$ was detected to undergo proteolytic processing specifically conducted by caspase-3. $p21^{WAFI/CIPI}$ translated in vitro was cleaved into a p14 fragment, when incubated with cell extracts obtained from either G-$Rh_2$- or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-cho, a specific caspase-3 inhibitor, but not by Ac-YVkD-cho, a specific caspase-l inhibitor. Similarly, $p21^{WAFI/CIPI}$ was efficiently leaved by recombinant caspase-3 overexpressed in E. coli. Moreover, the endogenous $p21^{WAFI/CIPI}$ of untreated-cell extracts was also cleaved by recombinant caspase-3. Mutation analysis allowed identification of two caspase-3 cleavage sites, $DHVD^{112}$/L and $SMTD^{149}$/F, which are located within, or near the interaction domains for cyclins, Cdks, and PCNA. Taken together, these results show that G-$Rh_2$ as well as staurosporine increases caspase-3 activity, which in turn directly cleaves $p21^{WAFI/CIPI}$ resulting in elevation of Cdk kinase activity in the early stages of apoptosis. We propose that proteolytic cleavage of $p21^{WAFI/CIPI}$ is a functionally relevant event that allows unleashing the cyclin/Cdk activity from the inhibitor seen in the earlier stage of apoptosis, the event of which may be associated with the triggering mechanism for the execution of apoptosis.