• Title, Summary, Keyword: caspase

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Identification of the novel substrates for caspase-6 in apoptosis using proteomic approaches

  • Cho, Jin Hwa;Lee, Phil Young;Son, Woo-Chan;Chi, Seung-Wook;Park, Byoung Chul;Kim, Jeong-Hoon;Park, Sung Goo
    • BMB Reports
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    • v.46 no.12
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    • pp.588-593
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    • 2013
  • Apoptosis, programmed cell death, is a process involved in the development and maintenance of cell homeostasis in multicellular organisms. It is typically accompanied by the activation of a class of cysteine proteases called caspases. Apoptotic caspases are classified into the initiator caspases and the executioner caspases, according to the stage of their action in apoptotic processes. Although caspase-3, a typical executioner caspase, has been studied for its mechanism and substrates, little is known of caspase-6, one of the executioner caspases. To understand the biological functions of caspase-6, we performed proteomics analyses, to seek for novel caspase-6 substrates, using recombinant caspase-6 and HepG2 extract. Consequently, 34 different candidate proteins were identified, through 2-dimensional electrophoresis/MALDI-TOF analyses. Of these identified proteins, 8 proteins were validated with in vitro and in vivo cleavage assay. Herein, we report that HAUSP, Kinesin5B, GEP100, SDCCAG3 and PARD3 are novel substrates for caspase-6 during apoptosis.

Hep88 mAb-Mediated Paraptosis-Like Apoptosis in HepG2 Cells via Downstream Upregulation and Activation of Caspase-3, Caspase-8 and Caspase-9

  • Mitupatum, Thantip;Aree, Kalaya;Kittisenachai, Suthathip;Roytrakul, Sittiruk;Puthong, Songchan;Kangsadalampai, Sasichai;Rojpibulstit, Panadda
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.5
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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.

Caspase-3 Specifically Cleaves $p21^{WAF1/CIP1}$ in the Earlier Stage of Apoptosis in SK-HEP-1 Human Hepatoma Cells

  • Park, Jeong-Ae;Kim, Kyu-Won;Kim, Shin-Il;Lee, Seung-Ki
    • Proceedings of the Ginseng society Conference
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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.

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Biochemical Changes in the Tissue of Mice Irradiated with LINAC (선형가속기를 이용한 방사선조사에서 생쥐조직의 생화학적 변화)

  • Choi, Seong-Kwan
    • The Journal of the Korea Contents Association
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    • v.16 no.3
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    • pp.661-666
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    • 2016
  • In this study, a linear accelerator (LINAC) through 3 Gy of radiation per body irradiated mice of the small intestine and the liver to produce in order to protect the cells after radiation exposure that caspase (caspase 3 &caspase 9) and NO (nitric oxide), and looked like to know cytokine of IL-6 and TNF-${\alpha}$, the result is as follows. First, caspase 3 & caspase 9 showed a noticeable increase in the radiation group than in the control group both small intestine and liver tissues (P <0.001). Second, NO are both intestine and liver tissue showed a marked increase in the radiation group than in the control group (P <0.001). Third, one of Cytokine IL-6 and TNF-${\alpha}$ showed a significant increase in the irradiated group than the control group both small intestine and liver tissues (P <0.001).

Caspase3-like Death Protease Is Activated in CTLL2 Cells by Interleukin-2 Deprivation

  • Lee, Sang-Han;Kwon, O-Yu
    • Journal of Life Science
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    • v.10 no.2
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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.

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The Caspase-3 and c-myc Expressions in Completely Resected Non-small Cell Lung Cancer and Its Prognostic Significance (완전 절제된 원발성 비소세포 폐암에서 Caspase-3와 c-myc 단백의 발현과 임상 예후)

  • Cho, Deog-Gon;Cho, Kyu-Do;Kang, Chul-Ung;Jo, Min-Seop;Yoo, Jin-Young;Ahn, Myeong-Im;Kim, Chi-Hong;Shim, Byoung-Yong;Kim, Sung-Whan;Kim, Hoon-Kyo
    • The Korean Journal of Thoracic and Cardiovascular Surgery
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    • v.41 no.4
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    • pp.447-456
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    • 2008
  • Background: Caspase-3 is a cysteine protease that plays a major role in the process of apoptotic cell death. The dysregulated expression of c-myc contributes to the tumorigenesis in a variety of human cancers. The aim of this study was to investigate the expressions of caspase-3 and c-myc and their significances as prognosis markers in patients with completely resected non-small cell lung cancer (NSCLC). Material and Method: A total 130 consecutive patients who had undergone complete resection without pre-operative radio-therapy or chemotherapy between May 1996 and December 2003 for NSCLC were retrospectively reviewed. The median follow-up period of the patients was 50 months (range: $3{\sim}128$ months). The expressions of caspase-3 and c-myc were immuno-histochemically examined, and these were correlated with the clinico-pathologic data. Result: The prevalence of caspase-3 and c-myc expressions in the patients was 68% (88/130) and 59% (77/130), respectively. Significant association was found between the frequency of the expressions of caspase-3 and c-myc (p=0.025). The caspase-3 and c-myc expressions were not significantly associated with the prognosis in all the patients. However, according to stages, a positive caspase-3 expression was significantly correlated with a favorable prognosis for patients with stage IIIa disease (median survival period: 35 months vs. 10 months, p=0.021). Multivariate analysis showed the pathologic stage to be significantly correlated with a good prognosis in all the patients (p=0.024), and with a positive caspase-3 expression, well differentiated tumor and negative neuronal invasion in the patients with stage llla disease (p=0.005, p=0.003, p=0.004, respectively). Conclusion: Caspase-3 and c-myc were frequently expressed in NSCLC, suggesting its possible involvement in tumor development. The caspase-3 expression, as determined with performing immunohistochemical staining, may be a favorable prognostic indicator in patients with completely resected NSCLC an advanced stage (IIIa).

Effects of 8-week Exercise on Bcl-2, Bax, Caspase-8, Caspase-3 and HSP70 in Mouse Gastrocnemius Muscle (8주간 운동이 생쥐의 gastrocnemius에서 Bcl-2, Bax, caspase-8, caspase-3와 HSP70에 미치는 영향)

  • Kim, Ki-Bum;Kim, Yong-An;Park, Jung-Jun
    • Journal of Life Science
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    • v.20 no.9
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    • pp.1409-1414
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    • 2010
  • The aim of this study was to investigate the effects of exercise on intrinsic and extrinsic apoptosis signaling pathways in skeletal muscle. ICR-type white male mice were divided into a control group (CON: n=10) and an exercise training group (EX: n=10) after a 1 week adaptation period. EX performed treadmill running at 16.4 m/min with a 4% incline, 40 min/day and 5 days/week for 8 weeks. Cervical dislocation was performed at 48 hours after the last bout of exercise, after which gastrocnemius skeletal muscles were immediately collected. The results of verifying the intrinsic apoptosis pathway showed that there were no significant differences in Bcl-2, Bax, or the ratio of Bax/Bcl-2 proteins between EX and CON. On the other hand, the results of verifying the extrinsic apoptosis pathway showed that caspase-8 proteins were significantly lower in EX than in CON (p<0.05). Apoptosis suppressing protein HSP70 was higher in EX than in CON. In addition, caspase-3, which is the final factor for apoptosis, was not activated. These results indicate that apoptosis did not develop since caspase-3 is non-cleaved by the effects of caspase-8 and HSP70 extrinsic pathways rather than Bcl-2 and Bax intrinsic pathways among signal pathways for apoptosis.

Caspase-3 Expression in the Submandibular Gland of Rats under Restraint Stress (스트레스에 의한 백서 악하선 조직에서의 caspase-3 변화에 관한 실험적 연구)

  • Chung, Woon-Bong;Jung, Sung-Hee;Chun, Yang-Hyun;Lee, Jin-Yong;Hong, Jung-Pyo
    • Journal of Oral Medicine and Pain
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    • v.25 no.3
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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.

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Screening of the Bufonis Venenum on Hep G2 Cells (섬여가 간암(肝癌) 세포주 Hep G2에 미치는 효과)

  • Kang, A-my;Kim, Bo-Ram;Kim, Sung-Uk;Lim, Seong-Woo
    • The Journal of Korean Medicine
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    • v.29 no.4
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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.

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