• Title, Summary, Keyword: caspase

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The effect of caspase-3 inhibition on interdigital tissue regression in explant cultures of developing mouse limbs

  • Kudelova, Judita;Tucker, Abigail S.;Dubska, Lenka;Chlastakova, Ivana;Doubek, Jaroslav;Matalova, Eva
    • Animal cells and systems
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    • v.16 no.4
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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.

A Natural L-Arginine Analog, L-Canavanine-Induced Apoptosis is Suppressed by Protein Tyrosine Kinase p56lck in Human Acute Leukemia Jurkat T Cells (인체 급성백혈병 Jurkat T 세포에 있어서 L-canavanine에 의해 유도되는 세포자살기전에 미치는 단백질 티로신 키나아제 p56lck의 저해 효과)

  • Park, Hae-Sun;Jun, Do-Youn;Woo, Hyun-Ju;Rue, Seok-Woo;Kim, Sang-Kook;Kim, Kyung-Min;Park, Wan;Moon, Byung-Jo;Kim, Young-Ho
    • Journal of Life Science
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    • v.19 no.11
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    • pp.1529-1537
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    • 2009
  • To elucidate further the antitumor effects of a natural L-arginine analogue, L-canavanine, the mechanism underlying apoptogenic activity of L-canavanine and its modulation by protein tyrosine kinase $p56^{lck}$ was investigated in human Jurkat T cells. When the cells were treated with 1.25 to 2.5 mM L-canavanine for 36 h, several apoptotic events including mitochondrial membrane potential (${\Delta\Psi}m$) loss, activation of caspase-9, -3, -8, and -7, poly (ADP-ribose) polymerase (PARP) degradation, and DNA fragmentation were induced without alteration in the levels of Fas or FasL. These apoptotic changes were more significant in $p56^{lck}$-deficient Jurkat clone JCaM1.6 than in $p56^{lck}$-positive Jurkat clone E6.1. The L-canavanine-induced apoptosis observed in $p56^{lck}$-deficient JCaM1.6 cells was significantly reduced by introducing $p56^{lck}$ gene into JCaM1.6 cells by stable transfection. Treatment of JCaM1.6/lck cells with L-canavanine caused a transient 1.6-fold increase in the kinase activity of $p56^{lck}$. Both FADD-positive wild-type Jurkat T cell clone A3 and FADD-deficient Jurkat T cell clone I2.1 exhibited a similar susceptibility to the cytotoxicity of L-canavanine, excluding involvement of Fas/FasL system in triggering L-canavanine-induced apoptosis. The L-canavanine-induced apoptotic sub-$G_1$ peak and activation of caspase-3, -8, and -7 were abrogated by pan-caspase inhibitor (z-VAD-fmk), whereas L-canavanine-induced activation of caspase-9 was not affected. These results demonstrated that L-canavanine caused apoptosis of Jurkat T cells via the loss of ${\Delta\Psi}m$, and the activation of caspase-9, -3, -8, and -7, leading to PARP degradation, and that the $p56^{lck}$ kinase attenuated the ${\Delta\Psi}m$ loss and activation of caspases, and thus contributed as a negative regulator to L-canavanine-induced apoptosis.

Ectopic expression of Bcl-2 or Bcl-xL suppresses p-fluorophenylalanine-induced apoptosis through blocking mitochondria-dependent caspase cascade in human Jurkat T cells (Jurkat T 세포에 있어서 ρ-fluorophenylalanine에 의해 유도되는 세포자살의 Bcl-2 및 Bcl-xL에 의한 저해 기전)

  • Han, Kyu-Hyun;Oh, Hyun-Ji;Jun, Do-Youn;Kim, Young-Ho
    • Journal of Life Science
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    • v.13 no.1
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    • pp.118-127
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    • 2003
  • $\rho$-Fluorophenylalanine (FPA), a phenylalanine analog, is able to induce apoptotic cell death of human acute leukemia Jurkat T cells. To better understand the mechanism by which FPA induces apoptotic cell death, the effect of ectopic expression of antiapoptotic proteins, Bcl-2 and Bcl-xL, on FPA-induced apoptosis was investigated by employing lurkat T cells transfected with Bcl-2 gene (JT/Bcl-2) or Bcl-xL gene (1/Bcl-xL) and Jurkat T cells transfected with vector (JT/Neo or J/Neo). When Jurkat T cells, JT/Neo or J/Neo, were exposed to FPA at concentrations ranging from 0.63 to 5.0 mM, the cell viability determined by MTT assay declined in a dose-dependent manner. In addition, apoptotic DNA fragmentation along with several apoptotic events such as caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, caspase-9 activation, caspase-3 activation, and degradation of PARP was induced. However, the FPA-induced cytotoxic effect, activation of caspase-8, and cleavage of Bid were significantly abrogated by ectopic expression of Bcl-2 or Bcl-xL. At the same time, there was marked reduction in the level of cytochrome c release from mitorhondria, caspase-9 activation, caspase-3 activation, and degradation of PARP. These results indicate that caspase-8 activation, Bid cleavage, and mitochondrial cytochrome c release with subsequent activation of the caspase cascade are negatively regulated by Bcl-2 or Bcl-xL, and are thus required for FPA-induced apoptosis in Jurkat T cells

Induction of Apoptosis in FRTL-5 Thyroid Cells by Okadaic Acid (Okadaic Acid에 의한 FRTL-5 갑상선 세포주의 Apoptosis 유도)

  • Cho Ji-Hyoung;Chung Ki-Yong;Park Jong-Wook
    • Korean Journal of Head & Neck Oncology
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    • v.18 no.2
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    • pp.142-149
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    • 2002
  • Objectve : Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 and 2A. In order to know the mechanism of apoptosis induced by okadaic acid, we treated FRTL-5 thyroid cells with okadaic acid and measured the changes of important proteins that are involved in apoptosis. Materials and Methods: We measured caspase 3 activity, $PLC-{\gamma}1$ degradation, the expression of XIAP, cIAP1, cIAP2, and cytochrome c release in okadaic acid-treated FRTL-5 thyroid cells. Results: Okadaic acid-induced caspase 3 activation and $PLC-{\gamma}1$ degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 80 nmol and time-dependent with a maximal effect at 24 hours after treatment. The elevated caspase 3 activity in okadaic acid treated FRTL-5 thyroid cells are correlated with down-regulation of XIAP and cIAP1, but not cIAP2. General and potent inhibitor of caspases, z-VAD-fmk. abolished okadaic acid-induced caspase 3 activity and $PLC-{\gamma}1$ degradation. The release of cytochrome c in okadaic acid-induced FRTL-5 thyroid cells was dose-dependent with a maximal effect at a concentration of 80 nmol. Conclusions: These findings suggest that mechanism of okadaic acid-induced apoptosis is associated with cytochrome c release and increase of caspase 3 activation in FRTL-5 thyroid cells.

Cathepsin B Is Implicated in Triglyceride (TG)-Induced Cell Death of Macrophage (중성지방에 의한 대식세포 사멸 과정에서 Cathepsin B의 영향)

  • Jung, Byung Chul;Lim, Jaewon;Kim, Sung Hoon;Kim, Yoon Suk
    • Korean Journal of Clinical Laboratory Science
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    • v.52 no.3
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    • pp.245-252
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    • 2020
  • Macrophage cell death contributes to the formation of plaque, leading to the development of atherosclerosis. The accumulation of triglyceride (TG) is also associated with the pathogenesis of atherosclerosis. A previous study reported that TG induces the cell death of macrophages. This study examined whether the cytoplasmic release of cathepsin B from lysosome is associated with the TG-induced cell death of macrophage. The release of cathepsin B was increased in the TG-treated THP-1 macrophages, but the TG treatment did not affect cathepsin B expression. Furthermore, the inhibition of cathepsin B by its inhibitor, CA-074 Me, partially inhibited the TG-induced cell death of macrophage. TG-triggered macrophage cell death is mediated by the activation of caspase-1, -2, and apoptotic caspases. Therefore, this study investigated whether cathepsin B is implicated in the activation of these caspases. The inhibition of cathepsin B blocked the activation of caspase-7, -8, and -1 but did not affect the activity of caspase-3, -9, and -2. Overall, these results suggest that TG-induced cytoplasmic cathepsin B causes THP-1 macrophage cell death by activating caspase-1, leading to subsequent activation of the extrinsic apoptotic pathway.

Design, Syntheses and Biological Evaluations of Nonpeptidic Caspase 3 Inhibitors

  • Kim, Eun-Sook;Yoo, Sung-Eun;Yi, Kyu-Yang;Lee, Sun-Kyung;Noh, Jae-Sung;Jung, Yong-Sam;Kim, Eun-Hee;Jeong, Nak-Chul
    • Bulletin of the Korean Chemical Society
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    • v.23 no.7
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    • pp.1003-1010
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    • 2002
  • Caspase 3, a member of cysteine protease family, is well known as a major apoptosis effector and is involved in cell death as a result of ischemic diseases such as stroke and myocardial infarction, therefore the inhibition of caspase 3 may protect those apoptotic cell damages. During the high-throughput screening of the compounds from the Korea Chemical Bank, berberine derivatives (A and B), an isoquinoline alkaloid, have been identified as potential inhibitors for caspase 3. Based on this finding we carried out molecular modeling study to identify the pharmacophoric elements of berberine structure which interact with a substrate-recognition binding site of caspase 3 and came up with several novel scaffolds. In this report, we will discuss the molecular modeling, syntheses and the enzyme inhibitory activities of these novel compounds.

Cysteine Participates in Cell Proliferation by Inhibiting Caspase3-like Death Protease

  • Lee, Sang-Han;Hong, Soon-Duck
    • Journal of Life Science
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    • v.9 no.1
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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.

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Expression of Survivin and Caspase 3 in Oral Squamous Cell Carcinoma and Peritumoral Tissue

  • Li, S.X.;Chai, L.;Cai, Z.G.;Jin, L.J.;Chen, Y.;Wu, H.R.;Sun, Z.
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.10
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    • pp.5027-5031
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    • 2012
  • This study was conducted to investigate the expression of survivin and caspase 3 in oral squamous cell carcinoma and peritumoral tissue, and possible pathogenesis mechanisms. We used ELISA and western blotting to detect the protein expression levels of survivin and caspase 3 in tissue. In situ hybridization and real-time PCR were applied to assess mRNA expression levels. In this study, 13 tumor samples and 13 peritumoral tissue samples were collected from oral squamous cell carcinoma patients and 10 normal tissue samples obtained from patients without tumor. The result showed that the protein and mRNA expression of survivin in carcinoma was the highest among three types of tissue; following was that in peritumoral tissue. No difference in caspase 3 zymogen between peritumoral tissue and normal tissue could be found, while it was evidently decreased in carcinoma tissue. Activated caspase 3 was detected in normal tissue but could not be identified in peritumoral or carcinoma tissue. Our results indicate that the expression of survivin is apparently elevated in tumoral and peritumoral tissue. Expression of activated caspase 3 was not detected in tumoral tissue and the expression of caspase 3 zymogen was decreased in tumoral tissue. Our findings suggest that survivin may inhibit both synthesis and activation of caspase 3, hence inhibiting cell apoptosis and facililitating eventual development of oral squamous cell carcinoma.

Mechanism of gemcitabine-induced apoptosis (Gemcitabine의 세포사멸 기전 연구)

  • Seol, Jae-Won;Lee, You-Jin;Kang, Dong-Won;Kang, Hyung-Sub;Kim, Nam-Soo;Kim, In-Shik;Park, Sang-Youel
    • Korean Journal of Veterinary Research
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    • v.45 no.4
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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.

Induction of apoptosis by a hexane extract of aged black garlic in the human leukemic U937 cells

  • Park, Cheol;Park, Sejin;Chung, Yoon Ho;Kim, Gi-Young;Choi, Young Whan;Kim, Byung Woo;Choi, Yung Hyun
    • Nutrition Research and Practice
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    • v.8 no.2
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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.