DOCSLIB.ORG

Synthetic Bichalcone TSWU-BR23 Induces Apoptosis of Human Colon Cancer HT-29 Cells by P53-Mediated Mitochondrial Oligomerization

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Synthetic Bichalcone TSWU-BR23 Induces Apoptosis of Human Colon Cancer HT-29 Cells by P53-Mediated Mitochondrial Oligomerization ANTICANCER RESEARCH 35: 5407-5416 (2015) Synthetic Bichalcone TSWU-BR23 Induces Apoptosis of Human Colon Cancer HT-29 Cells by p53-Mediated Mitochondrial Oligomerization of BAX/BAK and Lipid Raft Localization of CD95/FADD MENG-LIANG LIN1, SHIH-SHUN CHEN2 and TIAN-SHUNG WU3 1Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, R.O.C.; 2Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan, R.O.C.; 3Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, R.O.C. Abstract. A synthetic bichalcone analog, (E)-1-(3-((4-(4- Apoptosis is a key regulatory mechanism of tissue and acetylphenyl)piperazin-1-yl)methyl)-4-hydroxy-5- cellular homeostasis that can be induced via activation of methoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU- either the extrinsic or the intrinsic signaling pathway (1). The BR23), has been shown to induce apoptosis in human colon extrinsic pathway is triggered by binding cluster of cancer HT-29 cells involving the induction of CD95 and FAS- differentiation 95 (CD95) ligand to CD95 receptor on the associated protein death domain (FADD), but its precise target cells, which in turn leads to recruitment of cytosolic mechanism of action has not been fully elucidated. Using adaptor FAS-associated death domain-containing protein cell-surface biotinylation and sucrose density-gradient-based (FADD) and pro-caspase-8. Upon co-localization with membrane flotation techniques, we showed that the disruption FADD, pro-caspase-8 undergoes auto-proteolytic cleavage, of TSWU-BR23-induced lipid raft localization of CD95/FADD releasing activated caspase-8 into the cytosol, where it causes by cholesterol-depleting agent (methyl-β-cyclodextrin) was cleavage of BH3-interacting domain death agonist (BID) into reversed by cholesterol replenishment. Blockade of p53 truncated BID (tBID) to engage the intrinsic pathway and expression by short-hairpin RNA (shRNA) suppressed initiates apoptosis by activating executioner caspase (1). The oligomeric Bcl-2-associated x protein (BAX)/Bcl-2 antagonist intrinsic pathway is closely linked to permeabilization of the killer 1 (BAK)-mediated mitochondrial apoptosis but did not mitochondrial outer membrane by the activation and inhibit lipid raft localization of CD95/FADD and pro- oligomerization of pro-apoptotic Bcl-2-associated x protein caspase-8 cleavage induced by TSWU-BR23. Co-expression and Bcl-2 antagonist killer 1, that lead to cytochrome c of p53 shRNA and dominant-negative mutant of FADD release from the mitochondria into the cytosol (2). Cytosolic completely inhibited TSWU-BR32-induced mitochondrial cytochrome c triggers the processing of pro-caspase-9, apoptotic cell death. Collectively, these data demonstrate that thereby initiating the formation of an apoptosome composed TSWU-BR23 leads to HT-29 cell apoptosis by inducing p53- of apoptotic protease-activating factor 1, dATP, caspase-9, mediated mitochondrial oligomerization of BAX/BAK and the and cytochrome c. Apoptosome formation leads to activation localization of CD95/FADD with lipid rafts at the cell of the executioner caspase-3, which causes apoptotic cell surface. death (1). Membrane lipid rafts are specialized, detergent-insoluble, cholesterol/glycosphingolipid-rich microdomains that can serve as recruitment platforms for membrane-associated Correspondence to: Dr. M.-L. Lin, Department of Medical receptors and downstream specific adaptors to affect signaling Laboratory Science and Biotechnology, China Medical University, transduction (3). CD95 and FADD concentration in lipid rafts No. 91, Hsueh-Shih Road, Taichung City 40402, Taiwan, R.O.C. can form apoptosis-promoting clusters (4). Evidence also Tel.: +886 422053366 ext. 7211, Fax: +886 422057414, e-mail: exists that recruitment of CD95 and FADD to lipid rafts can [email protected] be driven not only by the interaction between CD95 ligand Key Words: Apoptosis, BAX/BAK, bichalcone analog, CD95, and their receptors at the cell surface, but also through FADD, human colon carcinoma HT-29 cells. induction of chemotherapeutic agent in a CD95 ligand- 0250-7005/2015 $2.00+.40 5407 ANTICANCER RESEARCH 35: 5407-5416 (2015) independent manner (4). Thus, it has garnered intense interest Assays for the detection of caspase-3, -8, and -9 activities. Caspase- as a potential agent that can induce CD95 ligand-independent 3, -8, and -9 activities were measured using the PhiPhiLux G1D2 activation of apoptosis signal transduction pathways. kit (OncoImmunin, College Park, MD, USA), Caspase-8 Colorimetric Assay Kit (Abcam), and Caspase-9 Colorimetric Assay We previously used a Mannich base reaction to synthesize Kit (Calbiochem, San Diego, CA, USA) according to the a new synthetic bichalcone analog (E)-1-(3-((4-(4- manufacturer’s protocols. For the detection of caspase-3 activity, the acetylphenyl)piperazin-1-yl)methyl)-4-hydroxy-5- TSWU-BR23- or vehicle-treated cells were incubated with the methoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one, or TSWU- PhiPhiLux fluorogenic Caspase substrate at 37˚C for 1 h and were BR23, with a single B-ring pyridyl moiety (5). Although we then analyzed using a FACSCount flow cytometer. For the detection found that TSWU-BR23 potently activated the caspase cascade of caspase-8 and -9 activities, lysates of treated cells were incubated and induced apoptosis of human colon cancer HT-29 cell line with Ile-Glu-Thr-Asp (IETD)-p-nitroanilide (p-NA) or Leu-Glu-His- Asp (LEHD)-p-NA substrate at 37˚C for 1 h and were then analyzed through the induction of CD95 and FADD, the mechanism by using a microtiter plate reader at 405 nm. which TSWU-BR23 causes HT-29 cells to undergo apoptotic cell death was not elucidated. In the present study, we Measurement of DNA fragmentation. Histone-associated DNA investigated the molecular mechanism by which TSWU-BR23 fragments were determined using the Cell Death Detection enzyme- induces apoptotic effects in HT-29 cells and how these effects linked immunosorbent assay (ELISA) kit (Roche Applied Science, are modulated by CD95/FADD and mitochondria. Mannheim, Germany). Briefly, vehicle- or TSWU-BR23-treated cells were incubated in hypertonic buffer for 30 min at room Materials and Methods temperature. After centrifugation, the cell lysates were transferred into an anti-histone–coated microplate to bind histone-associated Cell culture. The human colon cancer HT-29 cell line was obtained DNA fragments. Plates were washed after 1.5 h of incubation, and as previously described (5). The HT-29 cell line was cultured nonspecific binding sites were saturated with blocking buffer. Plates routinely in Roswell Park Memorial Institute 1640 (RPMI-1640) were then incubated with peroxidase-conjugated anti-DNA for 1.5 h medium supplemented with 5% fetal bovine serum (FBS) and at room temperature. To determine the amount of retained grown in 10-cm tissue culture dishes at 37˚C in a humidified peroxidase, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate) was added as a substrate, and a spectrophotometer (Thermo Labsystems incubator containing 5% CO2. Multiskan Spectrum, Frankin, MA, USA) was used to measure the Chemicals, reagents and plasmids. Bismaleimidohexane (BMH), absorbance at 405 nm. Brij 98, Tris-HCl, cyclosporine A, methyl-β-cyclodextrin, and Triton X-100, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Western blot analysis. Treated or transfected cells were lysed in lysis The TSWU-BR23 was dissolved in and diluted with dimethyl buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 μg/ml aprotinin, sulfoxide (DMSO) and then stored at −20˚C as a 100 mM stock. 100 mM Na3VO4, 50 mM NaF, 0.5% NP-40). Protein concentration DMSO and potassium phosphate were purchased from Merck was determined by the Bradford method (Bio-Rad, Hercules, CA, (Darmstadt, Germany). Lipofectamine 2000 was obtained from USA). Proteins were separated by electrophoresis on a 10% sodium Invitrogen (Carlsbad, CA, USA). RPMI-1640, FBS, trypsin-EDTA, dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and glutamine were obtained from Gibco BRL (Grand Island, NY, and then transferred to polyvinylidene difluoride membranes USA). The caspase-3 activity assay kit was purchased from (Immobilon-P; Millipore, Bedford, MA, USA). Membranes were OncoImmunin (Gaithersburg, MD, USA). The plasmids encoding blocked overnight with phosphate buffered saline (PBS) containing p53 shRNA, green-fluorescent protein (GFP) shRNA, or dominant- 3% skim milk and then incubated with primary antibody against negative mutant form (dn) FADD were obtained from Addgene CD95, FADD, CD55, CD71, caspase-8, caspase-9, BAX, BAK, (Cambridge, MA, USA). BAX small-interfering RNA (siRNA), calnexin, BAX, BAK, BID, tBID, caspase-3, caspase-8, caspase-9, BAK siRNA, control siRNA, and western blotting luminol reagent cytochrome c, or COX2. Proteins were detected with horseradish were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, peroxidase-conjugated goat anti-mouse, goat anti-rabbit, or donkey USA). The BAX siRNA, BAK siRNA, and control siRNA were anti-goat antibodies and western Blotting Luminol Reagent. To dissolved in RNase-free water. confirm equal protein loading, β-actin was measured. Antibodies. The antibodies against BAK, BAX, and cytochrome c Cell surface biotinylation. This assay was performed as previously were obtained from BD Pharmingen (San Diego, CA, USA). described (6). Briefly, treated cells were washed twice in ice-cold Caspase-3, caspase-8, and caspase-9 antibodies were purchased PBS and incubated with 0.5 mg/ml of EZ-Link Sulfo-NHS-SS- from Calbiochem (San Diego, CA, USA). Anti-cycle-oxygenase Biotin (Pierce, Rockford, IL, USA) for 30 min at 4˚C. Biotinylated subunit II (COX2) and calnexin antibodies were obtained from cells were washed twice in ice-cold PBS and treated with 50 mM Abcam (Cambridge, MA, USA). The antibodies against p53 and - NH4Cl for 10 min at 4˚C to stop the biotinylation reaction. The phospho (p)-p53 (Ser 15) were purchased from Santa Cruz avidin-agarose beads (Pierce, Rockford, IL, USA) were then added Biotechnology.
Load more

Recommended publications
  • Combination of the Natural Compound Periplocin and TRAIL Induce
    Han et al. Journal of Experimental & Clinical Cancer Research (2019) 38:501 https://doi.org/10.1186/s13046-019-1498-z RESEARCH Open Access Combination of the natural compound Periplocin and TRAIL induce esophageal squamous cell carcinoma apoptosis in vitro and in vivo: Implication in anticancer therapy Lujuan Han1†, Suli Dai1†, Zhirong Li1, Cong Zhang1, Sisi Wei1, Ruinian Zhao1, Hongtao Zhang2, Lianmei Zhao1* and Baoen Shan1* Abstract Background: Esophageal cancer is one of the most common malignant tumors in the world. With currently available therapies, only 20% ~ 30% patients can survive this disease for more than 5 years. TRAIL, a natural ligand for death receptors that can induce the apoptosis of cancer cells, has been explored as a therapeutic agent for cancers, but it has been reported that many cancer cells are resistant to TRAIL, limiting the potential clinical use of TRAIL as a cancer therapy. Meanwhile, Periplocin (CPP), a natural compound from dry root of Periploca sepium Bge, has been studied for its anti-cancer activity in a variety of cancers. It is not clear whether CPP and TRAIL can have activity on esophageal squamous cell carcinoma (ESCC) cells, or whether the combination of these two agents can have synergistic activity. Methods: We used MTS assay, flow cytometry and TUNEL assay to detect the effects of CPP alone or in combination with TRAIL on ESCC cells. The mechanism of CPP enhances the activity of TRAIL was analyzed by western blot, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay. The anti-tumor effects and the potential toxic side effects of CPP alone or in combination with TRAIL were also evaluated in vivo.
    [Show full text]
  • XIAP's Profile in Human Cancer
    biomolecules Review XIAP’s Profile in Human Cancer Huailu Tu and Max Costa * Department of Environmental Medicine, Grossman School of Medicine, New York University, New York, NY 10010, USA; [email protected] * Correspondence: [email protected] Received: 16 September 2020; Accepted: 25 October 2020; Published: 29 October 2020 Abstract: XIAP, the X-linked inhibitor of apoptosis protein, regulates cell death signaling pathways through binding and inhibiting caspases. Mounting experimental research associated with XIAP has shown it to be a master regulator of cell death not only in apoptosis, but also in autophagy and necroptosis. As a vital decider on cell survival, XIAP is involved in the regulation of cancer initiation, promotion and progression. XIAP up-regulation occurs in many human diseases, resulting in a series of undesired effects such as raising the cellular tolerance to genetic lesions, inflammation and cytotoxicity. Hence, anti-tumor drugs targeting XIAP have become an important focus for cancer therapy research. RNA–XIAP interaction is a focus, which has enriched the general profile of XIAP regulation in human cancer. In this review, the basic functions of XIAP, its regulatory role in cancer, anti-XIAP drugs and recent findings about RNA–XIAP interactions are discussed. Keywords: XIAP; apoptosis; cancer; therapeutics; non-coding RNA 1. Introduction X-linked inhibitor of apoptosis protein (XIAP), also known as inhibitor of apoptosis protein 3 (IAP3), baculoviral IAP repeat-containing protein 4 (BIRC4), and human IAPs like protein (hILP), belongs to IAP family which was discovered in insect baculovirus [1]. Eight different IAPs have been isolated from human tissues: NAIP (BIRC1), BIRC2 (cIAP1), BIRC3 (cIAP2), XIAP (BIRC4), BIRC5 (survivin), BIRC6 (apollon), BIRC7 (livin) and BIRC8 [2].
    [Show full text]
  • Essential Role of Survivin, an Inhibitor of Apoptosis Protein, in T Cell
    Essential Role of Survivin, an Inhibitor of Apoptosis Protein, in T Cell Development, Maturation, and Homeostasis Zheng Xing,1 Edward M. Conway,2 Chulho Kang,1 and Astar Winoto1 1Department of Molecular and Cell Biology, Division of Immunology and Cancer Research Laboratory, University of California at Berkeley, Berkeley, CA 94720 2Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, B-3000 Leuven, Belgium Abstract Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell–specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre–T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development. Key words: proliferation • apoptosis • T cell development • survivin • IAP Introduction The thymus is the major organ of T lymphocyte maturation CD4 CD8 or CD4 CD8 (single positive [SP]) cells. and differentiation. During development, T cells have to These mature cells then migrate to the peripheral immune confront sequentially fateful decisions: the pre-TCR organs where they carry out their major function in defend- checkpoint, TCR chain rearrangements, positive selection, ing the body against foreign invasion.
    [Show full text]
  • Survivin-3B Potentiates Immune Escape in Cancer but Also Inhibits the Toxicity of Cancer Chemotherapy
    Published OnlineFirst July 15, 2013; DOI: 10.1158/0008-5472.CAN-13-0036 Cancer Molecular and Cellular Pathobiology Research Survivin-3B Potentiates Immune Escape in Cancer but Also Inhibits the Toxicity of Cancer Chemotherapy Fred erique Vegran 1,5, Romain Mary1, Anne Gibeaud1,Celine Mirjolet2, Bertrand Collin4,6, Alexandra Oudot4, Celine Charon-Barra3, Laurent Arnould3, Sarab Lizard-Nacol1, and Romain Boidot1 Abstract Dysregulation in patterns of alternative RNA splicing in cancer cells is emerging as a significant factor in cancer pathophysiology. In this study, we investigated the little known alternative splice isoform survivin-3B (S-3B) that is overexpressed in a tumor-specific manner. Ectopic overexpression of S-3B drove tumorigenesis by facilitating immune escape in a manner associated with resistance to immune cell toxicity. This resistance was mediated by interaction of S-3B with procaspase-8, inhibiting death-inducing signaling complex formation in response to Fas/ Fas ligand interaction. We found that S-3B overexpression also mediated resistance to cancer chemotherapy, in this case through interactions with procaspase-6. S-3B binding to procaspase-6 inhibited its activation despite mitochondrial depolarization and caspase-3 activation. When combined with chemotherapy, S-3B targeting in vivo elicited a nearly eradication of tumors. Mechanistic investigations identified a previously unrecognized 7-amino acid region as responsible for the procancerous properties of survivin proteins. Taken together, our results defined S-3B as an important functional actor in tumor formation and treatment resistance. Cancer Res; 73(17); 1–11. Ó2013 AACR. Introduction that can induce the expression of five different transcripts with D Alternative splicing is an important mechanism for the different functions: survivin, survivin- Ex3, survivin-2B (5), a generation of the variety of proteins indispensable for cell survivin-3B (S-3B; ref.
    [Show full text]
  • Cordycepin Induces Apoptosis in Human Liver Cancer Hepg2 Cells Through Extrinsic and Intrinsic Signaling Pathways
    ONCOLOGY LETTERS 12: 995-1000, 2016 Cordycepin induces apoptosis in human liver cancer HepG2 cells through extrinsic and intrinsic signaling pathways LE-WEN SHAO1, LI-HUA HUANG1, SHENG YAN2, JIAN-DI JIN3 and SHAO-YAN REN3 1Nursing Department; Departments of 2Hepato-Biliary-Pancreatic Surgery and 3Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China Received March 6, 2015; Accepted April 12, 2016 DOI: 10.3892/ol.2016.4706 Abstract. Cordycepin, also termed 3'-deoxyadenosine, is a to have natural medicinal properties, such as anti-angiogenic, nucleoside analogue from Cordyceps sinensis and has been anti‑tumor, anti‑viral, anti‑inflammatory and hypoglycemic reported to demonstrate numerous biological and pharma- effects (1-8). cological properties. Our previous study illustrated that the Cordycepin, also termed 3'-deoxyadenosine, is a nucleo- anti-tumor effect of cordycepin may be associated with apop- side analogue from C. militaris (9) and has been reported tosis. In the present study, the apoptotic effect of cordycepin to demonstrate numerous notable biological and pharma- on HepG2 cells was investigated using 4',6-diamidino-2-phe- cological properties, including immunological stimulation, nylindole, tetraethylbenzimidazolylcarbocyanine iodide and anti-cancer and anti-viral effects (10-15), a stimulating effect propidium iodide staining analysis and flow cytometry. The on interlukin-10 production as an immune modulator (16) and results showed that cordycepin exhibited the ability to inhibit preventing hyperlipidermia (17). HepG2 cells in a time- and dose-dependent manner when cells Apoptosis, also termed programmed cell death, is a key produced typical apoptotic morphological changes, including regulator of tissue homeostasis and is characterized by chromatin condensation, the accumulation of sub-G1 cells and typical morphological and biochemical hallmarks, including change mitochondrial permeability.
    [Show full text]
  • Therapeutic Small Molecules Target Inhibitor of Apoptosis Proteins In
    Published OnlineFirst December 30, 2016; DOI: 10.1158/1078-0432.CCR-16-2172 Review Clinical Cancer Research Therapeutic Small Molecules Target Inhibitor of Apoptosis Proteins in Cancers with Deregulation of Extrinsic and Intrinsic Cell Death Pathways Adeeb Derakhshan, Zhong Chen, and Carter Van Waes Abstract TheCancerGenomeAtlas(TCGA)hasunveiledgenomic the results of targeting IAPs in preclinical models of HNSCC deregulation of various components of the extrinsic and intrin- using SMAC mimetics. Synergistic activity of SMAC mimetics sic apoptotic pathways in different types of cancers. Such together with death agonists TNFa or TRAIL occurred in vitro, alterations are particularly common in head and neck squa- whereas their antitumor effects were augmented when com- mous cell carcinomas (HNSCC), which frequently display bined with radiation and chemotherapeutic agents that induce amplification and overexpression of the Fas-associated via TNFa in vivo. In addition, clinical trials testing SMAC mimetics death domain (FADD) and inhibitor of apoptosis proteins as single agents or together with chemo- or radiation therapies (IAP) that complex with members of the TNF receptor family. in patients with HNSCC and solid tumors are summarized. Second mitochondria-derived activator of caspases (SMAC) As we achieve a deeper understanding of the genomic altera- mimetics, modeled after the endogenous IAP antagonist tions and molecular mechanisms underlying deregulated SMAC, and IAP inhibitors represent important classes of novel death and survival pathways in different cancers, the role small molecules currently in phase I/II clinical trials. Here we of SMAC mimetics and IAP inhibitors in cancer treatment will review the physiologic roles of IAPs, FADD, and other com- be elucidated.
    [Show full text]
  • Roles of Bid and XIAP
    Cell Death and Differentiation (2012) 19, 42–50 & 2012 Macmillan Publishers Limited All rights reserved 1350-9047/12 www.nature.com/cdd Review Fas death receptor signalling: roles of Bid and XIAP T Kaufmann*,1, A Strasser2,3 and PJ Jost4 Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.
    [Show full text]
  • Caspase Activation & Apoptosis
    RnDSy-lu-2945 Caspase Activation & Apoptosis Extrinsic & Intrinsic Pathways of Caspase Activation CASPASE CLEAVAGE & ACTIVATION Caspases are a family of aspartate-specific, cysteine proteases that serve as the primary mediators of Pro-Domain Large Subunit (p20) Small Subunit (p10) apoptosis. Mammalian caspases can be subdivided into three functional groups, apoptotic initiator TRAIL Pro-Domain α chain β chain caspases (Caspase-2, -8, -9, -10), apoptotic effector caspases (Caspase-3, -6, -7), and caspases involved Asp-x Asp-x Proteolytic cleavage in inflammatory cytokine processing (Caspase-1, -4, -5, 11, and -12L/12S). All caspases are synthesized α chain as inactive zymogens containing a variable length pro-domain, followed by a large (20 kDa) and a small Fas Ligand TRAIL R1 Pro-Domain β chain TRAIL R2 Heterotetramer Formation (10 kDa) subunit. FADD FADD α chain α chain Pro-caspase-8, -10 Active caspase Pro-caspase-8, -10 TWEAK β chain Apoptotic caspases are activated upon the receipt of either an extrinsic or an intrinsic death signal. The Fas/CD95 extrinsic pathway (green arrows) is initiated by ligand binding to cell surface death receptors (TNF RI, Fas/ MAMMALIAN CASPASE DOMAINS & CLEAVAGE SITES FADD FADD TNF-α APOPTOTIC CASPASES CD95, DR3, TRAIL R1/DR4, TRAIL R2/DR5) followed by receptor oligomerization and cleavage of Pro- Extrinsic Pathway DR3 (or another TWEAK R) INITIATOR CASPASES 152 316 331 Pro-caspase-8, -10 Pro-caspase-8, -10 1 435 caspase-8 and -10. Activation of Caspase-8 and Caspase-10 results in the cleavage of BID and Caspase-2 CARD TRADD FLIP TRADD downstream effector caspases.
    [Show full text]
  • Regulation of Caspase-8 Activity at the Crossroads of Pro-Inflammation
    International Journal of Molecular Sciences Review Regulation of Caspase-8 Activity at the Crossroads of Pro-Inflammation and Anti-Inflammation Jun-Hyuk Han 1, Jooho Park 1,2, Tae-Bong Kang 1,3,* and Kwang-Ho Lee 1,3 1 Department of Applied Life Sciences, Graduate School, BK21 Program, Konkuk University, Chungju 27478, Korea; [email protected] (J.-H.H.); [email protected] (J.P.); [email protected] (K.-H.L.) 2 Department of Biomedical Chemistry, College of Biomedical & Health Science, Konkuk University, Chungju 27487, Korea 3 Department of Biotechnology, College of Biomedical & Health Science, Konkuk University, Chungju 27487, Korea * Correspondence: [email protected]; Tel.: +82-43-840-3904 Abstract: Caspase-8 has been classified as an apoptotic caspase, and its initial definition was an initiator of extrinsic cell death. During the past decade, the concept of caspase-8 functioning has been changed by findings of its additional roles in diverse biological processes. Although caspase-8 was not originally thought to be involved in the inflammation process, many recent works have determined that caspase-8 plays an important role in the regulatory functions of inflammatory processes. In this review, we describe the recent advances in knowledge regarding the manner in which caspase-8 modulates the inflammatory responses concerning inflammasome activation, cell death, and cytokine induction. Keywords: caspase-8; inflammasome; inflammation; necroptosis; pyroptosis; apoptosis Citation: Han, J.-H.; Park, J.; Kang, T.-B.; Lee, K.-H. Regulation of Caspase-8 Activity at the Crossroads 1. Introduction of Pro-Inflammation and Anti-Inflammation. Int. J. Mol. Sci. Mammalian caspases have classically been divided into inflammatory and apoptotic 2021, 22, 3318.
    [Show full text]
  • YM155 Sensitizes Hela Cells to TRAIL‑Mediated Apoptosis Via Cflip and Survivin Downregulation
    ONCOLOGY LETTERS 20: 72, 2020 YM155 sensitizes HeLa cells to TRAIL‑mediated apoptosis via cFLIP and survivin downregulation ARUN PANDIAN CHANDRASEKARAN1*, NARESH POONDLA1*, NA RE KO2,3, SEUNG JUN OH3 and SURESH RAMAKRISHNA1,4 1Graduate School of Biomedical Science and Engineering, Department of Biomedical Science, Hanyang University, Seoul 04763; 2Biomedical Research Center, Asan Institute for Life Sciences; 3Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; 4College of Medicine, Department of Genetics, Hanyang University, Seoul 04763, Republic of Korea Received February 19, 2020; Accepted June 16, 2020 DOI: 10.3892/ol.2020.11933 Abstract. Tumor necrosis factor‑related apoptosis inducing results suggested that the combination treatment significantly ligand (TRAIL)‑mediated apoptosis is a safe method for reduced cell viability, invasion and migration of HeLa cells. the treatment of various types of cancer. However, TRAIL Overall, the present findings indicated that the low dosage of therapy is less effective in certain types of cancer, including YM155 sensitized HeLa cells to TRAIL‑induced apoptosis cervical cancer. To address this problem, a combinatorial via a mechanism involving downregulation of cFLIP and approach was employed to sensitize cervical cancer at low survivin. The results indicated the importance of combination dosages. YM155, a survivin inhibitor, was used at low dosages drug treatment and reveal an effective therapeutic alternative along with TRAIL to induce apoptosis in HeLa cells. The for TRAIL therapy in human cervical cancer. effects of the individual treatment with TRAIL and YM155 on apoptosis were assessed by propidium iodide assay. In Introduction addition, to validate the DNA damage exhibited by the combi‑ nation treatment, the phosphorylation status of γH2A histone Cervical cancer is the fourth most prevalent type of cancer family member X was investigated by immunofluorescence in females globally and a significant cause of mortality in and western blot analysis.
    [Show full text]
  • TRAF2 Is a Biologically Important Necroptosis Suppressor
    Cell Death and Differentiation (2015) 22, 1846–1857 & 2015 Macmillan Publishers Limited All rights reserved 1350-9047/15 www.nature.com/cdd TRAF2 is a biologically important necroptosis suppressor SL Petersen1, TT Chen1, DA Lawrence1, SA Marsters1, F Gonzalvez1 and A Ashkenazi*,1 Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor- interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.
    [Show full text]
  • Or FADD-Binding Proteins Fas/FLICE Receptor
    Cutting Edge: Apoptosis Induced by a Chimeric Fas/FLICE Receptor: Lack of Requirement for Fas- or FADD-Binding Proteins This information is current as Sarfraz A. Memon, Jing-zhou Hou, M. Belen Moreno and Charles of September 28, 2021. M. Zacharchuk J Immunol 1998; 160:2046-2049; ; http://www.jimmunol.org/content/160/5/2046 Downloaded from References This article cites 15 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/160/5/2046.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. c Cutting Edge: Apoptosis Induced by a Chimeric Fas/FLICE Receptor: Lack of Requirement for Fas- or FADD-Binding Proteins Sarfraz A. Memon, Jing-zhou Hou, M. Belen Moreno, and Charles M. Zacharchuk1 stream substrates. This model is supported by the ability of caspase Current models for Fas (CD95)-mediated apoptosis suggest inhibitors and dominant negative variants of FADD and FLICE to that FLICE/caspase-8 is recruited and activated, which results block Fas-mediated cell death (4, 5).
    [Show full text]