Artigo Acesso aberto Revisado por pares

The Marine Product Cephalostatin 1 Activates an Endoplasmic Reticulum Stress-specific and Apoptosome-independent Apoptotic Signaling Pathway

2006; Elsevier BV; Volume: 281; Issue: 44 Linguagem: Inglês

10.1074/jbc.m607904200

ISSN

1083-351X

Autores

Nancy López-Antón, Anita Rudy, Nicole D. Barth, M. Lienhard Schmitz, George R. Pettit, Klaus Schulze‐Osthoff, Verena M. Dirsch, Angelika M. Vollmar,

Tópico(s)

Redox biology and oxidative stress

Resumo

Cephalostatin 1, a bis-steroidal marine natural product, has been reported to induce apoptosis without the requirement of an active caspase-8 or mitochondrial cytochrome c release and apoptosome formation. Here we show that despite the absence of these events, caspase-9 activation is essential for cephalostatin 1-induced apoptosis. Cephalostatin 1 initiates a rapid endoplasmic reticulum stress response characterized by phosphorylation of eukaryotic initiation factor-2 α-subunit and increased expression of the chaperone immunoglobulin heavy chain-binding protein GRP78 as well as the transcription factor C/EBP homologous protein (CHOP)/GADD153. Cephalostatin 1 activates apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (JNK). However, this pathway does not play a major role in cephalostatin 1-induced apoptosis, as assessed by stable expression of a dominant negative apoptosis signal-regulating kinase 1. Importantly, the endoplasmic reticulum-associated caspase-4 is required and as shown by biochemical and genetic inhibition experiments, acts upstream of caspase-9 in cephalostatin-induced apoptosis. Cephalostatin 1, a bis-steroidal marine natural product, has been reported to induce apoptosis without the requirement of an active caspase-8 or mitochondrial cytochrome c release and apoptosome formation. Here we show that despite the absence of these events, caspase-9 activation is essential for cephalostatin 1-induced apoptosis. Cephalostatin 1 initiates a rapid endoplasmic reticulum stress response characterized by phosphorylation of eukaryotic initiation factor-2 α-subunit and increased expression of the chaperone immunoglobulin heavy chain-binding protein GRP78 as well as the transcription factor C/EBP homologous protein (CHOP)/GADD153. Cephalostatin 1 activates apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (JNK). However, this pathway does not play a major role in cephalostatin 1-induced apoptosis, as assessed by stable expression of a dominant negative apoptosis signal-regulating kinase 1. Importantly, the endoplasmic reticulum-associated caspase-4 is required and as shown by biochemical and genetic inhibition experiments, acts upstream of caspase-9 in cephalostatin-induced apoptosis. Apoptosis dysregulation represents an important contribution to the development of cancer as well as chemoresistance. Moreover, a wide range of chemotherapeutic drugs induces death in malignant cells by inducing apoptosis (1Kim R. Cancer. 2005; 103: 1551-1560Crossref PubMed Scopus (259) Google Scholar, 2Herr I. Debatin K.M. Blood. 2001; 98: 2603-2614Crossref PubMed Scopus (683) Google Scholar). Two major pathways leading to apoptosis have been elucidated: one triggered by tumor necrosis factor (TNF) 3The abbreviations used are: TNF, tumor necrosis factor; Apaf-1, apoptosis activating factor-1; ASK1, apoptosis signal-regulating kinase 1; BiP, immunoglobulin heavy chain-binding protein; BiP/GRP78, 78 kDa glucose-regulated protein; CHOP, C/EBP homologous protein; GADD153, growth arrest and DNA damage-inducible gene 153; TG, thapsigargin; ETO, etoposide; eIF2α, eukaryotic initiation factor 2 α-subunit; ER, endoplasmic reticulum; IRE1, inositol requiring 1; TM, tunicamycin; JNK, c-Jun N-terminal kinase; siRNA, small interfering RNA; TRAF2, TNF receptor-associated factor 2; UPR, unfolded protein response; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone. 3The abbreviations used are: TNF, tumor necrosis factor; Apaf-1, apoptosis activating factor-1; ASK1, apoptosis signal-regulating kinase 1; BiP, immunoglobulin heavy chain-binding protein; BiP/GRP78, 78 kDa glucose-regulated protein; CHOP, C/EBP homologous protein; GADD153, growth arrest and DNA damage-inducible gene 153; TG, thapsigargin; ETO, etoposide; eIF2α, eukaryotic initiation factor 2 α-subunit; ER, endoplasmic reticulum; IRE1, inositol requiring 1; TM, tunicamycin; JNK, c-Jun N-terminal kinase; siRNA, small interfering RNA; TRAF2, TNF receptor-associated factor 2; UPR, unfolded protein response; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone./Fas family receptors and the other mediated by mitochondrial release of cytochrome c and other proteins (1Kim R. Cancer. 2005; 103: 1551-1560Crossref PubMed Scopus (259) Google Scholar, 3Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1613) Google Scholar). However, damage or stress in many organelles (besides mitochondria) may trigger apoptosis through mechanisms, which remain partially unclear (4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1291) Google Scholar). In this respect, a pathway of apoptosis induction has been linked to stress in the endoplasmic reticulum (ER) (5Breckenridge D.G. Germain M. Mathai J.P. Nguyen M. Shore G.C. Oncogene. 2003; 22: 8608-8618Crossref PubMed Scopus (647) Google Scholar, 6Kadowaki H. Nishitoh H. Ichijo H. J. Chem. Neuroanat. 2004; 28: 93-100Crossref PubMed Scopus (119) Google Scholar, 7Momoi T. J. Chem. Neuroanat. 2004; 28: 101-105Crossref PubMed Scopus (184) Google Scholar, 8Rao R.V. Ellerby H.M. Bredesen D.E. Cell Death Differ. 2004; 11: 372-380Crossref PubMed Scopus (811) Google Scholar). ER stress leads to the activation of genes possessing an unfolded protein response (UPR) element, which controls the levels of molecular chaperones, such as Mr 78,000 glucose-regulated stress protein (BiP/GRP78) involved in protein folding in the ER (9Schroder M. Kaufman R.J. Mutat. Res. 2005; 569: 29-63Crossref PubMed Scopus (1367) Google Scholar). Furthermore, the eukaryotic initiation factor-2 (eIF2) is phosphorylated by the PKR-like ER-localized eIF2α kinase (PERK) in response to ER stress leading to an attenuation of translational initiation and protein synthesis (8Rao R.V. Ellerby H.M. Bredesen D.E. Cell Death Differ. 2004; 11: 372-380Crossref PubMed Scopus (811) Google Scholar). When these stress modulators are unable to rescue cells, various apoptotic pathways are activated. Recruitment of TNF receptor-associated factor 2 (TRAF2) to activated stress sensor proteins, called IREs, induces the apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) cascade (6Kadowaki H. Nishitoh H. Ichijo H. J. Chem. Neuroanat. 2004; 28: 93-100Crossref PubMed Scopus (119) Google Scholar). In addition, ER-specific caspases, such as caspase-12 in the murine system, seem to aggregate at the ER membrane surface through TRAF2 proteins resulting in their cleavage and activation (7Momoi T. J. Chem. Neuroanat. 2004; 28: 101-105Crossref PubMed Scopus (184) Google Scholar). In humans, caspase-4 has been proposed to play a role as ER stress-specific caspase similar to caspase-12 (10Hitomi J. Katayama T. Eguchi Y. Kudo T. Taniguchi M. Koyama Y. Manabe T. Yamagishi S. Bando Y. Imaizumi K. Tsujimoto Y. Tohyama M. J. Cell Biol. 2004; 165: 347-356Crossref PubMed Scopus (730) Google Scholar). Furthermore, the UPR increases the transcription of the transcription factor C/EBP homologous protein (CHOP), which is closely associated with cell death (11Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1665) Google Scholar, 12Oyadomari S. Mori M. Cell Death Differ. 2004; 11: 381-389Crossref PubMed Scopus (2193) Google Scholar). ER stress pathways linked to apoptosis have been reported in pathological states such as ischemia-reperfusion injury and diabetes as well as in neurodegenerative diseases such as Alzheimer and Parkinson, where abnormalities in protein folding have been identified (8Rao R.V. Ellerby H.M. Bredesen D.E. Cell Death Differ. 2004; 11: 372-380Crossref PubMed Scopus (811) Google Scholar, 9Schroder M. Kaufman R.J. Mutat. Res. 2005; 569: 29-63Crossref PubMed Scopus (1367) Google Scholar, 13Paschen W. Cell Calcium. 2003; 34: 365-383Crossref PubMed Scopus (168) Google Scholar). The role of ER stress in tumor development and therapy is unclear at present, although supposedly ER stress response is important for regulating the balance between tumor cell death and its growth as well as for the sensitivity to chemotherapeutic agents (14Ma Y. Hendershot L.M. Nat. Rev. Cancer. 2004; 4: 966-977Crossref PubMed Scopus (591) Google Scholar).Because most of the cancer chemotherapeutic drugs signal through mitochondrial cytochrome c release, it is very difficult to distinguish as to whether the ER may play a role as stress sensor for chemotherapeutics that reroute the signal directly through mitochondria and the apoptosome or whether the ER is able to activate its own death pathway (2Herr I. Debatin K.M. Blood. 2001; 98: 2603-2614Crossref PubMed Scopus (683) Google Scholar, 3Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1613) Google Scholar, 4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1291) Google Scholar, 8Rao R.V. Ellerby H.M. Bredesen D.E. Cell Death Differ. 2004; 11: 372-380Crossref PubMed Scopus (811) Google Scholar). In this respect, it is important to characterize specific elements of the ER stress response because they could represent novel targets for the development of new cancer chemotherapeutic strategies.We recently characterized an experimental anticancer agent, cephalostatin 1, that showed promise to be a helpful tool in this respect. Cephalostatin 1 is a bis-steroidal marine natural product, which induces a novel pathway of apoptosis in leukemia T cells. Cephalostatin 1 triggers cell death in a CD95- and caspase-8-independent manner. Even more intriguingly, without triggering cytochrome c release from mitochondria and apoptosome formation, cephalostatin-induced apoptosis is accompanied by caspase-9 activation (15Dirsch V.M. Muller I.M. Eichhorst S.T. Pettit G.R. Kamano Y. Inoue M. Xu J.P. Ichihara Y. Wanner G. Vollmar A.M. Cancer Res. 2003; 63: 8869-8876PubMed Google Scholar). Apoptosome-independent cell death induction has been described in the literature before (16Marsden V.S. O'Connor L. O'Reilly L.A. Silke J. Metcalf D. Ekert P.G. Huang D.C. Cecconi F. Kuida K. Tomaselli K.J. Roy S. Nicholson D.W. Vaux D.L. Bouillet P. Adams J.M. Strasser A. Nature. 2002; 419: 634-637Crossref PubMed Scopus (486) Google Scholar, 17Henderson C.J. Aleo E. Fontanini A. Maestro R. Paroni G. Brancolini C. Cell Death Differ. 2005; 12: 1240-1254Crossref PubMed Scopus (45) Google Scholar), but in these cases, apoptosis occurred independently of caspase-9 activation.In search of the mechanisms responsible for apoptosome-independent caspase-9 activation, we hypothesized that cephalostatin 1 induces ER stress. Thus, we investigated several markers of ER stress induced by cephalostatin 1 and identified ER-specific molecular players such as caspase-4 and the ASK1/JNK cascade being involved in apoptosome-independent execution of cell death.EXPERIMENTAL PROCEDURESCompounds—Cephalostatin 1 was isolated from the marine worm Cephalodiscus gilchristi as described previously (18Pettit G.R. Inoue M. Kamano Y. Herald D.L. Arm C. Dufresne C. Christie N.D. Schmidt J.M. Doubek D.L. Krupa T.S. J. Am. Chem. Soc. 1988; 110: 2006-2007Crossref Scopus (193) Google Scholar). Purity of the compound was 98% as judged by high performance liquid chromatography. Before application, cephalostatin 1 was dissolved and further diluted in Me2SO. Final Me2SO concentration did not exceed 1%, a concentration verified not to interfere with the experiments performed. Propidium iodide, thapsigargin (TG), tunicamycin (TM), and human TNF-α were from Sigma (Deisenhofen, Germany); etoposide (ETO) and the specific JNK inhibitor SP600125 were from Calbiochem; and the selective caspase-4 inhibitor benzyloxycarbonyl-LEVD-fluoromethyl ketone (Z-LEVD-fmk) was from MBL (Woburn, MA).Cell Culture—Human leukemia Jurkat T cells (J16) (kindly provided by P.H. Krammer and H. Walczak, Heidelberg, Germany), Jurkat T cells lacking caspase-9 (Casp.9–/–), deficient cells stably retransfected with full-length caspase-9 (Casp. 9+/+) (19Samraj A.K. Keil E. Ueffing N. Schulze-Osthoff K. Schmitz I. J. Biol. Chem. 2006; 281: 29652-29659Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), Bak-deficient Jurkat cells (Bak–/–, JCaM1.6), deficient cells reconstituted with Bak (Bak+/+) (20Samraj A.K. Stroh C. Fischer U. Schulze-Osthoff K. Oncogene. 2006; 25: 186-197Crossref PubMed Scopus (40) Google Scholar) as well as a Jurkat cell line expressing an inactive form of ASK1 (ASK1-DN, clones A2–1 and A2–3) (21Hofmann T.G. Moller A. Hehner S.P. Welsch D. Droge W. Schmitz M.L. Int. J. Cancer. 2001; 93: 185-191Crossref PubMed Scopus (22) Google Scholar) were cultured (37 °C and 5% CO2) in RPMI 1640 medium containing 2 mm l-glutamine (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Cölbe, Germany) and 1% pyruvate (Merck, Darmstadt, Germany). Medium of ASK1-DN transfected cells was supplemented with 1 mg/ml G418 (PAA Laboratories, Cölbe, Germany) every fifth passage.Quantification of Apoptosis—Quantification of apoptosis was performed according to Nicoletti et al. (22Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4406) Google Scholar). Briefly, cells were incubated in a hypotonic buffer (0.1% sodium citrate, 0.1% Triton X-100, and 50 μg/ml propidium iodide) overnight at 4 °C and analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, Heidelberg, Germany). Nuclei to the left of the G1 peak containing hypodiploid DNA were considered apoptotic.Western Blot Analysis—Cells were collected by centrifugation, washed with ice-cold phosphate-buffered saline, and lysed for 30 min either in 1% Triton X-100, 150 mm NaCl, 2 mm EDTA, and 30 mm Tris-HCl, pH 7.5 with the protease inhibitor Complete™ (Roche, Mannheim, Germany) (for Apaf-1, BiP, CHOP, and caspases); in 2 mm EDTA, 137 mm NaCl, 10% glycerol, 2 mm tetrasodium pyrophosphate, 20 mm Tris, 1% Triton X-100, 20 mm sodium glycerophosphate hydrate, 10 mm NaF, 2 mm sodium orthovanadate, and 1 mm phenylmethylsulfonyl fluoride supplemented with Complete™ (for p-JNK, p-eIF2α, and JNK); or in 100 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm NaF, 20 mm tetrasodium pyrophosphate, 2 mm sodium orthovanadate, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, and 10 mm Tris, pH 7.4 with Complete™ (for p-ASK1 and ASK1). Lysates were homogenized with an ultrasonic device and centrifuged at 10,000 × g for 10 min at 4 °C. Equal amounts of protein were separated by SDS-PAGE (7.5% for Apaf-1 and ASK1; 10% for JNK; 12% for BiP, CHOP, p-eIF2α, and caspases) and transferred to nitrocellulose membranes (Hybond™ ECL™; Amersham Biosciences). Membranes were blocked with 5% fat-free milk powder in phosphate-buffered saline containing 0.05% Tween 20 (1 h) and incubated with specific antibodies against Apaf-1 (mouse IgG1; BD Transduction Laboratories), Bak (mouse IgG2a; Calbiochem), BiP (mouse IgG2a; BD Transduction Laboratories), CHOP (rabbit polyclonal antibody; Sigma), p-eIF2α (rabbit polyclonal antibody; Cell Signaling, Frankfurt, Germany), p-ASK1 (Thr-845) (rabbit polyclonal antibody; Cell Signaling), ASK1 (rabbit polyclonal antibody; Cell Signaling), p-JNK (Thr-183/Tyr-185) (mouse IgG1 monoclonal antibody; Cell Signaling), JNK (rabbit polyclonal antibody; Cell Signaling), caspase-4 (mouse IgG1κ, clone 4B9; MBL), or caspase-9 (rabbit polyclonal antibody; Cell Signaling) overnight at 4 °C. Specific proteins were visualized by secondary antibodies conjugated to horseradish peroxidase and the ECL Plus™ Western blotting detection reagent (Amersham Biosciences). Membranes were exposed to x-ray film for the appropriate time periods and subsequently developed in a tabletop film processor (Curix 60; Agfa, Cologne, Germany). Equal protein loading was controlled by Ponceau S staining of membranes.Apaf-1 siRNA—Sense and antisense small interfering RNA (siRNA) oligonucleotides corresponding to nucleotides 978–998 of Apaf-1 (AATTGGTGCACTTTTACGTGA)(23Lassus P. Opitz-Araya X. Lazebnik Y. Science. 2002; 297: 1352-1354Crossref PubMed Scopus (657) Google Scholar) and oligonucleotides corresponding to a scramble sequence were purchased from biomers.net GmbH (Ulm, Germany) and annealed to create the double-stranded siRNAs. Jurkat cells were transfected with 3 μg of scramble or Apaf-1 siRNAs using the Nucleofector™ II (Amaxa, Cologne, Germany) according to the manufacturer's instructions.Clonogenic Assay—Caspase-9-deficient Jurkat cells and cells retransfected with full-length caspase-9 were left untreated or treated with cephalostatin 1 for 2 h. Subsequently, cells were washed with phosphate-buffered saline and resuspended in culture medium (5 × 105 cells/ml). Cell suspensions were diluted 1:10 with methylcellulose (0.52%) medium containing 40% fetal calf serum. Cells were seeded in 96-well plates (100 μl), and colonies were scored after 7 days of culture.Caspase-9 Activity Assay—Caspase-9 activity was measured using the Caspase-Glo™ 9 assay from Promega GmbH (Mannheim, Germany) following the manufacturer's protocol. Jurkat T cells grown in 96-well plates were left untreated or treated with cephalostatin 1 for 4 h with or without preaddition of caspase-4 inhibitor. After stimulation, Caspase-Glo™ 9 reagent was added to each well and gently mixed. Luminescence was measured immediately in a plate-reading multifunction photometer (SPECTRAFluor Plus; Tecan, Crailsheim, Germany) every 10 min for 2 h. Background luminescence corresponding to the culture medium was subtracted from experimental values.Plasmid Construction—A siRNA-expressing sequence for targeting the caspase-4 gene was cloned into a psiRNA-h7SKneo G1 expression vector system (InvivoGen, San Diego, CA) to generate siRNA. Forward target sequences of the caspase-4 siRNA hairpin transcripts were 5′-acctcAAGTGGCCTCTTCACAGTCATtcaagagATGACTGTGAAGAGGCCACTTtt-3′ and 5′-acctcAAGATTTCCTCACTGGTGTTTccaagagAAACACCAGTGAGGAAATCTTtt-3′, respectively (10Hitomi J. Katayama T. Eguchi Y. Kudo T. Taniguchi M. Koyama Y. Manabe T. Yamagishi S. Bando Y. Imaizumi K. Tsujimoto Y. Tohyama M. J. Cell Biol. 2004; 165: 347-356Crossref PubMed Scopus (730) Google Scholar) (uppercase letters denote the double-stranded region corresponding to the caspase-4 sequence targeted). Oligonucleotides encoding a scramble sequence (universal control from InvivoGen) were used as a control. After hybridization, oligonucleotides were cloned into the BbsI site of psiRNA-h7SKneo G1 expression vector, and the plasmids were sequenced and amplified. The recombinant plasmid was transformed into Escherichia coli LyoComp GT116 strain, and the resultant cells were cultured in LB-kanamycin-containing medium. The recombinant plasmids in the selected colonies were extracted, digested with SpeI, and run on 0.8% agarose gel to isolate the cells containing psiRNA-h7SKneo G1 caspase-4 siRNA plasmids.Plasmid Transfection—Jurkat cells were transfected by electroporation with the Nucleofector™ II (Amaxa) according to the manufacturer's protocol. 4 × 106 Jurkat T cells in exponential growing phase were transfected with 3 μg of psiRNA-h7SKneo, 3 μg of psiRNA-h7SKnScr (a control plasmid containing a scramble sequence), or 3–4 μgofa1:1 mixture of the siRNA constructs. Efficiency of RNA interference was checked by Western blot analysis using antibodies against caspase-4.To study the influence of caspase-4 down-regulation in apoptosis, caspase-4 siRNA-generating plasmids were transfected into Jurkat cells as described above, and cells were incubated for 24 h before induction of apoptosis. To investigate caspase-9 activation in cells with silenced caspase-4 expression, 1 mg/ml G418 was added to the culture medium 2 days after transfection, and experiments were performed 48 h later. In both experiments, cells containing psiRNA-h7SKneo and psiRNA-h7SKnScr vectors were used as controls.Statistical Analysis—All experiments were performed at least three times in triplicate. Results are expressed as mean value ± S.E. Student's unpaired two-tailed t test was performed using GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA). p values <0.05 were considered significant.RESULTSCaspase-9 Is Essential in Apoptosome-independent Apoptosis Induced by Cephalostatin 1—Cephalostatin 1 activates caspase-9 and induces apoptosis without cytochrome c release and apoptosome formation (15Dirsch V.M. Muller I.M. Eichhorst S.T. Pettit G.R. Kamano Y. Inoue M. Xu J.P. Ichihara Y. Wanner G. Vollmar A.M. Cancer Res. 2003; 63: 8869-8876PubMed Google Scholar). To further support this unique finding two strategies were used: 1) Bak-deficient Jurkat cells (Bak–/–) were exposed to cephalostatin 1 as well as etoposide as positive control. Because Jurkat cells do not express Bax (24Brimmell M. Mendiola R. Mangion J. Packham G. Oncogene. 1998; 16: 1803-1812Crossref PubMed Scopus (147) Google Scholar), the Bak–/– Jurkat cells are resistant to apoptotic stimuli such as etoposide that use the intrinsic mitochondrial pathway hallmarked by release of cytochrome c (Fig. 1A). Importantly, Bak–/– cells are equally sensitive to cephalostatin as Bak-reconstituted control cells (Fig. 1A), further pointing to a cytochrome c-independent signaling pathway. 2) To confirm cephalostatin 1 induction of apoptosis without apoptosome formation, Apaf-1 was silenced via siRNA in Jurkat cells. Cephalostatin 1 killed cells transfected with Apaf-1 siRNA to the same extent as cells transfected with a scramble siRNA sequence (Fig. 1B, left). The apoptotic response to etoposide, however, was significantly blunted in Apaf-1-silenced cells. Moreover, caspase-9 was activated to the same extent in Apaf-1 as in scramble siRNA-transfected cells upon treatment with cephalostatin 1 (Fig. 1B, right), confirming that activation of caspase-9 occurred independently of the apoptosome complex. Apoptosis induction and more intriguingly, activation of caspase-9 independently from the classical mitochondrial signaling raises the question of the role and activation pathway of caspase-9 in cephalostatin 1-induced apoptosis. For this purpose, we used a Jurkat cell line deficient in caspase-9. Interestingly, apoptosis induced by cephalostatin 1 was almost completely inhibited in caspase-9-deficient cells, whereas cells stably retransfected with full-length caspase-9 died normally when exposed to cephalostatin (Fig. 1C, left). Moreover, significant differences between both cell lines were still observed in a 7-day clonogenic assay (Fig. 1C, right). These data ask for clarifying the mechanism underlying the apoptosome-independent activation of caspase-9 by cephalostatin 1.Cephalostatin 1 Induces ER Stress in Jurkat Leukemia T Cells—In search of the initial event leading to caspase-9 activation and apoptosis, cephalostatin 1 was hypothesized to induce ER stress. In fact, the expressions of an ER stress sensor (BiP/GRP78) and an ER stress-induced cell death modulator (CHOP/GADD153) were affected by cephalostatin 1. As shown in Fig. 2, cephalostatin 1 (1 μm) increased expression of the ER chaperone BiP/GRP78 and the transcription factor CHOP/GADD153 as did the known ER stress-inducer tunicamycin. The increase of BiP showed a biphasic pattern with an early increase (30 min to 1 h) followed by a plateau and a second strong elevation after 16–24 h. CHOP protein level increased rapidly (15 min to 1 h) and decreased slowly thereafter. The eukaryotic initiation factor-2 is phosphorylated by the PKR-like ER-localized eIF2α kinase in response to ER stress leading to an attenuation of translational initiation and protein synthesis and activation of pathways leading to cell death or survival (8Rao R.V. Ellerby H.M. Bredesen D.E. Cell Death Differ. 2004; 11: 372-380Crossref PubMed Scopus (811) Google Scholar). Cephalostatin 1 induced a very early and strong phosphorylation of the α-subunit of eIF2α as did the known ER stress-inducer thapsigargin (Fig. 2), further supporting the assumption of ER stress induction by cephalostatin. Similar results were obtained in HeLa and MCF-7 cells (data not shown).FIGURE 2Cephalostatin 1 induces ER stress in Jurkat leukemia T cells. Cells were either left untreated (CO) or treated with cephalostatin 1 (CPH; 1 μm) or as a positive control with TM (1 μg/ml) or TG (3 μm, 2 h) for the indicated times. Western blot analysis was performed as described under "Experimental Procedures" using antibodies against BiP, CHOP, and the phosphorylated form of eIF2α. One representative blot of three is shown. Equal protein loading was controlled by staining membranes with Ponceau S (a representative section of the stained membrane is shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cephalostatin 1 Activates the ASK1-JNK Pathway, Which Is Involved in Cell Death—The ASK1 and the downstream JNK cascade are known to be activated under conditions of ER stress-induced apoptosis (6Kadowaki H. Nishitoh H. Ichijo H. J. Chem. Neuroanat. 2004; 28: 93-100Crossref PubMed Scopus (119) Google Scholar). As observed in Fig. 3A, both kinases were activated very early upon cephalostatin 1 treatment. Phosphorylation of ASK1 at Thr-845, which is correlated with ASK1 activity, was observed after only 15 min and maintained up to at least 2 h of treatment. Its downstream kinase JNK was also already phosphorylated after 15 min, and the intensity of phosphorylation increased further in the course of time.FIGURE 3The ASK1-JNK pathway is important for cephalostatin 1-induced apoptosis. A, Jurkat T cells were left untreated (CO) or stimulated with cephalostatin 1 (CPH; 1 μm) or with TG (3 μm) for the indicated times (p-JNK/JNK, stimulation with TG for 2 h). Western blot experiments were performed using antibodies against the active phosphorylated forms of JNK (Thr-183/Tyr-185) and ASK1 (Thr-845). Membranes were stripped and reprobed with antibodies recognizing the total level of the proteins. B, Jurkat J16 and Jurkat ASK1-DN cells (clone A2–1 and A2–3) were treated for 24 h with cephalostatin 1(CPH; 1 μm) or human TNF (10 nm), and apoptotic cells were quantified by flow cytometry as described under "Experimental Procedures." Inset, the indicated cells were lysed, and equal amounts of protein were separated by SDS-PAGE and further analyzed by Western blot using anti-ASK1 antibodies. The lower arrow points to the endogenous and overexpressed ASK1 proteins, whereas the upper band detects a nonspecific (n.s.) band. C, Jurkat cells and ASK1-DN clone A2–3 were investigated for the activation of JNK after treatment with cephalostatin 1 (CPH; 1 μm) and TNF-α (10 nm). After treatment for 30 min, cell lysates were prepared, and the phosphorylated form of JNK was detected using Western blot analysis. Equal protein loading was controlled by staining membranes with Ponceau S (a representative section of the stained membrane is shown). All experiments were performed three times with consistent results. Bars, mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01 (unpaired two-tailed t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To evaluate the role of the ASK1-JNK pathway in cephalostatin 1-induced apoptosis, two Jurkat clones expressing a dominant negative ASK1 (ASK1-DN) (21Hofmann T.G. Moller A. Hehner S.P. Welsch D. Droge W. Schmitz M.L. Int. J. Cancer. 2001; 93: 185-191Crossref PubMed Scopus (22) Google Scholar) were employed. In both clones of ASK1-DN cells (A2–1, A2–3), only a partial reduction of cephalostatin 1 (1 μm, 24 h)-induced apoptosis was observed as compared with the parental J16 cells (Fig. 3B). TNF-α (10 nm, 24 h) was used as a positive control because TNF-induced apoptosis also partially depends on the activation of ASK1 (25Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (988) Google Scholar). To verify the lack of ASK1 activity of our ASK1-DN clones, J16 and A2–3 clones were treated with cephalostatin 1 or TNF-α and investigated for JNK phosphorylation by Western blot. As expected, JNK phosphorylation was severely impaired in A2–3 cells in both cases compared with control J16 cells (Fig. 3C). In summary, Fig. 3 shows that the ASK1-JNK cascade seems not to play a major role in cephalostatin 1-induced apoptosis.Cephalostatin 1-induced Activation of Caspase-4 Is Necessary for Apoptosis—In humans caspase-4 seems to play a similar role as caspase-12 in mice being localized predominantly to the ER and involved specifically in ER stress-induced apoptosis (10Hitomi J. Katayama T. Eguchi Y. Kudo T. Taniguchi M. Koyama Y. Manabe T. Yamagishi S. Bando Y. Imaizumi K. Tsujimoto Y. Tohyama M. J. Cell Biol. 2004; 165: 347-356Crossref PubMed Scopus (730) Google Scholar). Fig. 4A shows that cephalostatin 1 as well as the well known ER stress inducers tunicamycin and thapsigargin activate caspase-4 time-dependently. A strong reduction of the proform was already observed after 4 h of cephalostatin 1 treatment. Employment of the specific caspase-4 inhibitor Z-LEVD-fmk led to a marked inhibition of cephalostatin 1-induced DNA fragmentation (Fig. 4B), demonstrating a crucial role of caspase-4 in the apoptotic pathway of cephalostatin 1. Caspase-4 contribution to tunicamycin and thapsigargin-induced apoptosis was similar to that seen for cephalostatin 1, further supporting its role in ER stress-induced apoptosis. To further prove the involvement of caspase-4 in cephalostatin 1-i

Referência(s)