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Apoptosis Induced by Proteasome Inhibition in Cancer Cells: Predominant Role of the P53/PUMA Pathway

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Apoptosis Induced by Proteasome Inhibition in Cancer Cells: Predominant Role of the P53/PUMA Pathway Oncogene (2007) 26, 1681–1692 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Apoptosis induced by proteasome inhibition in cancer cells: predominant role of the p53/PUMA pathway CG Concannon1, BF Koehler1,2, Claus Reimertz2, BM Murphy1, C Bonner1, N Thurow2, MW Ward1, AVillunger 3, AStrasser 4,DKo¨ gel2,5 and JHM Prehn1,5 1Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland; 2Experimental Neurosurgery, Centre for Neurology and Neurosurgery, Johann Wolfgang Goethe University Clinics, Theodor-Stern-Kai 7, Frankfurt/Main, Germany; 3Division of Experimental Pathophysiology and Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria and 4The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia The proteasome has emerged as a novel target for Introduction antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous The correct functioning of the ubiquitin-proteasome system. To identify cell death pathways activated in pathway is essential for the degradation of the majority response to inhibition of the proteasome system in cancer of intracellular proteins. Several key regulatory proteins cells, we treated human SH-SY5Y neuroblastoma cells involved in cell proliferation and differentiation are with the selective proteasome inhibitor (PI) epoxomicin regulated by proteasome-mediated proteolysis resulting (Epoxo). Prolonged exposure to Epoxo was associated in the activation or inhibition of specific cell signaling with increased levels of poly-ubiquitinylated proteins and pathways (Adams, 2004a). The proteasome is also p53, release of cytochrome c from the mitochondria, and central to the regulation of cell death and apoptosis. activation of caspases. Analysis of global gene expression The proapoptotic Bcl-2 family proteins Bim, Bik and using high-density oligonucleotide microarrays revealed Bid, which regulate the release of proapoptotic factors that Epoxo triggered transcriptional activation of the two such as cytochrome c and Smac from mitochondria, are Bcl-2-homology domain-3-only (BH3-only) genes p53 known substrates for targeted ubiquitination and upregulated modulator of apoptosis (PUMA) and Bim. degradation (Breitschopf et al., 2000; Marshansky Subsequent studies in PUMA- and Bim-deficient cells et al., 2001; Ley et al., 2003). Indeed, caspases, the key indicated that Epoxo-induced caspase activation and proteases activated during apoptosis, are also regulated apoptosis was predominantly PUMA-dependent. Further by the proteasome. An endogenous caspase inhibitor characterization of the transcriptional response to Epoxo family of proteins, the inhibitor of apoptosis (IAPs), not in HCT116 human colon cancer cells demonstrated that only inhibit active caspases but also target them for PUMA induction was p53-dependent; with deficiency in destruction by acting as ubiquitin ligases and promoting either p53 or PUMA significantly protected HCT116 polyubiquitination of activate caspases (Huang et al., cells against Epoxo-induced apoptosis. Our data suggest 2000; Suzuki et al., 2001). that p53 activation and the transcriptional induction of In recent years modulation of proteasomal function its target gene PUMA play an important role in with specific inhibitors has evolved as a novel target for the sensitivity of cancer cells to apoptosis induced by the treatment of cancers such as multiple myeloma proteasome inhibition, and imply that antineoplastic (Chauhan et al., 2005) as well as lung, colon and therapies with PIs might be especially useful in cancers prostate cancer (Aghajanian et al., 2002; Papandreou with functional p53. et al., 2004; Chauhan et al., 2005). Proteasome Oncogene (2007) 26, 1681–1692. doi:10.1038/sj.onc.1209974; inhibitors (PIs) have been demonstrated to overcome published online 18 September 2006 chemoresistance of tumor cells by enhancing chemo- sensitivity and even acting in synergy with other agents Keywords: proteasome; apoptosis; epoxomicin; Bcl-2 to induce apoptotic cell death of tumor cells (Adams, family; BH3-only protein; p53 2002; Park and Lenz, 2004). Indeed, clinical efficiency of Bortezomib (PS-341/Velcade), a reversible PI has recently been observed in multiple myeloma patients (Richardson et al., 2003). Inhibitors with a broader Correspondence: Professor JHM Prehn, Department of Physiology specificity and irreversible binding have likewise been and Medical Physics, Royal College of Surgeons in Ireland, 123 identified and are currently being tested in preclinical St Stephen’s Green, Dublin 2, Ireland. trials (Melino, 2005). One such inhibitor is epoxomicin E-mail: [email protected] 0 0 5These authors share equal senior authorship. (Epoxo), a naturally found a , b epoxyketone peptide, Received 29 March 2006; revised 27 July 2006; accepted 1 August 2006; which is a highly selective and irreversible inhibitor of published online 18 September 2006 the chymotryptic-, tryptic- and post-glutamyl peptidyl PUMA and proteasome inhibition induced apoptosis CG Concannon et al 1682 hydrolytic-like activities of the proteasome (Meng et al., Results 1999), and was first identified and isolated from a species of actinomycetes on the basis of its antitumor activity Inhibition of proteasome function using Epoxo induces (Hanada et al., 1992). Unlike several widely used PIs, caspase activity and apoptosis Epoxo is highly specific for the proteasome without To investigate the ability of Epoxo to inhibit proteaso- affecting the activity of other non-proteasomal pro- mal activity and induce apoptosis in human SH-SY5Y teases including calpain, trypsin and cathepsin B (Meng neuroblastoma cells we treated cells with 50 nM Epoxo et al., 1999). for varying time periods. Western blot analysis of mono- In addition to the post-translational modification and poly-ubiquitinylated proteins demonstrated a time- of proapoptotic proteins, inhibition of proteasomal dependent increase in the levels of ubiquitinylated function may also regulate several transcription-depen- proteins, characteristic of proteasome dysfunction and dent processes. Indeed, the activity of several transcrip- stress, especially following 16 and 24 h treatment with tion factors, including p53 and nuclear factor kappa B 50 nM Epoxo (Figure 1a). The increased levels of (NF-kB) (Chowdary et al., 1994; Palombella et al., ubiquitinylated proteins was followed by an activation 1994), are known to be modulated by proteasome of DEVDase activity, indicative of the activation of activity. This study was undertaken to analyse the the effector caspases -3 and -7 during this process underlying transcriptional mechanisms leading to (Figure 1b). Epoxo also led to the release of cytochrome apoptosis triggered by inhibition of the proteasome c from mitochondria (Figure 1c) and increased levels of system in cancer cells. Our data demonstrates apoptosis as detected by annexin V binding (Figure 1d). that although the two Bcl-2-homology domain 3-only Further analysis using Hoechst staining revealed con- (BH3-only) genes p53 upregulated modulator of apoptosis densation and fragmentation of most nuclei. Coincu- (PUMA) and Bim are transcriptionally activated by bation with the pan-caspase inhibitor, zVAD.fmk, proteasome inhibition, p53-dependent activation of inhibited these nuclear morphological changes indicat- PUMAplays a predominant role in this type of cell ing that caspases are critically involved in proteasome death. inhibition-induced apoptosis (Figure 1e). Figure 1 Epoxo induces proteasomal stress and apoptosis in human SH-SY5Y neuroblastoma cells. (a) Increase in the level of ubiquitinylated proteins following Epoxo treatment. SH-SY5Y cells were treated with vehicle (DMSO) for 24 h or Epoxo (50 nM) for the indicated time periods. Whole-cell lysates were analysed by Western blotting. Membranes were probed with an antibody recognizing mono- or poly-ubiquitinylated proteins. a-Tubulin served as a loading control. Similar results were obtained in two separate experiments. (b) Time course of Epoxo induced caspase-3 like activity. SH-SY5Y cells were treated with vehicle (DMSO) for 24 h or Epoxo (50 nM) for indicated time periods. Following treatment cells were harvested and whole-cell lysate prepared and used to monitor caspase-3 like activity by measuring cleavage of the fluorogenic substrate Ac-DEVD-AMC (10 mM). Data are means7s.e. from n ¼ 3 separate experiments. Different from control (Con): *, Po0.05. (c) Immunofluoresence showing the redistribution of cytochrome c from mitochondria following treatment with 50 nM Epoxo for 24 h. (d) Quantification of cells undergoing apoptosis following treating with 50 nM Epoxo for the indicated time periods. Cells were stained with using Annexin V-FITC and PI and analysed by flow cytometry. (e) Hoechst staining of SH-SY5Y cells treated with DMSO, Epoxo (50 nM) with or without or pre-treatment with zVAD.fmk (100 mM). Condensation and fragmentation of the nuclear chromatin is clearly evident in the Epoxo-treated cells. Oncogene PUMA and proteasome inhibition induced apoptosis CG Concannon et al 1683 Microarray analysis of transcriptional response to Table 1 (continued ) proteasome inhibition by Epoxo Target genes Affymetrix Up/down- In an effort to identify apoptotic pathways involved in accession no. regulated Epoxo-induced cell death we utilized high-density microarrays to investigate the transcriptional changes NAG-1 (TGF-beta occurring after Epoxo treatment. A16
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