[Paraptosis]
Cancer, a large mass of tissue called tumor, is the uncontrolled growth of abnormal cells anywhere in a body. In many cancer patients, chemotherapy is one of the reasonable choices for survival, but various cancer cells are resistant to chemotherapeutic-drug-induced apoptosis. Numerous data indicate that defects in apoptotic signaling pathways contribute to the development of cancer and to therapy resistance in many types of malignant tumors (Roth, 2009). Therefore, cancer cells that have acquired resistance to apoptosis need novel strategies for inducing non-apoptotic cell death. Non-apoptotic cell death may have considerable merit for the treatment of cancer cells which have acquired resistance to apoptosis (Bröker et al., 2005). Paraptosis characteristically lacks the apoptotic features (paraptosis; para = next to or related to, and apoptosis). It typically does not involve the activation of caspases, the formation of apoptotic bodies, or other characteristics of apoptotic morphologies; it is insensitive to apoptotic inhibitors (e.g., caspase inhibitors and Bcl-xL) and requires protein synthesis (Sperandio et al., 2000; Sperandio et al., 2004). Paraptosis is characterized by a requirement for new gene transcription and translation and AIP-1/Alix (a protein that interacts with the cell death-related calcium-binding protein, ALG-2) as an inhibitor of paraptosis (Sperandio et al., 2004; Valamanesh et al., 2007). Recently, paraptosis was shown to be associated with the activation of the mitogen-activated protein kinase pathways as well as generation of reactive oxidant species (ROS) and Ca2+ (Yoon et al., 2012; Lee et al., 2015; Wang et al., 2012; Kar et al., 2009; Li et al., 2011).
However, the underlying mechanism of paraptosis, in particular the signals responsible for triggering mitochondria and ER dilation, have not yet been fully determined.
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[ER stress]
The vast majority of proteins that a cell secretes or displays on the cell surface first enter the ER, where they fold and mature. To ascertain fidelity in protein folding, cells regulate the protein folding capacity in the ER according to need (Walter & Ron, 2011). Disruption of balance between protein load and folding capacity induces the ER stress. Upon ER stress, cells activate a series of complementary adaptive mechanisms to cope with protein-folding alteration, which together are known as UPR (Hetz, 2012). The UPR monitors condition in the ER, sensing an insufficiency in the ER’s protein folding capacity and transduces information about the protein-folding status in the ER lumen to the nucleus and cytosol to buffer fluctuation in unfolded protein load (Ron & Walter, 2007;
Schröder & Kaufman, 2005; Hetz et al., 2011). Initiation of the UPR is mediated by three ER transmembrane sensors: activating transcription factor 6α (ATF6α), inositol-requiring enzyme 1α (IRE1α), and PKR-like endoplasmic reticulum kinase (PERK) (Ron & Walter, 2007). The UPR results in global changes in gene expression to restore the ER homeostasis (Huber et al., 2013): While ATF6α activates genes to increase the protein-folding capacity through the upregulation of chaperone proteins, IRE1α (via RNase activity) and PERK (via kinase activity) both branches decrease the load of proteins entering the ER through activation of degradation and inhibition of translation. In unstressed ER, Bip/Grp78 binds to the ER luminal domains of these sensor proteins and keeps them inactive (Szegezdi et al., 2006). Upon sensing the accumulation of misfolded protein in the ER lumen, Bip/Grp78 dissociates from its clients and translocates to the ER lumen to help protein folding (Bertolotti et al., 2000; Liu & Kaufman, 2003). Improperly folded proteins in the ER lumen are delivered for proteasomal degradation after retro-translocation into the cytosol, a process called ER-associated degradation (ERAD) (Smith et al., 2011). Therefore, accumulation of misfolded protein is important for the ER stress, and the ERAD pathway is required for maintenance of the ER
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homeostasis. But, prolonged activity of the UPR, an indication that ER stress cannot be mitigated and homeostasis cannot be reestablished, correlates with cell death (Walter & Ron, 2011). During the induction of paraptosis, dilation of the ER seems to be mediated by prolonged UPR or the ER stress At least, the ER dilation is accompanied by the increase in the ER stress marker proteins.
[The ubiquitin proteasome system (UPS)]
Like all intracellular components, the proteome is in a dynamic state of biogenesis and proteolysis (Glickman & Ciechanover, 2002). Proteolysis is the enzymatic process that is associated with the breakdown of proteins into their component polypeptide or amino acids. This process is served by a diverse group of proteases, protein hydrolysis enzymes, involving the proteasome, which is multifunctional proteolytic complex. Proteasome is the 26S proteasome complex, macromolecular machinery, which consists of two 19S regulatory subunits and one 20S catalytic subunit (Gallastegui et al., 2010). Proteasome differs from typical proteases in many characteristics, including ATP dependency, systematic process, and unique recognizing-molecule, etc. (Kisselev & Goldberg, 2001). In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins (Grigoreva et al., 2015). Because studies on the proteasome inhibitors have shown that the bulk of cellular proteins, 80–90%, are degraded by the proteasome, ubiquitin-proteasome system (UPS) is important for the maintenance of protein homeostasis via protein degradation (Voges et al., 1999; Rock et al., 1994; Craiu et al., 1997; Crawford & Irvine, 2013). The UPS is tightly regulated to prevent the intracellular environment from accumulation of poly-ubiquitinated proteins, involving abnormal and damaged proteins (Ciechanover, 2005). UPS is accomplished in multi-steps protein degradation: 1) poly-ubiquitination of proteins, 2) recognition through poly-ubiquitin chains by the 19S proteasome, and 3)
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degradation within 20S proteasome (Ciechanover, 2005). For the poly-ubiquitination, three different enzymes are involved in the cascade reaction for covalent conjugation of ubiquitin protein to the target proteins (Hershko &
Ciechanover 1998): Ub-activating enzymes (E1), Ub-conjugating or -carrier enzymes (E2), and Ub-protein ligases (E3). In next step, poly-ubiquitin chain plays as a signal peptide to target the substrate to the proteasome for degradation. Poly-ubiquitin chain is recognized through several subunits in 19S proteasome prior to translocation into 20S core complex (Finley, 2009; Hao et al., 2013). In the last steps, translocated protein is cleaved by several proteolytic subunits in 20S proteasome. 20S proteasome has barrel-like shape and is comprised of two outer layers containing seven α-subunits (α1- α7 subunits) and two inner rings made up seven β-subunits (β1- β7 subunits) (Lander et al., 2012). Some of them, β1, β2, and β5 subunits have a proteolytic activity: β1 is related with a caspase-like activity, β2 with trypsin-like activity, and β5 with chymotrypsin-like activity (Grigoreva et al., 2015; Groll et al., 1999; Kisselev et al., 2003; Britton et al., 2009). UPS plays a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. (Voges et al., 1999; Grigoreva et al., 2015; D'Arcy et al., 2015). Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target (Grigoreva et al., 2015; D'Arcy et al., 2015).
[Bortezomib]
Proteasome specific inhibitors have been regarded to be positive clinical benefits for cancer therapy. Bortezomib (PS341, Velcade), the first FDA-approved proteasome inhibitors (PIs), is now used in the clinic for the treatment of newly diagnosed and relapsed multiple myeloma and mantle cell lymphoma (Kane et al.,
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2006). Bortezomib inhibits mainly β5 and partially β1 of proteasome complex (Crawford et al. 2006, Chen et al., 2011) and results in the disruption of protein homeostasis and induction of ER stress, contributing to its cytotoxicity (Obeng et al., 2006). Since proteasome activity is required for the retrotranslocation of misfolded proteins across the ER membrane into the cytoplasm and subsequent degradation of these proteins, proteasome inhibitors induce accumulation of misfolded protein in the ER lumen, followed by the stress to ER (Kostova Z &
Wolf, 2003; Obeng et al., 2006). Under this stress condition, cells utilize the unfolded protein response (UPR) signaling for recovery. Prolonged ER stress induces the terminal UPR associated with ER-stress-mediated cell death, although transient UPR initially increases the machinery for resolving these problems (Hetz et al. 2015; Schröder & Kaufman, 2005). However, recent studies have shown that when bortezomib was used as a single agent in newly diagnosed multiple myeloma patients, approximately 50% did not achieve a partial response or better (Ruschak et al., 2011; Dispenzieri et al., 2010). Moreover, the clinical response to bortezomib in other hematologic malignancies and solid tumors remain unsatisfactory (Cortes J et al., 2004; Kale AJ & Moore BS, 2012). Therefore, there is a growing challenge for overcoming the resistance to bortezomib and for the improvement of the survival of patients.
[Nutlin-3]
Nutlin-3 is a small molecule inhibitor of the MDM2/p53 interaction, which leads to the non-genotoxic p53 stabilization, activation of cell cycle arrest and apoptosis pathways (Secchiero et al., 2011). MDM2 protein is an ubiquitin-E3 ligase to be able to degrade p53 (Chène, 2003). But, recently several research groups have shown that the nutlin-3 has another function in the cells, independently of p53 (Kurokawa et al., 2013; Valentine et al., 2011).
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Here, we show that the nutlin-3 effectively overcomes the resistance of various cancer cells with defective p53 to bortezomib, and thus through p53-independent mechanism. Interestingly, combined treatment with subtoxic doses of bortezomib and nutlin-3 induced severe vacuolization, which was derived of the dilation of both the ER and mitochondria, prior to cell death. In this process, accumulation of poly-ubiquitinated proteins and the proteins associated with ER stress, including CHOP, as well as mitochondria Ca2+ influx was observed. We found that CHOP induction plays a critical role in the ER dilation and mitochondrial Ca2+ influx contributes to the cell death induced by the combined treatment with bortezomib and nultlin-3. Combined regimen of bortezomib and nultin-3 may offer an effective therapeutic strategy to overcome the resistance of cancer cells to bortezomib. In this study, we found that nutlin-3 effectively overcomes the resistance of various cancer cells to bortezomib, independently of p53, via induction of paraptosis-like cell death and explored its underlying mechanisms.
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